WO2006078318A2 - Immunogenic compositions for gram positive bacteria such as streptococcus agalactiae - Google Patents

Immunogenic compositions for gram positive bacteria such as streptococcus agalactiae Download PDF

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WO2006078318A2
WO2006078318A2 PCT/US2005/027239 US2005027239W WO2006078318A2 WO 2006078318 A2 WO2006078318 A2 WO 2006078318A2 US 2005027239 W US2005027239 W US 2005027239W WO 2006078318 A2 WO2006078318 A2 WO 2006078318A2
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gbs
gas
proteins
strain
protein
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PCT/US2005/027239
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French (fr)
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WO2006078318A3 (en
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John L. Telford
Guido Grandi
Peter Lauer
Marirosa Mora
Immaculada Margarit Y. Ros
Domenico Maione
Guiliano Bensi
Daniela Rinaudo
Vega Masignani
Michelle Barocchi
Rino Rappuloi
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Novartis Vaccines And Diagnostics Inc.
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Priority to US11/658,842 priority Critical patent/US20090317420A1/en
Priority to CA002575548A priority patent/CA2575548A1/en
Priority to JP2007523880A priority patent/JP2008508320A/en
Priority to EP05856900A priority patent/EP1784211A4/en
Publication of WO2006078318A2 publication Critical patent/WO2006078318A2/en
Publication of WO2006078318A3 publication Critical patent/WO2006078318A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the identification of adhesin islands within the genome Streptococcus agalactiae ("GBS") and the use of adhesin island amino acid sequences encoded by these adhesin islands in compositions for the treatment or prevention of GBS infection. Similar sequences have been identified in other Gram positive bacteria.
  • the invention further includes immunogenic compositions comprising adhesin island amino acid sequences of Gram positive bacteria for the treatment or prevention of infection of Gram positive bacteria.
  • Preferred immunogenic compositions of the invention include an adhesin island surface protein which may be formulated or purified in an oligomeric or pilus form.
  • GBS has emerged in the last 20 years as the major cause of neonatal sepsis and meningitis that affects 0.5 - 3 per 1000 live births, and an important cause of morbidity among older age groups affecting 5 — 8 per 100,000 of the population.
  • Current disease management strategies rely on intrapartum antibiotics and neonatal monitoring which have reduced neonatal case mortality from >50% in the 1970's to less than 10% in the 1990's. Nevertheless, there is still considerable morbidity and mortality and the management is expensive. 15 — 35% of pregnant women are asymptomatic carriers and at high risk of transmitting the disease to their babies. Risk of neonatal infection is associated with low serotype specific maternal antibodies and high titers are believed to be protective.
  • invasive GBS disease is increasingly recognized in elderly adults with underlying disease such as diabetes and cancer.
  • the "B” in “GBS” refers to the Lancef ⁇ eld classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate.
  • Lancefield identified 13 types of C carbohydrate, designated A to O, that could be serologically differentiated.
  • the organisms that most commonly infect humans are found in groups A, B, D, and G.
  • strains can be divided into at least 9 serotypes (Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII) based on the structure of their polysaccharide capsule.
  • serotypes Ia, Ib, II, and III were equally prevalent in normal vaginal carriage and early onset sepsis in newborns.
  • Type V GBS has emerged as an important cause of GBS infection in the USA, however, and strains of types VI and VIII have become prevalent among Japanese women.
  • Gram positive bacteria are frequent human pathogens and include Staphylococcus (such as S. aureus), Streptococcus (such as S. pyogenes (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutatis), Enterococcus (such as E.faecalis and E. faeciuin), Clostridium (such as C. difficile), Listeria (such as L. monocytogenes) and Cory neb act erium (such as C. diphtheria).
  • Staphylococcus such as S. aureus
  • Streptococcus such as S. pyogenes (GBS), S. py
  • compositions for providing immunity against disease and/or infection of Gram positive bacteria.
  • the compositions are based on the identification of adhesin islands within Streptococcal genomes and the use of amino acid sequences encoded by these islands in therapeutic or prophylactic compositions.
  • the invention further includes compositions comprising immunogenic adhesin island proteins within other Gram positive bacteria in therapeutic or prophylactic compositions.
  • GBS Adhesin Island 1 "AI-I” or “GBS AI-I”
  • This adhesin island is thought to encode surface proteins which are important in the bacteria's virulence.
  • Applicants have discovered that surface proteins within GBS Adhesin Islands form a previously unseen pilus structure on the surface of GBS bacteria. Amino acid sequences encoded by such GBS Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of GBS infection.
  • a preferred immunogenic composition of the invention comprises an AI-I surface protein, such as GBS 80, which may be formulated or purified in an oligomeric (pilus) form.
  • the oligomeric form is a hyperoligomer. Electron micrographs depicting some of the first visualizations of this pilus structure in a wild type GBS strain are shown in Figures 16, 17, 49, and 50.
  • Applicants have transformed a GBS strain with a plasmid comprising the AI surface protein GBS 80 which resulted in increased production of that AI surface protein.
  • the electron micrographs of this mutant GBS strain in Figures 13 - 15 reveal long, hyper-oligomeric structures comprising GBS 80 which appear to cover portions of the surface of the bacteria and stretch far out into the supernatant.
  • These hyper-oligomeric pilus structures comprising a GBS AI surface protein may be purified or otherwise formulated for use in immunogenic compositions.
  • GBS AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("AI-I proteins").
  • AI-I includes polynucleotide sequences encoding for two or more of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648.
  • One or more of the AI-I polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the AI-I open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF. f'" !
  • AI-I surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the AI surface proteins of the invention may affect the ability of the GBS bacteria to adhere to and invade epithelial cells.
  • AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer.
  • one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • AI-I may encode at least one surface protein.
  • AI-I may encode at least two surface proteins and at least one sortase.
  • AI-I encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif or other sortase substrate motif.
  • the GBS AI-I protein of the composition may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648.
  • GBS AI-I surface proteins GBS 80 and GBS 104 are preferred for use in the immunogenic compositions of the invention.
  • AI-I may also include a divergently transcribed transcriptional regulator such as araC ⁇ i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). It is believed that araC may regulate the expression of the GBS AI operon. (See Korbel et al., Nature Biotechnology (2004) 22(7): 911 - 917 for a discussion of divergently transcribed regulators in E. col ⁇ ).
  • araC may regulate the expression of the GBS AI operon.
  • a second adhesin island "Adhesin Island-2", “AI-2” or “GBS AI-2”, has also been identified in numerous GBS serotypes. Amino acid sequences encoded by the open reading frames of AI-2 may also be used in immunogenic compositions for the treatment or prevention of GBS infection.
  • GBS AI-2 comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, AI-2 includes open reading frames encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525. The GBS AI-2 sequences may be divided into two subgroups. In one embodiment, AI-2 includes open reading frames encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406. This collection of open reading frames may be generally referred to as GBS AI-2 subgroup 1.
  • AI-2 may include open reading frames encoding for two or more of 01520, 01521, 01522, 01523, 01523, 01524 and 01525.
  • This collection of open reading frames may be generally referred to as GBS AI-2 subgroup 2.
  • One or more of the AI-2 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the AI-2 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
  • ril ,, Qne,.or typically include an LPXTG motif (such as LPXTG
  • AI-2 may encode for at least one surface protein.
  • AI-2 may encode for at least two surface proteins and at least one sortase.
  • AI-2 encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif.
  • the AI-2 protein of the composition may be selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525.
  • AI- 2 surface proteins GBS 67, GBS 59, and 01524 are preferred AI-2 proteins for use in the immunogenic compositions of the invention.
  • GBS 67 or GBS 59 is particularly preferred.
  • GBS AI-2 may also include a divergently transcribed transcriptional regulator such as a RofA like protein (for example rogB). As in AI-I, rogB is thought to regulate the expression of the AI-2 operon.
  • a divergently transcribed transcriptional regulator such as a RofA like protein (for example rogB).
  • rogB is thought to regulate the expression of the AI-2 operon.
  • the GBS AI proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against GBS infection.
  • the invention may include an immunogenic composition comprising one or more GBS AI-I proteins and one or more GBS AI-2 proteins.
  • the immunogenic compositions may also be selected to provide protection against an increased range of GBS serotypes and strain isolates.
  • the immunogenic composition may comprise a first and second GBS AI protein, wherein a full length polynucleotide sequence encoding for the first GBS AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second GBS AI protein.
  • each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple GBS serotypes and strain isolates.
  • each antigen is presnt in the genomes of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) GBS strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5 or more) GBS serotypes.
  • GBS AI-I Group B Streptococcus surface exposure of GBS 104 is dependent on the concurrent expression of GBS 80. It is thought that GBS 80 is involved in the transport or localization of GBS 104 to the surface of the bacteria.
  • the two proteins may be oligomerized or otherwise chemically or physically associated. It is possible that this association involves a conformational change in GBS 104 that facilitates its transition to the surface of the GBS bacteria.
  • one or more AI sortases may also be involved in this surface localization and chemical or physical association. Similar relationships are thought to exist within GBS AI-2.
  • the compositions of the invention may therefore include at least two AI proteins, wherein the two AI proteins are physically or chemically associated.
  • the two AI proteins form an oligomer.
  • one or more of the AI proteins are in a hyper-oligomeric form.
  • the associated AI proteins may be purified or isolated from a GBS bacteria or recombinant host cell. It 1 Is .alsp.ao.pbiect Qflh ⁇ jraftsnti ⁇ n to provide further and improved compositions for p Ii il / U !!:::i ⁇ ILIi H:::i ⁇ / c:; ./ n:;;:: somehow3 h» providing prophylactic or therapeutic protection against disease and/or infection of Gram positive bacteria.
  • compositions are based on the identification of adhesin islands within Streptococcal genomes and the use of amino acid sequences encoded by these islands in therapeutic or prophylactic compositions.
  • the invention further includes compositions comprising immunogenic adhesin island proteins within other Gram positive bacteria in therapeutic or prophylactic compositions.
  • Preferred Gram positive adhesin island proteins for use in the invention may be derived from Staphylococcus (such as S. aureus), Streptococcus (such as S. agalactiae (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutans), Enterococcus (such as E.faecalis and E.
  • the Gram positive adhesin island surface proteins are in oligomeric or hyperologimeric form.
  • adhesin islands within the genomes of several Group A Streptococcus serotypes and isolates. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus.
  • Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fasciitis.
  • poststreptococcal autoimmune responses are still a major cause of cardiac pathology in children.
  • Group A Streptococcal infection of its human host can generally occur in three phases.
  • the first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces. Generally this attachment phase begins in the throat or the skin. The deeper the tissue level infected, the more severe the damage that can be caused.
  • the bacteria secretes a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. This toxin binds to susceptible host cell receptors and triggers innappropropriate immune responses by these host cells, resulting in pathology. Because the toxin can diffuse throughout the host, the necrosis directly caused by the GAS toxins may be physically located in sites distant from the bacterial infection.
  • the final phase of GAS infection can occur long after the original bacteria have been cleared from the host system. At this stage, the host's previous immune response to the GAS bacteria due to cross reactivity between epitopes of a GAS surface protein, M, and host tissues, such as the heart.
  • a general review of GAS infection can be found in Principles of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
  • an effective vaccine against GAS will preferably facilitate host elimination of the bacteria during the initial attachment and invasion stage.
  • Isolates of Group A Streptococcus are historically classified according to the M surface protein described abo ⁇ e.
  • the M protein is surface exposed trypsin-sensitive protein generally capitis!' ⁇ .. ⁇ Slffli ⁇ H ⁇ WS ⁇ M ⁇ in an al P ha helical formation.
  • the carboxyl terminus is anchored in the cytoplasmic membrane and is highly conserved among all group A streptococci.
  • the amino terminus which extend through the cell wall to the cell surface, is responsible for the antigenic variability observed among the 80 or more serotypes of M proteins.
  • a second layer of classification is based on a variable, trypsin-resistant surface antigen, commonly referred to as the T-antigen.
  • T serological typing Decades of epidemiology based on M and T serological typing have been central to studies on the biological diversity and disease causing potential of Group A Streptococci. While the M-protein component and its inherent variability have been extensively characterized, even after five decades of study, there is still very little known about the structure and variability of T-antigens. Antisera to define T types is commercially available from several sources, including Sevapharma (http://www.sevapharma.cz/en).
  • T-antigen T-type 6
  • M 6 strain of GAS M 6 strain of GAS
  • FCT Fibronectin-binding, Collagen-binding T-antigen
  • GAS AI sequences can be identified in numerous M types, Applicants have surprisingly pilus subunits from the four different GAS AI types and specific T classifications. While other trypsin-resistant surface exposed proteins are likely also implicated in the T classification designations, the discovery of the role of the GAS adhesin islands (and the associated hyper-oligomeric pilus like structures) in T classification and GAS serotype variance has important implications for prevention and treatment of GAS infections. Applicants have identified protein components within each of the GAS adhesin islands which are associated with the pilus formation. These proteins are believed to be involved in the bacteria's initial adherence mechanisms. Immunological recognition of these proteins may allow the host immune response to slow or prevent the bacteria's transition into the more pathogenic later stages of infection.
  • GBS pili structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysaccharide matrix).
  • Biofilms are generally associated with bacterial resistance, as antibiotic treatments and host immune response are frequently unable to erradicate all of the bacteria components of the biofilm.
  • Direction of a host immune response against surface proteins exposed during the first steps of bacterial attachment is preferable.
  • the invention therefore provides for improved immunogenic compositions against GAS infection which may target GAS bacteria during their initial attachment efforts to the host epithelial cells and may provide protection against a wide range of GAS serotypes.
  • the immunogenic compositions of the invention include GAS AI surface proteins which may be formulated in an oligomeric, or hyperoligomeric (pilus) form.
  • the immunogenic compositions of the invention may include one or more GAS AI surface proteins.
  • the invention also includes combinations of GAS AI surface proteins. Combinations of GAS AI surface proteins may be selected from the same adhesin island or they may be selected from different GAS adhesin islands.
  • Amino acid sequence encoded by such GAS Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of GAS infection.
  • Preferred immunogenic compositions of the invention comprise a GAS AI surface protein which has been formulated or purified in an oligomeric (pilus) form.
  • the oligomeric fo ⁇ n is a hyperoligomer.
  • GAS Adhesin Islands generally include a series of open reading frames within a GAS genome that encode for a collection of surface proteins and sortases.
  • a GAS Adhesin Island may encode for an amino acid sequence comprising at least one surface protein.
  • the Adhesin Island therefore, may encode at least one surface protein.
  • a GAS Adhesin Island may encode for at least two surface proteins and at least one sortase.
  • a GAS Adhesin Island encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • GAS AI surface proteins may participate in the formation of a pilus structure on the surface of the Gram positive bacteria.
  • B- I , j i-.. 'GAS- Adfeesjm Islanfepfiitheiiinipption preferably include a divergently transcribed transcriptional regulator.
  • the transcriptional regulator may regulate the expression of the GAS AI operon. Examples of transcriptional regulators found in GAS AI sequences include RofA and Nra.
  • the GAS AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen.
  • One or more of the GAS AI surface proteins may comprise a f ⁇ mbrial structural subunit.
  • One or more of the GAS AI surface proteins may include an LPXTG motif or other sortase substrate motif.
  • the LPXTG motif may be followed by a hydrophobic region and a charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. See Barnett, et al, J. Bacteriology (2004) 186 (17): 5865-5875.
  • GAS AI sequences may be generally categorized as Type 1, Type 2, Type 3, or Type 4, depending on the number and type of sortase sequences within the island and the percentage identity of other proteins (with the exception of RofA and cpa) within the island.
  • Schematics of the GAS adhesin islands are set forth in FIGURE 51A and FIGURE 162.
  • "GAS Adhesin Island-1 or "GAS AI- 1” comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (“GAS AI-I proteins").
  • GAS AI-I preferably comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator.
  • GAS AI-I surface proteins may include a fibronectin binding protein, a collagen adhesion protein and a f ⁇ mbrial structural subunit.
  • the f ⁇ mbrial structural subunit also known as tee ⁇
  • the collagen adhesion protein is thought to act as an accessory protein facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule.
  • GAS AI-I includes polynucleotide sequences encoding for two or more of M6_SpyO157, M6_SpyO158, M6_SpyO159, M6_SpyO16O, M6_SpyO161.
  • the GAS AI-I may also include polynucleotide sequences encoding for any one of CDC SS 410_f ⁇ mbrial, ISS3650_fimbrial, DSM2071J ⁇ mbrial
  • a preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which may be formulated or purified in an oligomeric (pilus) form.
  • the oligomeric form is a hyperoligomer.
  • the immunogenic composition of the invention may alternatively comprise an isolated GAS AI-I surface protein in oligomeric (pilus) form.
  • the oligomer or hyperoHgomeric pilus structures comprising GAS AI-I surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • One or more of the GAS AI-I polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-I open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
  • GAS AI-I surface proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI- 1 may enojOide
  • GAS AI-I may encode for at least two surface proteins and at least one sortase.
  • GAS AI-I encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif.
  • GAS AI-I preferably includes a srtB sortase.
  • GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
  • the GAS AI-I protein of the composition may be selected from the group consisting of M6_SpyO157, M6_SpyO158, M6_SpyO159, M6_Spy0160 M6_Spy0161, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_f ⁇ mbrial.
  • GAS AI-I surface proteins M6_S ⁇ yO157 (a fibronectin binding protein), M6_S ⁇ yO159 (a collagen adhesion protein, Cpa), M6_Spy0160 (a fimbrial structural subunit, tee ⁇ ), CDC SS 410_fimbrial (a fimbrial structural subunit), ISS3650_f ⁇ mbrial (a fimbrial structural subunit), and DSM2071_fimbrial (a fimbrial structural subunit) are preferred GAS AI-I proteins for use in the immunogenic compositions of the invention.
  • the fimbrial structural subunit tee6 and the collagen adhesion protein Cpa are preferred GAS AI -1 surface proteins.
  • each of these GAS AI-I surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with serine).
  • LPXTG SEQ ID NO: 122
  • LPXSG SEQ ID NO: 134
  • GAS AI-I may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the GAS AI protein open reading frames, but it transcribed in the opposite direction).
  • rofA a divergently transcribed transcriptional regulator
  • the GAS AI-I surface proteins may be used alone, in combination with other GAS AI-I surface proteins or in combination with other GAS AI surface proteins.
  • the immunogenic compositions of the invention include the GAS AI-I fimbrial structural subunit (tee ⁇ ) and the GAS AI-I collagen binding protein. Still more preferably, the immunogenic compositions of the invention include the GAS AI-I fimbrial structural subunit (tee ⁇ ).
  • a second GAS adhesion island "GAS Adhesin Island-2" or “GAS AI-2,” has also been identified in GAS serotypes.
  • Amino acid sequences encoded by the open reading frames of GAS AI- 2 may also be used in immunogenic compositions for the treatment or prevention of GAS infection.
  • a preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer.
  • a preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-2 surface protein in oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising GAS AI-2 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • GAS AI-2 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-2 proteins"). Gjf iSfAT-p p ⁇ - ⁇ U? ⁇ d
  • GAS AI-2 includes polynucleotide sequences encoding for two or more of GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SpyO133, and GAS20.
  • One or more of the GAS AI-2 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-2 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
  • GAS AI-2 surface proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI-2 may encode for at least one surface protein.
  • GAS AI-2 may encode for at least two surface proteins and at least one sortase.
  • GAS AI-2 encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif.
  • GAS AI-2 preferably includes a srtB sortase and a srtCl sortase.
  • GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
  • GAS srtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO: 167) motif.
  • GAS srtCl may be differentially regulated by rofA.
  • the GAS AI-2 protein of the composition may be selected from the group consisting of
  • GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SpyO133, and GAS20 are preferred for use in the immunogenic compositions of the invention.
  • GAS 16 is thought to form the shaft portion of the pilus like structure, while GAS 15 (the collagen adhesion protein Cpa) and GAS 18 are thought to act as accessory proteins facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule.
  • each of these GAS AI-2 surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VVXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
  • LPXTG sortase substrate motif such as LPXTG (SEQ ID NO: 122), VVXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
  • GAS AI-2 may also include a divergently transcribed transcriptional regulator such as rofA ⁇ i.e., the transcriptional regulator is located near or adjacent to the GAS AI protein open reading frames, but it transcribed in the opposite direction).
  • the GAS AI-2 surface proteins may be used alone, in combination with other GAS AI-2 surface proteins or in combination with other GAS AI surface proteins.
  • the immunogenic compositions of the invention include the GAS AI-2 fimbrial protein (GAS 16), the GAS AI-2 collagen binding protein (GAS 15) and GAS 18 (Ml_SpyO13O).
  • the immunogenic compositions of the invention include the GAS AI-2 fimbrial protein (GAS 16).
  • GAS Adhesin Island-3 or “GAS AI-3,” has also been identified in numerous GAS serotypes. Amino acid sequences encoded by the open reading frames of compositions for the treatment or prevention of GAS infection.
  • a preferred immunogenic composition of the invention comprises a GAS AI-3 surface protein which may be formulated or purified in an oligomeric (pilus) form.
  • the oligomeric form is a hyperoligomer.
  • a preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-3 surface protein in oligomeric (pilus) form.
  • the oligomer or hyperoligomeric pilus structures comprising GAS AI-3 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • GAS AI-3 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-3 proteins").
  • GAS AI-3 preferably comprises surface proteins, a srtC2 sortase, and a Negative transcriptional regulator (Nra) divergently transcribed transcriptional regulator.
  • GAS AI-3 surface proteins may include a collagen binding protein, a f ⁇ mbrial protein, and a F2 like fibronectin-binding protein.
  • GAS AI-3 surface proteins may also include a hypothetical surface protein. The f ⁇ mbrial protein is thought to form the shaft portion of the pilus like structure, while the collagen adhesion protein (Cpa) and the hypothetical surface protein are thought to act as accessory proteins facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule.
  • Preferred AI-3 surface proteins include the fimbrial proein, the collagen binding protein and the hypothetical protein.
  • each of these GAS AI-3 surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
  • LPXTG sortase substrate motif such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
  • GAS AI-3 includes polynucleotide sequences encoding for two or more of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SpsOlOO, SpsOlOl, Sps0102, Sps0103, Sps0104, Sps0105, SpsO106, orf78, orf79, orf80, orfSl, orf82, orf83, orf84, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_O13O, spyM18_0131, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoMO 1000152, SpyoM01000151, SpyoMO 1000150, SpyoMO 1000
  • GAS AI-3 may include open reading frames encoding for two or more of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104.
  • GAS AI-3 may include open reading frames encoding for two or more of SpsOlOO, SpsOlOl, Sps01O2, Sps0103, Sps0104, Sps0105, and SpsOlO ⁇ .
  • GAS AI-3 may include open reading frames encoding for two or more of orf78, orf79, orfSO, orf ⁇ l, orf82, orf83, and orf84.
  • GAS AI-3 may include open reading frames encoding for two or more of s ⁇ yM18_0126, spyM18_0127, s ⁇ yM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132.
  • GAS AI-3 may include open reading frames encoding for two or more of SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and SpyoM01000149.
  • GAS AI-I may also include polynucleotide sequences encoding for any one of ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
  • ⁇ iucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-3 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
  • One or more of the GAS AI-3 surface proteins typically include an LPXTG motif (such as
  • GAS AI-3 may encode for at least one surface protein.
  • GAS AI-3 may encode for at least two surface proteins and at least one sortase.
  • GAS AI-3 encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif.
  • GAS AI-3 preferably includes a srtC2 type sortase.
  • GAS srtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail.
  • GAS SrtC2 may be differentially regulated by Nra.
  • the GAS AI-3 protein of the composition may be selected from the group consisting of
  • GAS AI-3 may also include a transcriptional regulator such as Nra.
  • GAS AI-3 may also include a LepA putative signal peptidase I protein.
  • the GAS AI-3 surface proteins may be used alone, in combination with other GAS AI-3 surface proteins or in combination with other GAS AI surface proteins.
  • the immunogenic compositions of the invention include the GAS AI-3 fimbrial protein, the GAS AI-3 collagen binding protein, the GAS AI-3 surface protein (such as S ⁇ yM3_0102, M3_Sps0104, M5_orf82, or spyM18_O130), and fibronectin binding protein PrtF2. More preferably, the immunogenic compositions of the invention include the GAS AI-3 fimbrial protein, the GAS AI-3 collagen binding protein, and the GAS AI-3 surface protein.
  • the immunogenic compositions of the invention include the GAS AI-3 fimbrial protein.
  • p £ '"p: ⁇ firybSWlM::fe ⁇ liJ ⁇ 'tf!tB l&S AI-3 fimbrial protein include SpyM3_0100, M3_Sps0102, M5_orf80, spyM18_128, SpyoM01000153, ISS3040_fimbrial, ISS3776_fimbrial, ISS4959Jimbrial.
  • GAS AI-3 collagen binding protein examples include SpyM3_0098, M3_S ⁇ s0100, M5_orf 78, s ⁇ yM18_0126, and SpyoMO 1000155.
  • GAS AI-3 fibronectin binding protein PrtF2 include SpyM3_0104, M3_Sps0106, M5_orf84 and spyM18_0132, and SpyoMO 1000149.
  • GAS Adhesin Island-4" or GAS AI-4 has also been identified in GAS serotypes.
  • Amino acid sequences encoded by the open reading frames of GAS AI- 4 may also be used in immunogenic compositions for the treatment or prevention of GAS infection.
  • a preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which may be formulated or purified in an oligomeric (pilus) form.
  • the oligomeric form is a hyperoligomer.
  • a preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-4 surface protein in oligomeric (pilus) form.
  • the oligomer or hyperoligomeric pilus structures comprising GAS AI-3 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • the oligomeric or hyperoligomeric pilus structures comprising GAS AI-4 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • GAS AI-4 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (“GAS AI-4 proteins").
  • This GAS adhesin island 4 (“GAS AI-4") comprises surface proteins, a srtC2 sortase, and a RofA regulatory protein.
  • GAS AI-4 surface proteins within may include a fimbrial protein, Fl and F2 like fibronectin-binding proteins, and a capsular polysaccharide adhesion protein (cpa).
  • GAS AI-4 surface proteins may also include a hypothetical surface protein in an open reading frame (orf).
  • each of these GAS AI-4 surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
  • GAS AI-4 includes polynucleotide sequences encoding for two or more of 19224134, 19224135, 19224136, 19224137, 19224138, 19224139, 19224140, and 19224141.
  • a GAS AI-4 polynucleotide may also include polynucleotide sequences encoding for any one of 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, ISS4538_fimbrial.
  • One or more of the GAS AI-4 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-4 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
  • p C ' ⁇ ' M&E ⁇ B1 ⁇ ' ⁇ ®W4M$M ⁇ e proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI-4 may encode for at least one surface protein.
  • GAS AI-4 may encode for at least two surface proteins and at least one sortase.
  • GAS AI-4 encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif.
  • GAS AI-4 includes a SrtC2 type sortase.
  • GAS SrtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail.
  • the GAS AI-4 protein of the composition may be selected from the group consisting of
  • GAS AI-4 surface proteins 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069__fimbrial, CDC SS 635_f ⁇ mbrial, ISS4883_fnnbrial, ISS4538_fimbrial are preferred proteins for use in the immunogenic compositions of the invention.
  • GAS AI-4 may also include a divergently transcribed transcriptional regulator such as RofA ⁇ i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction.
  • GAS AI-4 may also include a LepA putative signal peptidase I protein and a MsmRL protein.
  • the GAS AI-4 surface proteins may be used alone, in combination with other GAS AI-4 surface proteins or in combination with other GAS AI surface proteins.
  • the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein (EftLSL or 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, or ISS4538_fimbrial), the GAS AI-4 collagen binding protein, the GAS AI-4 surface protein (such as M12 isolate A735 orf 2), and fibronectin binding protein PrtFl and PrtF2.
  • the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein, the GAS AI-4 collagen binding protein, and the GAS AI-4 surface protein. Still more preferably, the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein.
  • the GAS AI proteins of the invention maybe used in immunogenic compositions for prophylactic or therapeutic immunization against GAS infection.
  • the invention may include an immunogenic composition comprising one or more GAS Al- 1 proteins and one or more of any of GAS AI-2, GAS AI-3, or GAS AI-4 proteins.
  • the invention includes an immunogenic composition comprising at least two GAS AI proteins where each protein is selected from a different GAS adhesin island.
  • the two GAS AI proteins may be selected from one of the following GAS AI combinations: GAS AM and GAS AI-2; GAS AI-I and GAS AI-3; GAS AI-I and GAS AI-4; GAS AI-2 and GAS AI-3; GAS AI-2 and GAS AI-4; and GAS AI 3 and GAS AI-4.
  • the combination includes fimbrial proteins from one or more GAS adhesin islands.
  • F" C ' T ⁇ ' ⁇ S ⁇ MdySiie'ci ⁇ ni&33i ⁇ is i' iiay also be selected to provide protection against an increased range of GAS serotypes and strain isolates.
  • the immunogenic composition may comprise a first and second GAS AI protein, wherein a full length polynucleotide sequence encoding for the first GAS AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second GAS AI protein.
  • each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple GAS serotypes and strain isolates.
  • each antigen is present in the genomes of at least two (i.e., 3,
  • each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5, or more) GAS serotypes.
  • Applicants have also identified adhesin islands within the genome of Streptococcus pneumoniae. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence. Amino acid sequence encoded by such S. pneumoniae Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of S. pneumoniae infection.
  • Preferred immunogenic compositions of the invention comprise a S. pneumoniae AI surface protein which has been formulated or purified in an oligomeric (pilus) form.
  • the oligomeric form is a hyperoligomer.
  • a preferred immunogenic composition of the invention alternatively comprises an isolated S. pneumoniae surface protein in oligomeric (pilus) form.
  • the oligomer or hyperoligomeric pilus structures comprising S. pneumoniae surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • the S. pneumoniae Adhesin Islands generally include a series of open reading frames within a
  • a S. pneumoniae Adhesin Island may encode for an amino acid sequence comprising at least one surface protein.
  • the S. pneumoniae Adhesin Island may encode for at least two surface proteins and at least one sortase.
  • a S. pneumoniae Adhesin Island encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPTXG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • One or more S. pneumoniae AI surface proteins may participate in the formation of a pilus structure on the surface of the S. pneumoniae bacteria.
  • the S. pneumoniae Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator.
  • the transcriptional regulator may regulate the expression of the S. pneumonaie AI operon.
  • An example of a transcriptional regulator found in S. pneumoniae AI sequences is HrA.
  • a schematic of the organization of a S. pneumoniae AI locus is provided in Figure 137.
  • the locus comprises open reading frames encoding a transcriptional regulator (rlrA), cell wall surface proteins (rrgA, rrgB, rrgC) and sortases (srt B, srtC, srtD).
  • rlrA transcriptional regulator
  • rrgA cell wall surface proteins
  • rrgC cell wall surface proteins
  • sortases srt B, srtC, srtD
  • S. pneumoniae AI sequences may be generally divided into two groups of homology, S. pneuamoniae AI-a and AI-b.
  • S. pneumoniae strains that comprise AI-a include 14 CSR 10, 19A 12, and 6B Spain 2.
  • S. pneumoniae AI strains that comprise AI-b include 19F Taiwan 14, 9V Spain 3, 23F Taiwan 15 and TIGR 4.
  • S. pneumoniae AI from TIGR4 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S 1 . pneumoniae AI proteins").
  • S. pneumoniae AI from TIGR4 includes polynucleotide sequences encoding for two or more of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, and SP0468.
  • One or more of the & pneumoniae AI from TIGR4 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the iS 1 . pneumoniae AI from TIGR4 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae strain 670 AI comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("_?. pneumoniae AI proteins"). Specifically, S. pneumoniae strain 670 AI includes polynucleotide sequences encoding for two or more of orfl_670, or ⁇ _670, orf4__670, orf5_670, orf6_670, orf7_670, and orf8_670.
  • One or more of the 5. pneumoniae strain 670 AI polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the iS. pneumoniae strain 670 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI from 14 CSRlO comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins").
  • S. pneumoniae AI from 14 CSRlO includes polynucleotide sequences encoding for two or more of ORF2 14CSR, ORF3J4CSR, ORF4_14CSR, ORF5__14CSR, ORF6J4CSR, ORF7_14CSR, and ORF8_14CSR.
  • S. pneumoniae AI from 14 CSRlO polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 14 CSRlO open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI from 19A Hungary 6 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S.
  • pneumoniae AI from 19A Hungary 6 includes polynucleotide sequences encoding for two or more of ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5J9AH, ORF6_19AH, ORF7_19AH, and ORF8_19AH.
  • One or more of the S. pneumoniae AI from 19A Hungary 6 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 19A Hungary 6 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • C ⁇ - 14 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ( U S. pneumoniae AI proteins").
  • S. pneumoniae AI from 19F Taiwan 14 includes polynucleotide sequences encoding for two or more of ORF2_19FTW, ORF3J9FTW, ORF4_19FTW, ORF5J9FTW, ORF6J9FTW, ORF7_19FTW, and ORF8J9FTW.
  • S. pneumoniae AI from 19F Taiwan 14 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 19F Taiwan 14 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI from 23F Poland 16 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S.
  • pneumoniae AI from 23F Poland 16 includes polynucleotide sequences encoding for two or more of ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, and ORF8_23FP.
  • One or more of the S. pneumoniae AI from 23F Tru 16 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 23F Poland 16 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI from 23F Taiwan 15 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, 6 1 . pneumoniae AI from 23F Taiwan 15 includes polynucleotide sequences encoding for two or more of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORP6_23FTW, ORF7_23FTW, and ORF8_23FTW.
  • One or more of the S. pneumoniae AI from 23F Taiwan 15 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 23F Taiwan 15 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI from 6B Finland 12 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 6B Finland 12 includes polynucleotide sequences encoding for two or more of ORF2 6BF, ORF3_6BF, ORF4 6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, and ORF8_6BF.
  • One or more of the S. pneumoniae AI from 6B Finland 12 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 6B Finland 12 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI from 6B Spain 2 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ('
  • One or more of the S. pneumoniae AI from 6B Spain 2 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 6B Spain 2 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae Al from 9V Spain 3 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("iS 1 . pneumoniae AI proteins").
  • S. pneumoniae AI from 9V Spain 3 includes polynucleotide sequences encoding for two or more of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP, and ORF8_9VSP.
  • One or more of the S. pneumoniae AI from 9V Spain 3 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae AI from 9V Spain 3 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • S. pneumoniae AI surface proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae AI may encode for at least one surface protein.
  • the Adhesin Island may encode at least one surface protein.
  • S. pneumoniae AI may encode for at least two surface proteins and at least one sortase.
  • S. pneumoniae AI encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif.
  • the S. pneumoniae AI protein of the composition may be selected from the group consisting of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, SP0468, orfl_670, orf3_670, orf4_670, orf5_670, orf6_670, orf7_670, orf8_670, ORF2_14CSR, ORF3 14CSR, ORF4_14CSR, ORF5J4CSR, ORF6J4CSR, ORF7J4CSR, ORF8_14CSR, ORF2_19AH, ORF3_19AH, ORF4J9AH, ORF5_19AH, ORF6J9AH, ORF7_19AH, ORF8J9AH, ORF2_19FTW, ORF3J9FTW, ORF4_19FTW, ORF5J9FTW, ORF6J9FTW, ORF7_19FTW, ORF8J9AH
  • S. pneumoniae AI surface proteins are preferred proteins for use in the immunogenic compositions of the invention.
  • the compositions of the invention comprise combinations of two or more S pneumoniae AI surface proteins. Preferably such combinations are sftefddFrp ⁇ H!t ⁇ i; €lffi ⁇ 'biffie l 'gi;;6ij,pcflhsisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF3_19AH, ORF4_19AH, ORF5J9AH, ORF3J9FTW, ORF4_19FTW, ORF5_19FTW, ORF3_23FP, ORF4 23FP, ORF5_23FP, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF3_6BF, ORF
  • S. pneumoniae AI may also include a transcriptional regulator.
  • the S. pneumoniae AI proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against S. pneumoniae infection.
  • the invention may include an immunogenic composition comprising one or more S. pneumoniae from TIGR4 AI proteins and one or more S. pneumoniae strain 670 proteins.
  • the immunogenic composition may comprise one or more AI proteins from any one or more of S. pneumoniae strains TIGR4, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, 23F Poland 16, and 670.
  • the immunogenic compositions may also be selected to provide protection against an increased range of S. pneumoniae serotypes and strain isolates.
  • the immunogenic composition may comprise a first and second S.
  • each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple S. pneumoniae serotypes and strain isolates.
  • each antigen is present in the genomes of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) S. pneumoniae strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5, or more) S. pneumoniae serotypes.
  • the immunogenic compositions may also be selected to provide protection against an increased range of serotypes and strain isolates of a Gram positive bacteria.
  • the immunogenic composition may comprise a first and second Gram positive bacteria AI protein, wherein a full length polynucleotide sequence encoding for the first Gram positive bacteria AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second Gram positive bacteria AI protein.
  • each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple serotypes and strain isolates of the Gram positive bacteria.
  • each antigen is present in the genomes of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram positive bacteria strain isolates.
  • each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5, or more) Gram positive bacteria serotypes.
  • One or both of the first and second AI proteins may preferably be in oligomeric or hyperoligomeric form.
  • Adhesin island surface proteins from two or more Gram positive bacterial genus or species may be combined to provide an immunogenic composition for prophylactic or therapeutic treatment ofecjji
  • the adhesin island surface proteins may be associated together in an oligomeric or hyperoligomeric structure.
  • the invention comprises adhesin island surface proteins from two or more Streptococcus species.
  • the invention includes a composition comprising a GBS AI surface protein and a GAS adhesin island surface protein.
  • the invention includes a composition comprising a GAS adhesin island surface protein and a S. pneumoniae adhesin island surface protein.
  • One or both of the GAS AI surface protein and the S. pneumoniae AI surface protein may be in oligomeric or hyperoligomeric form.
  • the invention includes a composition comprising a GBS adhesin island surface protein and a S. pneumoniae adhesin island surface protein.
  • the invention comprises an adhesin island surface protein from two or more Gram positive bacterial genus.
  • the invention includes a composition comprising a Streptococcus adhesin island protein and a Corynebacterium adhesin island protein.
  • One or more of the Gram positive bacteria AI surface proteins may be in an oligomeric or hyperoligomeric form.
  • the AI polynucleotides and amino acid sequences of the invention may also be used in diagnostics to identify the presence or absence of GBS (or a Gram positive bacteria) in a biological sample.
  • AI polynucleotides and amino acid sequences of the invention may also be used to identify small molecule compounds which inhibit or decrease the virulence associated activity of the AI.
  • FIGURE 1 presents a schematic depiction of Adhesin Island 1 ("AI-I") comprising open reading frames for GBS 80, GBS 52, SAG0647, SAG0648 and GBS 104.
  • FIGURE 2 illustrates the identification of AI-I sequences in several GBS serotypes and strain isolates (GBS serotype V, strain isolate 2603; GBS serotype III, strain isolate nem316; GBS serotype II, strain isolate 18RS21; GBS serotype V, strain isolate CJBl I l; GBS serotype III, strain isolate COHl and GBS serotype Ia, strain isolate A909). (An AI-I was not identified in GBS serotype Ib, strain isolate H36B or GBS serotype Ia, strain isolate 515).
  • FIGURE 3 presents a schematic depiction of the correlation between AI-I and the Adhesin
  • AI-2 within the GBS serotype V, strain isolate 2603 genome.
  • This AI-2 comprises open reading frames for GBS 67, GBS 59, SAG1406, SAG1405 and GBS 150).
  • FIGURE 4 illustrates the identification of AI-2 comprising open reading frames encoding for GBS 67, GBS 59, SAG1406, SAG1404 and GBS 150 (or sequences having sequence homology thereto) in several GBS serotypes and strain isolates (GBS serotype V, strain isolate 2603; GBS serotype III, strain isolate NEM316; GBS serotype Ib, strain isolate H36B; GBS serotype V, strain isolate CJBl 11; GBS serotype II, strain isolate 18RS21; and GBS serotype Ia, strain isolate 515).
  • GBS serotype V strain isolate 2603
  • GBS serotype III strain isolate NEM316
  • GBS serotype Ib strain isolate H36B
  • GBS serotype V strain isolate CJBl 11
  • GBS serotype II strain isolate 18RS21
  • GBS serotype Ia strain isolate 515
  • Figure 4 also illustrates the identification of AI-2 comprising open reading frames encoding for 01520 (f SC ⁇ VO ⁇ J2B OSS2.,(aior:tasi
  • FIGURE 5 presents data showing that GBS 80 binds to fibronectin and fibrinogen in ELISA.
  • FIGURE 6 illustrates that all genes in AI-I are co-transcribed as an operon.
  • FIGURE 7 presents schematic depictions of in-frame deletion mutations within AI-I .
  • FIGURE 8 presents FACS data showing that GBS 80 is required for surface localization of GBS 104.
  • FIGURE 9 presents FACS data showing that sortases SAG0647 and SAG0648 play a semi- redundant role in surface exposure of GBS 80 and GBS 104.
  • FIGURE 10 presents Western Blots of the in-frame deletion mutants probed with anti-GBS80 and anti-GBS 104 antisera.
  • FIGURE 11 Electron micrograph of surface exposed pili structures in Streptococcus agalactiae containing GBS 80.
  • FIGURE 12 PHD predicted secondary structure of GBS 067.
  • FIGURE 13, 14 and 15 Electron micrograph of surface exposed pili structures of strain isolate COHl of Streptococcus agalactiae containing a plasmid insert encoding GBS 80.
  • FIGURE 16 and 17 Electron micrograph of surface exposed pili structure of wild type strain isolate COHl of Streptococcus agalactiae.
  • FIGURE 18 Alignment of polynucleotide sequences of Al-I from serotype V, strain isolates 2603 and CJB 111; serotype II, strain isolate 18RS21 ; serotype III, strain isolates COHl and NEM316; and serotype Ia, strain isolate A909.
  • FIGURE 19 Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolates 2603 and CJBl 11; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype Ia, strain isolate 515.
  • FIGURE 20 Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolate
  • FIGURE 21 Alignment of polynucleotide sequences of AI-2 from serotype III, strain isolate COHl and serotype Ia, strain isolate A909.
  • FIGURE 22 Alignment of amino acid sequences of AI-I surface protein GBS 80 from serotype V, strain isolates 2603 and CJBl 11; serotype Ia, strain isolate A909; serotype III, strain isolates COHl and NEM316.
  • FIGURE 23 Alignment of amino acid sequences of AI-I surface protein GBS 104 from serotype V, strain isolates 2603 and CJB 111; serotype III, strain isolates COHl and NEM316; and serotype II, strain isolate 18RS21.
  • FIGURE 24 Alignment of amino acid sequences of AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJBl 11; serotype Ia, strain isolate 515; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype III, strain isolate NEM316.
  • FIGURE 26 Illustrates GBS infection of MEl 80 cells.
  • FIGURE 27 Illustrates that GBS 80 recombinant protein does not bind to epithelial cells.
  • FIGURE 28 Illustrates that deletion of GBS 80 does not effect the capacity of GBS strain
  • FIGURE 29 Illustrates binding of recombinant GBS 104 protein to epithelial cells.
  • FIGURE 30 Illustrates that deletion of GBS 104 in the GBS strain COHl, reduces the capacity of GBS to adhere to MEl 80 cervical epithelial cells.
  • FIGURE 31 Illustrates that GBS 80 knockout mutant strain partially loses the ability to translocate through an epithelial cell monolayer.
  • FIGURE 32 Illustrates that deletion of GBS 104, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cell line.
  • FIGURE 33 Illustrates that GBS 104 knockout mutant strain translocates through an epithelial monolayer less efficiently than the isogenic wild type.
  • FIGURE 34 Negative stained electron micrographs of GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80.
  • FIGURE 35 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles).
  • FIGURE 36 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles).
  • FIGURE 37 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 n m gold particles).
  • FIGURE 38 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 104 antibodies or preimmune sera (visualized with 10 nm gold particles).
  • FIGURE 39 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles) and anti-GBS 104 antibodies (visualized with 10 nm gold particles).
  • FIGURE 40 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles) and anti-GBS 104 antibodies (visualized with 10 nm gold particles).
  • p C " f 1,0XlMIQ S ⁇ xjst ' rgK : r ⁇ ⁇ 3r ⁇ 8O is necessary for polymer formation and GBS 104 and sortase SAG0648 are necessary for efficient assembly of pili.
  • FIGURE 42 Illustrates that GBS 67 is part of a second pilus and that GBS 80 is polymerized in strain 515.
  • FIGURE 43 Illustrates that two macro-molecules are visible in Cohl, one of which is the
  • FIGURE 44 Illustrates pilin assembly.
  • FIGURE 45 Illustrates that GBS 52 is a minor component of the GBS pilus.
  • FIGURE 46 Illustrates that the pilus is found in the supernatant of a bacterial culture.
  • FIGURE 47 Illustrates that the pilus is found in the supernatant of bacterial cultures in all phases.
  • FIGURE 48 Illustrates that in Cohl, only the GBS 80 protein and one sortase (sagO647 or sagO648) is required for polymerization.
  • FIGURE 49 IEM image of GBS 80 staining of a GBS serotype VIII strain JM9030013 that express pili.
  • FIGURE 50 IEM image of GBS 104 staining of a GBS serotype VIII strain JM9030013 that express pili.
  • FIGURE 5 IA Schematic depiction of open reading frames comprising a GAS AI-2 serotype Ml isolate, GAS AI-3 serotype M3, M5, M18, and M49 isolates, a GAS AI-4 serotype M12 isolate, and an GAS AI-I serotype M6 isolate.
  • FIGURE 51B Amino acid alignment of SrtCl-type sortase of a GAS AI-2 serotype Ml isolate, SrtC2-type sortases of serotype M3, M5, M18, and M49 isolates, and a SrtC2-type sortase of a GAS AI-4 serotype M12 isolate.
  • FIGURE 52 Amino acid alignment of the capsular polysaccharide adhesion proteins of GAS AI-4 serotype M12 (A735), GAS AI-3 serotype M5 (Manfredo), S. pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI-I serotype M3, S. pyogenes strain MGAS8232 serotype M3, and GAS AI-2 serotype Ml.
  • FIGURE 53 Amino acid alignment of F-like fibronectin-binding proteins of GAS AI-4 serotype M12 (A735) and S. pyogenes strain MGAS10394 serotype M6.
  • FIGURE 54 Amino acid alignment of F2-like fibronectin-binding proteins of GAS AI-4 serotype M12 (A735), S. pyogenes strain MGAS8232 serotype M3, GAS AI-3 strain M5 (Manfredo), S. pyogenes strain SSI serotype M3, and S. pyogenes strain MGAS315 serotype M3.
  • FIGURE 55 Amino acid alignment of fimbrial proteins of GAS AI-4 serotype M12 (A735), GAS AI-3 serotype M5 (Manfredo), S. pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI serotype M3, S. pyogenes strain MGAS 8232 serotype M3, and S. pyogenes Ml GAS serotype Ml. jp. I 1 ; 1 ;; "
  • FIGURE 57 Results of FASTA homology search for amino acid sequences that align with the collagen adhesion protein of GAS AI- 1 serotype M6 (MGAS 10394) .
  • FIGURE 58 Results of FASTA homology search for amino acid sequences that align with the fimbrial structural subunit of GAS AI-I serotype M6 (MGAS 10394).
  • FIGURE 59 Results of FASTA homology search for amino acid sequences that align with the hypothetical protein of GAS AI-2 serotype Ml (SF370).
  • FIGURE 60 Specifies pilin and E box motifs present in GAS type 3 and 4 adhesin islands.
  • FIGURE 61 Illustrates that surface expression of GBS 80 protein on GBS strains COH and JM9130013 correlates with formation of pili structures.
  • Surface expression of GBS 80 was determined by FACS analysis using an antibody that cross-hybridizes with GBS 80. Formation of pili structures was determined by immunogold electron microscopy using gold-labelled anti-GBS 80 antibody.
  • FIGURE 62 Illustrates that surface exposure is capsule-dependent for GBS 322 but not for GBS 80.
  • FIGURE 63 Illustrates the amino acid sequence identity of GBS 59 proteins in GBS strains.
  • FIGURE 64 Western blotting of whole GBS cell extracts with anti-GBS 59 antibodies.
  • FIGURE 65 Western blotting of purified GBS 59 and whole GBS cell extracts with anti-
  • FIGURE 66 FACS analysis of GBS strains CJBl 11, 7357B, 515 using GBS 59 antiserum.
  • FIGURE 67 Illustrates that anti-GBS 59 antibodies are opsonic for CJBl 11 GBS strain serotype V.
  • FIGURE 68 Western blotting of GBS strain JM9130013 total extracts.
  • FIGURE 69 Western blotting of GBS stain 515 total extracts shows that GBS 67 and GBS 150 are parts of a pilus.
  • FIGURE 70 Western blotting of GBS strain 515 knocked out for GBS 67 expression
  • FIGURE 71 FACS analysis of GBS strain 515 and GBS strain 515 knocked out for GBS 67 expression using GBS 67 and GBS 59 antiserum.
  • FIGURE 72 Illustrates complementation of GBS 515 knocked out for GBS 67 expression with a construct overexpressing GBS 80.
  • FIGURE 73 FACS analysis of GAS serotype M6 for spyM6_0159 surface expression.
  • FIGURE 74 FACS analysis of GAS serotype M6 for spyM6_0160 surface expression.
  • FIGURE 75 FACS analysis of GAS serotype Ml for GAS 15 surface expression.
  • FIGURE 76 FACS analysis of GAS serotype Ml for GAS 16 surface expression using a first anti-GAS 16 antiserum.
  • FIGURE 78 FACS analysis of GAS serotype Ml for GAS 18 surface expression using a second anti-GAS 18 antiserum.
  • FIGURE 79 FACS analysis of GAS serotype Ml for GAS 16 surface expression using a second anti-GAS 16 antisera.
  • FIGURE 80 FACS analysis of GAS serotype M3 for spyM3_0098 surface expression.
  • FIGURE 81 FACS analysis of GAS serotype M3 for spyM3_0100 surface expression.
  • FIGURE 82 FACS analysis of GAS serotype M3 for spyM3_0102 surface expression.
  • FIGURE 83 FACS analysis of GAS serotype M3 for spyM3_0104 surface expression.
  • FIGURE 84 FACS analysis of GAS serotype M3 for spyM3_0106 surface expression.
  • FIGURE 85 FACS analysis of GAS serotype M12 for 19224134 surface expression.
  • FIGURE 86 FACS analysis of GAS serotype M12 for 19224135 surface expression.
  • FIGURE 87 FACS analysis of GAS serotype M12 for 19224137 surface expression.
  • FIGURE 88 FACS analysis of GAS serotype M12 for 19224141 surface expression.
  • FIGURE 89 Western blot analysis of GAS 15 expression on GAS Ml bacteria.
  • FIGURE 90 Western blot analysis of GAS 15 expression using GAS 15 immune sera.
  • FIGURE 91 Western blot analysis of GAS 15 expression using GAS 15 pre-immune sera.
  • FIGURE 92 Western blot analysis of GAS 16 expression on GAS Ml bacteria.
  • FIGURE 93 Western blot analysis of GAS 16 expression using GAS 16 immune sera.
  • FIGURE 94 Western blot analysis of GAS 16 expression using GAS 16 pre-immune sera.
  • FIGURE 95 Western blot analysis of GAS 18 on GAS Ml bacteria.
  • FIGURE 96 Western blot analysis of GAS 18 using GAS 18 immune sera.
  • FIGURE 97 Western blot analysis of GAS 18 using GAS 18 pre-immune sera.
  • FIGURE 98 Western blot analysis of M6_S ⁇ yO159 expression on GAS bacteria.
  • FIGURE 99 Western blot analysis of 19224135 expression on M12 GAS bacteria.
  • FIGURE 100 Western blot analysis of 19224137 expression on M12 GAS bacteria.
  • FIGURE 101 Full length nucleotide sequence of an S. pneumoniae strain 670 AL
  • FIGURE 102 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 2580.
  • FIGURE 103 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 2913.
  • FIGURE 104 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 3280.
  • FIGURE 105 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain
  • FIGURE 106 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain
  • FIGURE 108 Western blot analysis of 19224135 and 19224137 in GAS M12 strain 2728.
  • FIGURE 109 Western blot analysis of 19224139 in GAS M12 strain 2728 using antisera raised against SpyM3_0102.
  • FIGURE 110 Western blot analysis of M6_SpyO159 and M6_Spy0160 in GAS M6 strain 2724.
  • FIGURE 111 Western blot analysis of M6_SpyO 159 and M6_SpyO 160 in GAS M6 strain SF370.
  • FIGURE 112 Western blot analysis of M6_Spyl60 in GAS M6 strain 2724.
  • FIGURES 113-115 Electron micrographs of surface exposed GAS 15 on GAS Ml strain SF370.
  • FIGURES 116-121 Electron micrographs of surface exposed GAS 16 on GAS Ml strain SF370.
  • FIGURES 122-125 Electron micrographs of surface exposed GAS 18 on GAS Ml strain
  • FIGURE 126 IEM image of a hyperoligomer on GAS Ml strain SF370 detected using anti- GAS 18 antisera.
  • FIGURES 127-132 IEM images of oligomeric and hyperoligomeric structures containing M6_Spy0160 extending from the surface of GAS serotype M6 3650.
  • FIGURE 133A and B Western blot analysis of L. lactis transformed to express GBS 80 with anti-GBS 80 antiserum.
  • FIGURES 134 Western blot analyses of L. lactis transformed to express GBS AI-I with anti-GBS 80 antiserum.
  • FIGURE 135 Ponceau staining of same acrylamide gel as used in Figure 134.
  • FIGURE 136A Western blot analysis of sonicated pellets and supematants of cultured L. lactis transformed to express GBS AI-I polypeptides using anti-GBS 80 antiserum.
  • FIGURE 136B Polyacrylamide gel electrophoresis of sonicated pellets and supematants of cultured L. lactis transformed to express GBS AI polypeptides.
  • FIGURE 137 Depiction of an example S. pneumoniae AI locus.
  • FIGURE 138 Schematic of primer hybridization sites within the S. pneumoniae AI locus of FIGURE 137.
  • FIGURE 139A The set of amplicons produced from the S. pneumoniae strain TIGR4 AI locus.
  • FIGURE 139B Base pair lengths of amplicons produced from FIGURE 139A primers in S. pneumoniae strain TIGR4.
  • FIGURE 140 CGH analysis of S. pneumoniae strains for the AI locus. 1"I' C;; 1 W ⁇ iBS €lt ilSpifi i$ ⁇ elr ⁇ ice alignment of polypeptides encoded by AI orf 2 in S. pneumoniae AI-positive strains.
  • FIGURE 142 Amino acid sequence alignment of polypeptides encoded by AI orf 3 in S. pneumoniae AI-positive strains.
  • FIGURE 143 Amino acid sequence alignment of polypeptides encoded by AI orf 4 in S. pneumoniae AI-positive strains.
  • FIGURE 144 Amino acid sequence alignment of polypeptides encoded by AI orf 5 in S. pneumoniae AI-positive strains.
  • FIGURE 145 Amino acid sequence alignment of polypeptides encoded by AI orf 6 in S. pneumoniae AI-positive strains.
  • FIGURE 146 Amino acid sequence alignment of polypeptides encoded by AI orf 7 in S. pneumoniae AI-positive strains.
  • FIGURE 147 Amino acid sequence alignment of polypeptides encoded by AI orf 8 in S. pneumoniae AI-positive strains.
  • FIGURE 148 Diagram comparing amino acid sequences of RrgA in S. pneumoniae strains.
  • FIGURE 149 Amino acid sequence comparison of RrgB S. pneumoniae strains.
  • FIGURE 150A SpO462 amino acid sequence.
  • FIGURE 150B Primers used to produce a clone encoding the SpO462 polypeptide.
  • FIGURE 15 IA Schematic depiction of recombinant SpO462 polypeptide.
  • FIGURE 151 B Schematic depiction of full-length SpO462 polypeptide.
  • FIGURE 152A Western blot probed with serum obtained from S. pneumoniae- infected patients for SpO462.
  • FIGURE 152B Western blot probed with GBS 80 serum for S ⁇ O462.
  • FIGURE 153 A SpO463 amino acid sequence.
  • FIGURE 153B Primers used to produce a clone encoding the SpO463 polypeptide.
  • FIGURE 154A Schematic depiction of recombinant SpO463 polypeptide.
  • FIGURE 154B Schematic depiction of full-length S ⁇ O463 polypeptide.
  • FIGURE 155 Western blot detection of recombinant SpO463 polypeptide.
  • FIGURE 156 Western blot detection of high molecular weight SpO463 polymers.
  • FIGURE 157A SpO464 amino acid sequence.
  • FIGURE 157B Primers used to produce a clone encoding the S ⁇ O464 polypeptide.
  • FIGURE 158 A Schematic depiction of recombinant SpO464 polypeptide.
  • FIGURE 158B Schematic depiction of full-length SpO464 polypeptide.
  • FIGURE 159 Western blot detection of recombinant SpO464 polypeptide.
  • FIGURE 160 Amplification products prepared for production of S ⁇ O462, SpO463, and
  • FIGURE 161 Opsonic killing by anti-sera raised against L. lactis expressing GBS AI 1 P C TtelMl::iO:iSolieiaaicftplcQg GAS adhesin islands GAS AI-I, GAS AI-2, GAS AI-3 and GAS AI-4.
  • FIGURES 163 A-D Immunoblots of cell-wall fractions of GAS strains with antisera specific for LPXTG proteins of M6_ ISS3650 (A), Ml_SF370 (B) 1 M5JSS4883 (C) and M12_20010296 (D).
  • FIGURES 163 E-H Immunoblots of cell-wall fractions of deletion mutants Ml_SF370 ⁇ 128
  • FIGURE 164 Schematic representation of the FCT region from 7 GAS strains
  • FIGURES 165 A-H Flow cytometry of GAS bacteria treated or not with trypsin and stained with sera specific for the major pilus component. Preimmune staining; black lines, untreated bacteria; green lines and trypsin treated bacteria; blue lines.
  • FIGURES 166 A-C Immunoblots of recombinant pilin components with polyvalent
  • FIGURES 166 D-G Immunoblots of pilin proteins with monovalent T-typing sera. The recombinant proteins are shown below the blot and the sera used above the blot.
  • Figure 166 H and I Flow cytometry analysis of strain Ml_SF370 (H) and the deletion strain
  • FIGURE 167 Chart describing the number and type of sortase sequences identified within GAS AIs.
  • FIGURE 168 A Immunogold-electronmicroscopy of L. lactis lacking an expression construct for GBS AI-I using anti-GBS 80 antibodies.
  • FIGURE 168 B and C Immunogold-electronmicroscopy detects GBS 80 in oligomeric (pilus) structures on surface of L. lactis transformed to express GBS AI-I
  • FIGURE 169 FACS analysis detects expression of GBS 80 and GBS 104 on the surface of L. lactis transformed to express GBS AI-I.
  • FIGURE 170 Phase contrast microscopy and immuno-electronmicroscopy shows that expression of GBS AI-I in L. lactis induces L. lactis aggregation.
  • FIGURE 171 Purification of GBS pili from L. lactis transformed to express GBS AI-I .
  • FIGURE 173 A-C Western blot analysis showing assembly of GAS pili in L. lactis expressing GAS AI-2 (Ml) (A), GAS AI-4 (M12) (B), and GAS AI-I (M6) (C).
  • FIGURE 174 FACS analysis of GAS serotype M6 for M6_SpyO157 surface expression.
  • FIGURE 175 FACS analysis of GAS serotype M12 for 19224139 surface expression.
  • FIGURE 176 A-E Immunogold electron microscopy using antibodies against M6_SpyO16O detects pili on the surface of M6 strain 2724.
  • FIGURE 176 F Immunogold electron microscopy using antibodies against M6_SpyO159 detects M6_SpyO159 surface expression on M6 strain 2724.
  • FIGURE 177 A-C Western blot analysis of Ml strain SF370 GAS bacteria individually deleted for Ml_130, SrtCl, or Ml_128 using anti-Ml_130 serum (A), anti-Ml_128 serum (B), and anti-Ml_126 serum (C).
  • FIGURE 178 A-C Immunogold electron microscopy using antibodies against Ml_128 to detect surface expression on wildtype strain SF370 bacteria (A), Ml_128 deleted SF370 bacteria (B), and SrtCl deleted SF370 bacteria (C).
  • FIGURE 179 A-C FACS analysis to detect expression of Ml_126 (A), Ml_128 (B), and Ml_130 (C) on the surface of wildtype SF370 GAS bacteria.
  • FIGURE 179 D-F FACS analysis to detect expression of Ml_126 (D), Ml_128 (E), and
  • FIGURE 179 G-I FACS analysis to detect expression of Ml_126 (G), Ml_128 (H), and Ml_130 (I) on the surface of SrtCl deleted SF370 GAS bacteria.
  • FIGURE 180 A and B FACS analysis of wildtype (A) and LepA deletion mutant (B) strains of SF370 bacteria for Ml surface expression.
  • FIGURE 181 Western blot analysis detects high molecular weight polymers in S. pneumoniae TIGR4 using anti-RrgB antisera.
  • FIGURE 182 Detection of high molecular weight polymers in S. pnuemoniae rlrA positive strains.
  • FIGURE 183 Detection of high molecular weight polymers in S. pneumoniae TIGR4 by silver staining and Western blot analysis using anti-RrgB antisera.
  • FIGURE 184 Deletion of S. pneumoniae TIGR4 adhesin island sequences interferes with the ability of S. pneumoniae to adhere to A549 alveolar cells.
  • FIGURE 185 Negative staining of S. pneumoniae strain TIGR4 showing abundant pili on the bacterial surface.
  • FIGURE 186 Negative staining of strain TIGR4 deleted for ixgA-srtD adhesin island sequences showing no pili on the bacterial surface I "1 ' C llGUffilO:ii:::K.egaH ⁇ e 1 ' sEi3rfiiof the TIGR4 mgrA mutant showing abundant pili on the bacterial surface.
  • FIGURE 188 Negative staining of the negative control TIGR4 mgrA mutant deleted for adhesin island sequences ixgA-srtD showing no pili on the bacterial surface.
  • FIGURE 189 Immuno-gold labelling of S. pneumoniae strain TIGR4 grown on blood agar solid medium using ⁇ -RrgB (5nm) and ⁇ -RrgC (lOnm). Bar represents 200nm.
  • FIGURE 190 A and B Detection of expression and purification of S. pneumoniae RrgA protein by SDS-PAGE (A) and Western blot analysis (B).
  • FIGURE 191 Detection of RrgB by antibodies produced in mice.
  • FIGURE 192 Detection of RrgC by antibodies produced in mice.
  • FIGURE 193 Purification of S. pneumoniae TIGR 4 pili by a cultivation and digestion method and detection of the purified TIGR4 pili.
  • FIGURE 194 Purification of S. pneumoniae TIGR 4 pili by a sucrose gradient centrifugation method and detection of the purified TIGR4 pili.
  • FIGURE 195 Purification of S. pneumoniae TIGR 4 pili by a gel filtration method and detection of the purified TIGR4 pili.
  • FIGURE 196 Alignment of full length S. pneumoniae adhesin island sequences from ten S. pneumoniae strains.
  • FIGURE 197 A Schematic of GBS AI-I coding sequences.
  • FIGURE 197 B Nucleotide sequence of intergenic region between AraC and GBS 80 (SEQ ID NO: 0
  • FIGURE 197 C FACS analysis results for GBS 80 expression in GBS strains having different length polyA tracts in the intergenic region between AraC and GBS 80.
  • FIGURE 198 Table comparing the percent identity of surface proteins encoded by a serotype M6 (harbouring a GAS AI-I) adhesin island relative to other GAS serotypes harbouring an adhesin island.
  • FIGURE 199 Table comparing the percent identity of surface proteins encoded by a serotype Ml (harbouring a GAS AI-2) adhesin island relative to other GAS serotypes harbouring an adhesin island.
  • FIGURE 200 Table comparing the percent identity of surface proteins encoded by serotypes
  • FIGURE 201 Table comparing the percent identity of surface proteins encoded by a serotype M 12 (harbouring a GAS AI-I) adhesin island- relative to other GAS serotypes harbouring an adhesin island.
  • FIGURE 202 GBS 80 recombinant protein does not bind to epithelial cells.
  • FIGURE 203 Deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade ME 180 cervical epithelial cells.
  • FIGURE 205 Deletion of GBS 104 protein, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cells
  • FIGURE 206 GBS 104 knockout mutant strains of bacteria translocate through an epithelial monolayer less efficiently that the isogenic wild type strain.
  • FIGURE 207 GBS 80 knockout mutant strains of bacteria partially lose the ability to translocate through an epithelial monolayer.
  • FIGURE 208 GBS adherence to HUVEC endothelial cells.
  • FIGURE 209 Strain growth rate of wildtype, GBS 80-deleted, or GBS 104 deleted COHl GBS.
  • FIGURE 210 Binding of recombinant GBS 104 protein to epithelial cells by FACS analysis.
  • FIGURE 211 Deletion of GBS 104 proteinin the " GBS strain COHl reduces the ability of GBS to adhere to MEl 80 cervical epithelial cells.
  • FIGURE 212 COHl strain GBS overexpressing GBS 80 protein has an impaired capacity to translocate through an epithelial monolayer.
  • FIGURE 213 Scanning electron microscopy shows that overexpression of GBS 80 protein on COHl strain GBS enhances the capacity of the COHl bacteria to form microcolonies on epithelial cells.
  • FIGURE 214 Confocal imaging shows that overexpression of GBS 80 proteins on COHl strain GBS enhances the capacity of the COHl bacteria to form microcolonies on epithelial cells.
  • FIGURE 215 Detection of GBS 59 on the surface of GBS strain 515 by immuno-electron microscopy.
  • FIGURE 216 Detection of GBS 67 on the surface of GBS strain 515 by immuno-electron microscopy.
  • FIGURE 217 GBS 67 binds to fibronectin.
  • FIGURE 218 Western blot analysis shows that deletion of both GBS AI-2 sortase genes abolishes assembly of the pilus.
  • FIGURE 219 FACS analysis shows that deletion of both GBS AI-2 sortase genes abolishes assembly of the pilus.
  • FIGURE 220 A-C Western blot analysis shows that GBS 59, GBS 67, and GBS 150 form high molecular weight complexes.
  • FIGURE 221 A-C Western blot analysis shows that GBS 59 is required for polymer formation of GBS 67 and GBS 150.
  • FIGURE 222 FACS analysis shows that GBS 59 is required for surface exposure of GBS 67.
  • FIGURE 223 Summary Western blots for detection of GBS 59, GBS 67, or GBS 150 in
  • FIGURE 224 Description of GBS 59 allelic variants. P only against a strain of GBS expressing a homologous GBS 59.
  • FIGURE 226 A and B Results of FACS analysis for surface expression of GBS 59 using antibodies specific for different GBS 59isoforms.
  • FIGURE 227 A and B Results of FACS analysis for surface expression of GBS 80, GBS
  • FIGURE 228 Results of FACS analysis for surface expression of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 on 41 strains of GBS bacteria obtained from the CDC.
  • FIGURE 229 Expected immunogenicity coverage of different combinations of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 across strains of GBS bacteria.
  • FIGURE 230 GBS 59 opsonophagocytic activity is comparable to that of a mixture of GBS 80, GBS 104, GBS 322 and GBS 67.
  • FIGURE 231 A-C Schematic presentation of example hybrid GBS AIs.
  • FIGURE 232 Schematic presentation of an example hybrid GBS AI.
  • FIGURE 233 A and B Western blot and FACS analysis detect expression of GBS 80 and
  • FIGURE 235 High magnification of S. pneumoniae strain TIGR4 pili double labeled with ⁇ - RrgB (5nm) and ⁇ -RrgC (lOnm). Bar represents lOOnm.
  • FIGURE 236 Immuno-gold labeling of the S. pneumoniae TIGR4 rrgA-srtD deletion mutant with no visible pili on the surface detectable by ⁇ -RrgB- and ⁇ -RrgC. Bar represents 200nm.
  • FIGURE 237 Variability in GBS 67 amino acid sequences between strains 2603 and H36B.
  • FIGURE 238 Strain variability in GBS 67 amino acid sequences of allele I (2603).
  • FIGURE 239 Stran variability in GBS 67 amino acid sequence of allele II (H36B).
  • GAS AI-2 sequences from Ml isolate SF370.
  • GAS AI-3 sequences from M3 isolate MGAS315).
  • GAS AI-3 sequences from M3 isolate SSI-I.
  • GAS AI-3 sequences from M18 isolate MGAS8232.
  • S. pneumoniae AI sequences from TIGR4 sequence S. pneumoniae AI sequences from TIGR4 sequence.
  • TABLE 22 Summary of FACS values for surface expression of GAS 16 using a second antisera.
  • TABLE 23 Summary of FACS values for surface expression of GAS 18.
  • an "Adhesin Island” or “AI” refers to a series of open reading frames within a bacterial genome, such as the genome for Group A or Group B Streptococcus or other gram positive bacteria, that encodes for a collection of surface proteins and sortases.
  • An Adhesin Island may eifibSe f5br-'amind : %C!i ⁇ "gfeqt ⁇ d ⁇ ee ⁇ (fomprisfng at least one surface protein.
  • the Adhesin Island may encode at least one surface protein.
  • an Adhesin Island may encode for at least two surface proteins and at least one sortase.
  • an Adhesin Island encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • One or more AI surface proteins may participate in the formation of a pilus structure on the surface of the gram positive bacteria.
  • Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction).
  • the transcriptional regulator may regulate the expression of the AI operon.
  • AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("AI-I proteins"). Specifically, AI-I includes open reading frames encoding for two or more (i.e., 2, 3, 4 or 5) of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648.
  • One or more of the AI-I open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the AI-I open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • AI-I typically resides on an approximately 16.1 kb transposon-like element frequently inserted into the open reading frame for trniA.
  • One or more of the AI-I surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) motif or other sortase substrate motif.
  • the AI surface proteins of the invention may affect the ability of the GBS bacteria to adhere to and invade epithelial cells.
  • AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer.
  • one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • the AI-I sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • AI-I may encode for at least one surface protein.
  • AI-I may encode for at least two surface exposed proteins and at least one sortase.
  • AI-I encodes for at least three surface exposed proteins and at least two sortases.
  • the AI-I protein preferably includes GBS 80 or a fragment thereof or a sequence having sequence identity thereto.
  • an LPXTG motif represents an amino acid sequence comprising at least five amino acid residues.
  • the motif includes a leucine (L) in the first amino acid position, a proline (P) in the second amino acid position, a threonine (T) in the fourth amino acid position and a glycine (G) in the fifth amino acid position.
  • the third position, represented by X, may be occupied by aiyimi ⁇ o. ⁇ iyyeiilMiiiPirefiraSifiiiiti ⁇ I SPis occupied by lysine (K), Glutamate (E), Asparagine (N), Glutamine (Q) or Alanine (A).
  • the X position is occupied by lysine (K).
  • one of the assigned LPXTG amino acid positions is replaced with another amino acid.
  • such replacements comprise conservative amino acid replacements, meaning that the replaced amino acid residue has similar physiological properties to the removed amino acid residue.
  • Genetically encoded amino acids may be divided into four families based on physiological properties: (1) acidic (asparatate and glutamate), (2) basic (lysine, arginine, histitidine), (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane) and (4) uncharged polar (glycine, asparagines, glutamine, cysteine, serine, threonine, and tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids.
  • the first amino acid position of the LPXTG motif may be replaced with another amino acid residue.
  • the first amino acid residue (leucine) is replaced with an alanine (A), valine (V), isoleucine (I), proline (P), phenylalanine (F), methionine (M), glutamic acid (E), glutamine (Q), or tryptophan (Y) residue.
  • the first amino acid residue is replaced with an isoleucine (I).
  • the second amino acid residue of the LPXTG motif may be replaced with another amino acid residue.
  • the second amino acid residue praline (P) is replaced with a valine (V) residue.
  • the fourth amino acid residue of the LPXTG motif may be replaced with another amino acid residue.
  • the fourth amino acid residue (threonine) is replaced with a serine (S) or an alanine (A).
  • an LPXTG motif may be represented by the amino acid sequence XXXXG, in which X at amino acid position 1 is an L, a V, an E, an I, an F, or a Q; X at amino acid position 2 is a P if X at amino acid position 1 is an L, an I, or an F; X at amino acid position 2 is a V if X at amino acid position 1 is a E or a Q; X at amino acid position 2 is a V or a P if X at amino acid position 1 is a V; X at amino acid position 3 is any amino acid residue; X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, I, F, or Q; and X at amino acid position 4 is a T, S, or A if X at amino acid position 1 is an L.
  • the LPXTG motif of a GBS AI protein may be represented by the amino acid sequence XPXTG, in which X at amino acid position 1 is L, I, or F, and X at amino acid position 3 is any amino acid residue.
  • Specific examples of LPXTG motifs in GBS AI proteins may include LPXTG (SEQ ID NO: 122) or IPXTG (SEQ ID NO: 133).
  • the threonine in the fourth amino acid position of the LPXTG motif may be involved in the formation of a bond between the LPXTG containing protein and a cell wall precursor.
  • the AI surface proteins of the invention may contain alternative sortase substrate motifs such as NPQTN (SEQ ID NO: 142), NPKTN (SEQ ID NO: 168), NPQTG (SEQ ID NO: 169), NPKTG (SEQ ID NO: 170), XPXTGG (SEQ ID NO: 143), LPXTAX (SEQ ID NO: 144), or LAXTGX (SEQ ID NO: 145). (Similar conservative amino acid substitutions can also be made to these membrane motifs).
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the AI surface proteins may be polymerized into pili by sortase-catalysed transpeptidation.
  • AI surface proteins include a pilin motif amino acid sequence which interacts with the sortase and LPXTG amino acid sequence.
  • the first lysine residue in a pilin motif can serve as an amino group acceptor of the cleaved LPXTG motif and thereby provide a covalent linkage between AI subunits to form pili.
  • the pilin motif can make a nucleophilic attack on the acyl enzyme providing a covalent linkage between AI subunits to form pili and regenerate the sortase enzyme.
  • pilin motifs may include ((YPKN(Xi 0 )K; SEQ ID NO: 146), (YPKN(Xg)K; SEQ ID NO: 147), (YPK(X 7 )K; SEQ ID NO: 148), (YPK(X n )K; SEQ ID NO: 149), or (PKN(X 9 )K; SEQ ID NO: 150)).
  • the AI surface proteins of the invention include a pilin motif amino acid sequence.
  • AI surface proteins of the invention will contain an N-terminal leader or secretion signal to facilitate translocation of the surface protein across the bacterial membrane.
  • Group B Streptococci are known to colonize the urinary tract, the lower gastrointestinal tract and the upper respiratory tract in humans.
  • Electron micrograph images of GBS infection of a cervical epithelial cell line (MEl 80) are presented in Figure 25. As shown in these images, the bacteria closely associate with tight junctions between the cells and appear to cross the monolayer by a paracellular route. Similar paracellular invasion of MEl 80 cells is also shown in the contrast images in Figure 26.
  • the AI surface proteins of the invention may effect the ability of the GBS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • AI-I surface protein GBS 104 can bind epithelial cells such as ME 180 human cervical cells, A549 human lung cells and Caco2 human intestinal cells (See F ⁇ iUKs ' 29''aiiy i i ⁇ Of3itr.thdiy!( ⁇ i ⁇ iioiyilhe GBS 104 sequence in a GBS strain reduces the capacity of GBS to adhere to ME180 cervical epithelial cells. (See Figures 30 and 211). Deletion of GBS 104 also reduces the capacity of GBS to invade J774 macrophage-like cells. (See Figures 32 and 205). Deletion of GBS 104 also causes GBS to translocate through epithelial monolayers less efficiently. See Figure 206. GBS 104 protein therefore appears to bind to ME180 epithelial cells and to have a role in adhesion to epithelial cells and macrophage cell lines.
  • GBS 80 knockout mutant strains also partially lose the ability to translocate through an epithelial monolayer. See Figure 207. Deletion of either GBS 80 or GBS 104 in COHl cells diminishes adherence to HUVEC endothelial cells. See Figure 208. Deletion of GBS 80 or GBS 104 in COHl does not, however, affect growth of COHl either with MEl 80 cells or in incubation medium (IM). See Figure 209. Both GBS 80 and GBS 104, therefore, appear to be involved in translocation of GBS through epithelial cells.
  • GBS 80 does not appear to bind to epithelial cells. Incubation of epithelial cells in the presence of GBS 80 protein followed by FACS analysis using an anti-GBS 80 polyclonal antibody did not detect GBS 80 binding to the epithelial cells. See Figure 202. Furthermore, deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade MEl 80 cervical epithelial cells. See Figure 203
  • one or more of the surface proteins may bind to one or more extracellular matrix (ECM) binding proteins, such as fibrinogen, fibronectin, or collagen.
  • ECM extracellular matrix
  • GBS 80 one of the AI-I surface proteins, can bind to the extracellular matrix binding proteins fibronectin and fibrinogen. While GBS 80 protein apparently does not bind to certain epithelial cells or affect the capacity of a GBS bacteria to adhere to or invade cervical epithelial cells (See Figures 27 and 28), removal of GBS 80 from a wild type strain decreases th'e ability of that strain to translocate through an epithelial cell layer (see Figure 31). GBS 80 may also be involved in formation of biofihns. COHl bacteria overexpressing GBS
  • FimA a major fimbrial subunit of a Gram positive bacteria A. naeslundii. FimA is thought to be involved in binding salivary proteins and may be a component in a f ⁇ mbrae on the surface of A. naeslundii. See Yeung et al. (1997) Infection & Immunity 65:2629-2639; Yeunge et al (1998) J. Bacteriol 66: 1482-1491; Yeung et al. (1988) J. Bacteriol 170:3803 - 3809; and Li et al (2001) Infection & Immunity 69:7224-7233.
  • the C. diphtheriae pilin subunit SpaA is thought to occur by sortase-catalyzed amide bond cross- linking of adjacent pilin subunits.
  • the conserved lysine within the SpaA pilin motif might function as an amino group acceptor of cleaved sorting signals, thereby providing for covalent linkages of the C diphtheria pilin subunits. See Figure 6(d) of Ton-That et al., Molecular Microbiology (2003) 50(4): 1429-1438.
  • E box comprising a conserved glutamic acid residue has also been identified in the C. diphtheria pilin associated proteins as important in C. diphtheria pilin assembly.
  • the E box motif generally comprises YxLxETxAPxGY (SEQ ID NO: 152; where x indicates a varying amino acid residue).
  • the conserved glutamic acid residue within the E box is thought necessary for C. diphtheria pilus formation.
  • the AI-I polypeptides of the immunogenic compositions comprise an E box motif.
  • E box motifs in the AI-I polypeptides may include the amino acid sequences YxLxExxxxxGY (SEQ ID NO: 153), YxLxExxxPxGY (SEQ ID NO: 154), or YxLxETxAPxGY (SEQ ID NO: 152).
  • the E box motif of the polypeptides may comprise the amino acid sequences YKLKETKAPEGY (SEQ ID NO: 155), YVLKEIETQSGY (SEQ ID NO: 156), or YKLYEISSPDGY (SEQ ID NO: 157).
  • GBS 80 As discussed in more detail below, a pilin motif containing a conserved lysine residue and an E box motif containing a conserved glutamic acid residue have both been identified in GBS 80.
  • FIG. 34 presents electron micrographs of GBS serotype III, strain isolate COHl with a plasmid insert to facilitate the overexpression of GBS 80. This EM photo was produced with a standard negative stain - no pilus structures are distinguishable.
  • AI surface proteins in immunogenic compositions for the treatment or prevention of infection against a Gram positive bacteria has not been previously described.
  • GBS 80 in surface exposed pilus formations visible in electron micrographs. These structures are only visible when the electron micrographs are specifically stained against an AI surface protein such as GBS 80. Examples of these electron micrographs are srlown in Figures 11, 16 and 17, which reveal the presence of pilus structures in wild type COHl Streptococcus agalactiae. Other examples of these electron ⁇ cilEgrSphs yeShSMin'Fiiurl!' It, .siffih reveals that GBS 80 is associated with pili in a wild type clinical isolate of S. agalactiae, JM9030013. (See figure 49.)
  • Applicants have also constructed mutant GBS strains containing a plasmid comprising the GBS 80 sequence resulting in the overexpression of GBS 80 within this mutant.
  • the electron micrographs of Figures 13 — 15 are also stained against GBS 80 and reveal long, oligomeric structures containing GBS 80 which appear to cover portions of the surface of the bacteria and stretch far out into the supernatant.
  • Figure 61 provides FAC analysis of GBS 80 surface levels on bacterial strains COHl and JM9130013 using an anti-GBS 80 antisera. Immunogold electron microscopy of the
  • GBS 80 The surface exposure of GBS 80 on GBS is generally not capsule-dependent.
  • Figure 62 provides FACS analysis of capsulated and uncapsulated GBS analyzed with anti-GBS 80 and anti- GBS 322 antibodies. Surface exposure of GBS 80, unlike GBS 322, is not capsule dependent.
  • Adhesin Island surface protein such as GBS 80 appears to be required for pili formation, as well as an Adhesin Island sortase.
  • Pili are formed in Cohl bacterial clones that overexpress GBS 80, but lack GBS 104, or one of the AI-I sortases sagO647 or sagO648.
  • pili are not formed in Cohl bacterial clones that overexpress GBS 80 and lack both sagO647 and sagO648.
  • at least GBS 80 and a sortase, sagO647 or sagO648, may be necessary for pili formation.
  • GBS 80 in GBS strain 515 which lacks an AI-I, also assembles GBS 80 into pili.
  • GBS strain 515 contains an AI-2, and thus AI-2 sortases.
  • the AI-2 sortases in GBS strain 515 apparently polymerize GBS 80 into pili.
  • Overexpression of GBS 80 in GBS strain 515 cell knocked out for GBS 67 expression also apparently polymerizes GBS 80 into pili. (See Figure 72.)
  • GBS 80 appears to be required for GBS AI-I pili formation
  • GBS 104 and sortase SAG0648 appears to be important for efficent AI-I pili assembly.
  • high-molecular structures are not assembled in isogenic COHl strains which lack expression of GBS 80 due to gene disruption and are less efficiently assembled in isogenic COHl strains which lack the expression of GBS 104 (see Figure 41).
  • This GBS strain comprises high molecular weight pili structures composed of covalently linked GBS 80 and GBS 104 subunits.
  • deleting SAG0648 in COHl bacteria interferes with assembly of some of the high molecular weight pili structures. Thus, indicating that SAG0648 plays a role in assembly of these pilin species. (See Figure 41).
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as GBS 80.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include one or both of a pilin motif comprising a conserved lysine residue and an E box motif comprising a conserved glutamic acid residue.
  • More than one AI surface protein may be present in the oligomeric, pilus-like structures of the invention.
  • GBS 80 and GBS 104 may be incorporated into an oligomeric structure.
  • GBS 80 and GBS 52 may be incorporated into an oligomeric structure, or GBS 80, GBS 104 and GBS 52 may be incorporated into an oligomeric structure.
  • the invention includes compositions comprising two or more AI surface proteins.
  • the composition may include surface proteins from the same adhesin island.
  • the composition may include two or more GBS AI-I surface proteins, such as GBS 80, GBS 104 and GBS 52.
  • the surface proteins may be isolated from Gram positve bacteria or they may be produced recombinantly.
  • the invention comprises a GBS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more GBS Adhesin Island 1 ("AI-I”) proteins and one or more GBS Adhesin Island 2 (“AI-2”) proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • AI-I GBS Adhesin Island 1
  • AI-2 GBS Adhesin Island 2
  • the oligomeric, pilus-like structures of the invention may be combined with one or more additional GBS proteins.
  • the oligomeric, pilus-like structures comprise one or more AI surface proteins in combination with a second GBS protein.
  • the second GBS protein may be a known GBS antigen, such as GBS 322 (commonly referred to as "sip") or GBS 276.
  • GBS 322 commonly referred to as "sip”
  • GBS 276 GBS antigen
  • Nucleotide and amino acid sequences of GBS 322 sequenced from serotype V isolated strain 2603 V/R are set ' Bifl'Md SEQ ID 8540 and in the present specification as SEQ ID NOs: 38 and 39.
  • a particularly preferred GBS 322 polypeptide lacks the N-terminal signal peptide, amino acid residues 1-24.
  • GBS 322 polypeptide is a 407 amino acid fragment and is shown in SEQ ID NO: 40.
  • Examples of preferred GBS 322 polypeptides are further described in PCTUS04/ , attorney docket number PP20665.002 filed September 15, 2004, hereby incorporated by reference, published as WO 2005/002619.
  • GBS proteins which may be combined with the GBS AI surface proteins of the invention are also described in WO 2005/002619. These GBS proteins include GBS 91, GBS 184, GBS 305, GBS 330, GBS 338, GBS 361, GBS 404, GBS 690, and GBS 691. Additional GBS proteins which may be combined with the GBS AI surface proteins of the invention are described in WO 02/34771.
  • GBS polysaccharides which may be combined with the GBS AI surface proteins of the invention are described in WO 2004/041157.
  • the GBS AI surface proteins of the invention may be combined with a GBS polysaccharides selected from the group consisting of serotype Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII.
  • the oligomeric, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an AI surface protein.
  • the invention therefore includes a method for manufacturing an oligomeric AI surface antigen comprising culturing a GBS bacterium that expresses the oligomeric AI protein and isolating the expressed oligomeric AI protein from the GBS bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed AI protein.
  • the AI protein is in a hyperoligomeric form. Macromolecular structures associated with oligomeric pili are observed in the supernatant of cultured GBS strain Cohl.
  • the oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing an AI surface protein.
  • the invention therefore includes a method for manufacturing an oligomeric Adhesin Island surface antigen comprising culturing a GBS bacterium adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the GBS bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed Adhesin Island protein.
  • the Adhesin Island protein is in a hyperoligomeric form.
  • the GBS bacteria are preferably adapted to increase AI protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels.
  • GBS bacteria may be adapted to increase AI protein expression by any means known in the art, including methods of increasing gene dosage and methods of gene upregulation. Such means include, for example, transformation of the GBS bacteria with a plasmid encoding the AI protein.
  • the plasmid may include a strong promoter or it may include multiple copies of the sequence encoding the the AI protein within the GBS bacterial genome may be deleted. Alternatively, or in addition, the promoter regulating the GBS Adhesin Island may be modified to increase expression.
  • GBS bacteria harbouring a GBS AI-I may also be adapted to increase AI protein expression by altering the number adenosine nucleotides present at two sites in the intergenic region between AraC and GBS 80.
  • Figure 197 A which is a schematic showing the organization of GBS AI-I and Figure 197 B, which provides the sequence of the intergenic region between AraC and GBS 80 in the AI.
  • the adenosine tracts which applicants have identified as influencing GBS 80 surface expression are at nucleotide positions 187 and 233 of the sequence shown in Figure 197 B (SEQ ID NO: 273).
  • FACS analysis of these strains using anti GBS 80 antiserum determined that an intergenic region with five adenosines at position 187 and six adenosines at position 233 had higher expression levels of GBS 80 on their surface than other stains. See Figure 197 C for results obtained from the FACS analysis. Therefore, manipulating the number of adenosines present at positions 187 and 233 of the AraC and GBS 80 intergenic region may further be used to adapt GBS to increase AI protein expression.
  • the invention further includes GBS bacteria which have been adapted to produce increased levels of AI surface protein.
  • the invention includes GBS bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein, such as GBS 80.
  • the Gram positive bacteria of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the invention further includes GBS bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface.
  • the GBS bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide.
  • Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pili proteins on the surface of Gram positive bacteria.
  • Increased expression of a leader peptidase in GBS may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation.
  • the GBS bacteria adapted to have increased levels of leader peptidase may additionally be adapted to express increased levels of at least one pili protein.
  • the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes” Infection and Immunity (2004) 72(6):3444-3450).
  • Streptococus gordonii See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors
  • nib ⁇ -liatiog ' e ⁇ ic'Bi-ain' ⁇ bS ⁇ ti ⁇ e W'aderiai refer to Gram positive bacteria which are compatible with a human host subject and are not associated with human pathogenisis.
  • the non-pathogenic bacteria are modified to express the AI surface protein in oligomeric, or hyper-oligomeric form. Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non-pathogenic Gram positive bacterial genome or inserted into a plasmid.
  • the non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the AI surface protein may be isolated or purified from a bacterial culture of the non-pathogenic Gram positive bacteria.
  • the AI surface protein may be isolated from cell extracts or culture supernatants.
  • the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria.
  • the non-pathogenic Gram positive bacteria may be used to express any of the Gram positive bacterial Adhesin Island proteins described herein, including proteins from a GBS Adhesin Island, a GAS Adhesin Island, or a S pneumo Adhesin Island.
  • the non-pathogenic Gram positive bacteria are transformed to express an Adhesin Island surface protein.
  • the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase.
  • the AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with a pathogenic Gram positive bacteria, such as GBS, GAS or Streptococcus pneumoniae.
  • the non-pathogenic Gram positive bacteria may express the Gram positive bacterial Adheshin Island proteins in oligomeric forms that further comprise adhesin island proteins encoded within the genome of the non-pathogenic Gram positive bacteria.
  • L. lactis was transformed with a construct encoding GBS 80 under its own promoter and terminator sequences.
  • the transformed L. lactis appeared to express GBS 80 as shown by Western blot analysis using anti-GBS 80 antiserum. See lanes 6 and 7 of the Western Blots provided in Figures 133 A and 133B (133A and 133B are two different exposures of the same Western blot). See also Example 13.
  • Applicants also transformed L. lactis with a construct encoding GBS AI-I polypeptides GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences.
  • L. lactis expressed high molecular weight structures that were immunoreactive with anti-GBS 80 in immunoblots. See Figure 134, lane 2, which shows detection of a GBS 80 monomer and higher molecular weight polymers in total transformed L. lactis extracts. Thus, it appeared that L. lactis is capable of expressing GBS 80 in oligomeric form. The high molecular weight polymers were not only detected in L. lactis extracts, but also in the culture supernatants. See Figure 135 at lane 4. See also Example 14. Thus, the GBS AI polypeptides in oligomeric form can be isolated and purified from either L. lactis cell extracts or culture supernatants.
  • oligomeric forms can, for instance, be isolated from cell extracts or culture supernatants by release by sonication. See Figure 136A and B. See also Figure 171, which shows purification of GBS pili from whole extracts of L. lactis expressing the GBS AI-I following sonication and gel filtration on a Sephacryl HR 400 column.
  • F C " f itfOMS&lUeX ilibSKIdi ' fSitned with the construct encoding GBS AI-I polypeptides GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences expressed the GBS AI-I polypeptides on its surface.
  • FACS analysis of these transformed L. lactis detected cell surface expression of both GBS 80 and GBS 104.
  • the surface expression levels of GBS 80 and GBS 104 on the transformed L. lactis were similar to the surface expression levels of GBS 80 and GBS 104 on GBS strains COHl and JM9130013, which naturally express GBS AI-I. See Figure 169 for FACS analysis data for L. lactis transformed with GBS AI-I and wildtype JM9130013 bacteria using anti-GBS 80 and GBS 104 antisera.
  • Table 40 provides the results of FACS analysis of transformed L. lactis, COHl, and JM9130013 bacteria using anti-GBS 80 and anti-GBS 104 antisera.
  • the numbers provided represent the mean fluorescence value difference calculated for immune versus pre-immune sera obtained for each bacterial strain.
  • Table 40 FACS analysis of Z. lactis and GBS bacteria strains expressing GBS AI-I
  • mice with L. lactis transformed with GBS AI-I were immunized with L. lactis transformed with GBS AI-I .
  • the immunized female mice were bred and their pups were challenged with a dose of GBS sufficient to kill 90% of non-immunized pups.
  • Detailed protocols for intranasal and subcutaneous immunization of mice with transformed L. lactis can be found in Examples 18 and 19, respectively.
  • Table 43 provides data showing that immunization of the female mice with L. lactis expressing GBS AI-I (LL-AI 1) greatly increased survival rate of challenged pups relative to both a negative PBS control (PBS) and a negative L. lactis control (LL 10 E9, which is wild type L. lactis not transformed to express GBS AI-I).
  • PBS negative PBS control
  • LL 10 E9 negative L. lactis control
  • Table 51 provides further evidence that immunization of mice with L. lactis transformed with GBS AI-I is protective against GBS.
  • mice with L. lactis expressing the GBS AI-I Protection of immunized mice with L. lactis expressing the GBS AI-I is at least partly due to a newly raised antibody response.
  • Table 46 provides anti-GBS 80 antibody titers detected in serum of the mice immunized with Z. lactis expressing the GBS AI-I as described above. Mice immunized with Z. lactis expressing the GBS AI-I have anti-GBS 80 antibody titres, which are not observed in mice immunized with L. lactis not transformed to express the GBS AI- 1. Further, as expected from the survival data, mice subcutaneously immunized with Z. lactis transformed to express the GBS AI-I have significantly higher serum anti-GBS 80 antibody titers than mice intranasally immunized with L. lactis transformed to express the GBS AI-I.
  • Anti-GBS 80 antibodies of the IgA isotype were specifically detected in various body fl ⁇ ids of the mice subcutaneously or intranasally immunized with Z. lactis expressing the GBS AI-I.
  • opsonophagocytosis assays also demonstrated that at least some of the antiserum produced against the Z. lactis expressing GBS AI 1 is opsonic for GBS. See Figure 161.
  • a hybrid GBS AI may be a GBS AI-I with a replacement of the GBS 104 gene with a GBS 67 gene.
  • a schematic of such a hybrid GBS AI is depicted in Figure 231 A.
  • a hybrid GBS AI may alternatively be a GBS AI-I with a replacement of the GBS 52 gene with a GBS 59 gene. See the schematic at Figure 231 B.
  • a hybrid GBS AI may be a GBS AI- 1 with a substitution of a GBS 59 polypeptide for the GBS 52 gene and a substitution of the GBS 104 gene for genes encoding GBS 59 and the two GBS AI-2 sortases.
  • Another example of a hybrid GBS AI is a GBS AI-I with the substitution of a GBS 59 gene for the GBS 52 gene and a GBS 67 for the GBS 104 gene. See the schematic at Figure 232.
  • a further example of a hybrid GBS AI is a GBS AI- 1 having a GBS 59 gene and genes encoding the GBS AI-2 sortases in place of the GBS 52 gene.
  • hybrid GBS AI is a GBS AI-I with a substitution of either GBS 52 or GBS 104 with a fusion protein comprising GBS 322 and one of GBS 59, GBS 67, or GBS 150.
  • Some of these hybrid GBS AIs may be prepared as briefly outlined in Figure 234 A-F.
  • Applicants have prepared a hybrid GBS AI having a GBS AI-I sequence with a substitution of a GBS 67 coding sequence for the GBS 104 gene as depicted in Figure 231 A. Transformation of L. lactis with the hybrid GBS AI-I resulted in L. lactis expression of high molecular weight polymers containing the GBS 80 and GBS 67 proteins. See Figure 233 A, which provides Western blot analysis of L. lactis transformed with the hybrid GBS AI depicted in Figure 231 A. When L. lactis transformed with the hybrid GBS AI were probed with antibodies to GBS 80 or GBS 67, high molecular weight structures were detected.
  • the oligomeric, pilus-like structures may be produced recombinantly.
  • the AI surface protein will preferably be expressed in coordination with the expression of one or more of the AI sortases of the invention.
  • AI sortases will facilitate oligomeric or hyperoligomeric formation of the AI surface protein subunits.
  • IP C l ⁇ ' oiAsife'l ⁇ ite- ⁇ inri'SwB ⁇ ' ⁇ pically have a signal peptide sequence within the first 70 amino acid residues. They may also include a transmembrane sequence within 50 amino acid residues of the C terminus.
  • the sortases may also include at least one basic amino acid residue within the last 8 amino acids.
  • the sortases have one or more active site residues, such as a catalytic cysteine and histidine.
  • AI-I includes the surface exposed proteins of GBS 80, GBS 52 and GBS 104 and the sortases SAG0647 and SAG0648. AI-I typically appears as an insertion into the 3' end of the trmA gene.
  • AI-I may also include a divergently transcribed transcriptional regulator such as araC (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). It is believed that araC may regulate the expression of the AI operon. (See Korbel et al., Nature Biotechnology (2004) 22(7): 911 — 917 for a discussion of divergently transcribed regulators in E. coli). AI-I may also include a sequence encoding a rho independent transcriptional terminator (see hairpin structure in Figure 1). The presence of this structure within the adhesin island is thought to interrupt transcription after the GBS 80 open reading frame, leading to increased expression of this surface protein.
  • araC i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction. It is believed that araC may regulate the expression of the AI operon. (See Korbel et al., Nature Biotechnology (2004) 22(7): 911
  • AI-I sequences were identified in GBS serotype V, strain isolate 2603; GBS serotype III, strain isolate NEM316; GBS serotype II, strain isolate 18RS21; GBS serotype V, strain isolate CJBl 11; GBS serotype III, strain isolate COHl and GBS serotype Ia, strain isolate A909. (Percentages shown are amino acid identity to the 2603 sequence). (An AI-I was not identified in GBS serotype Ib, strain isolate H36B or GBS serotype Ia, strain isolate 515). An alignment of AI-I polynucleotide sequences from serotype V, strain isolates 2603 and
  • GBS 80 As shown in this figure, the full length of surface protein GBS 80 is particularly conserved among GBS serotypes V (strain isolates 2603 and CJBIII), III (strain isolates NEM316 and COHl), and Ia (strain isolate A909).
  • the GBS 80 surface protein is missing or fragmented in serotypes II (strain isolate 18RS21), Ib (strain isolate H36B) and Ia (strain isolate 515).
  • FIGURE 30 Polynucleotide and amino acid sequences for AraC are set forth in FIGURE 30. GiMAcfc ⁇ nyiMlEii / iS! 7 ⁇ 3 ⁇
  • a second adhesin island "Adhesin Island 2" or “AI-2” or “GBS AI-2” has also been identified in numerous GBS serotypes.
  • a schematic depicting the correlation between AI-I and AI-2 within the GBS serotype V, strain isolate 2603 is shown in Figure 3.
  • Homology percentages in Figure 3 represent amino acid identity of the AI-2 proteins to the AI-I proteins.
  • Alignments of AI-2 polynucleotide sequences are presented in Figures 20 and 21 ( Figure 20 includes sequences from serotype V, strain isolate 2603 and serotype III, strain isolate NEM316.
  • Figure 21 includes sequences from serotype III, strain isolate COHl and serotype Ia, strain isolate A909).
  • AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJBl 11; serotype Ia, strain isolate 515; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype III, strain isolate NEM316 is presented in Figure 24.
  • Preferred AI-2 polynucleotide and amino acid sequences are conserved among two or more GBS serotypes or strain isolates.
  • AI-2 comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, AI-2 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5 or more) of GBS 67, GBS 59, GBS 150,
  • AI-2 includes open reading frames encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406.
  • AI-2 may include open reading frames encoding for two or more of 01520, 01521, 01522, 01523, 01523, 01524 and 01525.
  • One or more of the surface proteins typically include an LPXTG motif (such as LPXTG (SEQ
  • GBS AI-2 sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GBS AI-2 may encode for at least one surface protein.
  • AI-2 may encode for at least two surface proteins and at least one sortase.
  • GBS AI-2 encodes for at least three surface proteins and at least two sortases.
  • One or more of the AI-2 surface proteins may include an LPXTG or other sortase substrate motif.
  • One or more of the surface proteins may also typically include pilin motif.
  • the pilin motif may be involved in pili formation. Cleavage of AI surface proteins by sortase between the threonine and glycine residue of an LPXTG motif yields a thioester-linked acyl intermediate of sortase.
  • the first lysine residue in a pilin motif can serve as an amino group acceptor of the cleaved LPXTG motif and thereby provide a covalent linkage between AI subunits to form pili.
  • the pilin motif can make a nucleophilic attack on the acyl enzyme providing a covalent linkage between AI subunits to form pili and regenerate the sortase enzyme.
  • pilin motifs that may be present in the GBS AI-2 proteins include ((YPKN(X 8 )K; SEQ ID NO: 158), (PK(X 8 )K; SEQ ID NO: 159), (YPK(X 9 )KjSEQ ID NO: 160), (PKN(X 8 )K; SEQ ID NO: 161), or (PK(Xi 0 )K; SEQ ID NO: 162)).
  • One or more of the surface protein may also include an E box motif.
  • the E box motif contains a conserved glutamic acid residue that is believed to be necessary for pilus formation.
  • Some examples of E box motifs may include the amino acid sequences YxLxETxAPxG (SEQ ID NO: 163), 165), or YxLxETxAPxGY
  • GBS AI-2 may include the surface exposed proteins of GBS 67, GBS 59 and GBS 150 and the sortases of SAG1406 and SAG1405.
  • GBS AI-2 may include the proteins 01521, 01524 and 01525 and sortases 01520 and 01522.
  • GBS 067 and 01524 are preferred AI-2 surface proteins.
  • AI-2 may also include a divergently transcribed transcriptional regulator such as a Ro fA like protein (for example rogB). As in AI-I, rogB is thought to regulate the expression of the AI-2 operon.
  • a divergently transcribed transcriptional regulator such as a Ro fA like protein (for example rogB).
  • rogB is thought to regulate the expression of the AI-2 operon.
  • FIG. 4 A schematic depiction of AI-2 within several GBS serotypes is depicted in Figure 4. (Percentages shown are amino acid identity to the 2603 sequence). While the AI-2 surface proteins GBS 59 and GBS 67 are more variable across GBS serotypes than the corresponding AI-I surface proteins, AI-2 surface protein GBS 67 appears to be conserved in GBS serotypes where the AI-I surface proteins are disrupted or missing.
  • the AI-I GBS 80 surface protein is fragmented in GBS serotype II, strain isolate 18RS21.
  • the GBS 67 surface protein has 99% amino acid sequence homology with the corresponding sequence in strain isolate 2603.
  • the AI-I GBS 80 surface protein appears to be missing in GBS serotype Ib, strain isolate H36B and GBS serotype Ia, strain isolate 515.
  • the GBS 67 surface protein has 97 — 99 % amino acid sequence homology with the corresponding sequence in strain isolate 2603.
  • GBS 67 appears to have two allelic variants, which can be divided according to percent homology with strains 2603 and H36B. See figures 237-239.
  • GBS 59 of GBS strain isolate 2603 shares 100% amino acid residue homology with GBS strain 18RS21, 62% amino acid sequence homology with GBS strain H36B, 48% amino acid residue homology with GBS strain 515 and GBS strain CJBl 11, and 47% amino acid residue homology with GBS strain NEM316.
  • the amino acid sequence homologies of the different GBS strains suggest that there are two isoforms of GBS 59. The first isoform appears to include the GBS 59 protein of GBS strains CJBl I l, NEM316, and 515. The second isoform appears to include the GBS 59 protein of GBS strains 18RS21, 2603, and H36B. (See Figures 63 and 224.)
  • the immunogenic composition of the invention comprises a first and a second isoform of the GBS 59 protein to provide protection across a wide range of GBS serotypes that express polypeptides from a GBS AI-2.
  • the first isoform may be the GBS 59 protein of GBS strain CJBl 11, NEM316, or 515.
  • the second isoform may be the GBS 59 protein of GBS strain 18RS21, 2603, or H36B.
  • GBS 59 The gene encoding GBS 59 has been identified in a high number of GBS isolates; the GBS 59 gene was detected in 31 of 40 GBS isolates tested (77.5%).
  • the GBS 59 protein also appears to be present as part of a pilus in whole extracts derived from GBS strains.
  • Figure 64 shows detection of high molecular weight GBS 59 polymers in whole extracts of GBS strains CJB 111 , 7357B, COH31 , D1363C, 5408, 1999, 5364, 5518, and 515 using antiserum raised against GBS 59 of GBS strain CJB 111.
  • Figure 65 also shows detection of these high molecular weight GBS 59 polymers in whole extracts of GBS strains D136C, 515, and CJBl 11 with anti-GBS 59 antiserum. (See also Figure 220 A for detection of GBS 59 high molecular weight polymers in strain 515.)
  • Figure 65 confirms the presence of different isoforms of GBS 59. Antisera raised against two different GBS 59 isoforms results in different patterns of immunoreactivity depending on the GBS strain origin of the whole extract.
  • Figure 65 further shows detection of GBS 59 monomers in purified GBS 59 preparations.
  • GBS 59 is also highly expressed on the surface of GBS strains.
  • GBS 59 was detected on the surface of GBS strains CJB 111 , DKl , DK8, Davis, 515, 2986, 5551 , 1169, and 7357B by FACS analysis using mouse antiserum raised against GBS 59 of GBS CJBl 11.
  • FACS analysis did not detect surface expression of GBS 59 in GBS strains SMU071, JM9130013, and COHl, which do not contain a GBS 59 gene. (See Figure 66.)
  • Further confirmation that GBS 59 is expressed on the surface of GBS is detection of GBS 59 by immuno-electron microscopy on the surface of GBS strain 515 bacteria. See Figure 215.
  • GBS 67 and GBS 150 also appear to be included in high molecular weight structures, or pili.
  • Figure 69 shows that anti-GBS 67 and anti-GBS 150 immunoreact with high molecular weight structures in whole GBS strain 515 extracts. (See also Figure 220 B and C.) It is also notable in Figure 69 that the anti-GBS 59 antisera, raised in a mouse following immunization with GBS 59 of GBS strain 2603, does not cross-hybridize with GBS 59 in GBS strain 515.
  • GBS 59 of GBS stain 515 is of a different isotype than GBS 59 of GBS stain 2603.
  • FIG. 70 provides Western blots showing that higher molecular weight structures in GBS strain 515 total e ⁇ iy ⁇ tslim ⁇ ik ⁇ l with' ' a ⁇ i"i'es l : ⁇ ' ll3li anti-GBS 150 antiserum.
  • anti-GBS 67 antiserum no longer immunoreacts with polypeptides in total extracts, while anti-GBS 150 antiserum is still able to cross-hybridze with high molecular weight structures.
  • formation of pili containing GBS 59 does not appear to require GBS 67 expression.
  • FACS detects GBS 67 cell surface expression on wildtype GBS strain 515, but not GBS strain 515 cells knocked out for GBS 67.
  • FACS analysis using anti-GBS 59 antisera detects GBS 59 expression on both the wildtype GBS strain 515 cells and the GBS strain 515 cells knocked out for GBS 67.
  • GBS 59 cell surface expression is detected on GBS stain 515 cells regardless of GBS 67 expression.
  • GBS 67 while present in pili, appears to be localized around the surface of GBS strain 515 cells. See the immuno-electron micrographs presented in Figure 216. GBS 67 binds to fibronectin. See Figure 217.
  • GBS AI-2 Formation of pili encoded by GBS AI-2 does require expression of GBS 59.
  • Deletion of GBS 59 from strain 515 bacteria eliminates detection of high molecular weight structures by antibodies that bind to GBS 59 ( Figure 221 A, lane 3), GBS 67 ( Figure 221 B, lane 3), and GBS 150 ( Figure 221 C, lane 3).
  • Western blot analysis of 515 bacteria with a deletion of the GBS 67 gene detects high molecular weight structures using GBS 59 ( Figure 221 A, lane 2) and GBS 150 ( Figure 221 C, lane 2) antisera.
  • GBS 59 be a third type of AI (Adhesin Island-3, AI-3, or GBS AI-3).
  • More than one AI surface protein may be present in the oligomeric, pilus-like structures of the invention.
  • GBS 59 and GBS 67 may be incorporated into an oligomeric structure.
  • GBS 59 and GBS 150 may be incorporated into an oligomeric structure, or GBS 59, GBS 150 and GBS 67 may be incoiporated into an oligomeric structure.
  • the invention includes compositions comprising two or more AI surface proteins.
  • the composition may include surface proteins from the same adhesin island.
  • the composition may include two or more GBS AI-2 surface proteins, such as GBS 59, GBS 67 and GBS 150.
  • the surface proteins may be isolated from Gram positve bacteria or they may be produced recombinantly.
  • Applicants have identified at least four different GAS Adhesin Islands. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus.
  • Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fascilitis.
  • post-streptococcal autoimmune responses are still a major cause of cardiac pathology in children.
  • Group A Streptococcal infection of its human host can generally occur in three phases.
  • the first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces. Generally this attachment phase begins in the throat or the skin.. The deeper the tissue level infected, the more severe the damage that can be caused.
  • the bacteria secretes a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. This toxin binds to susceptible host cell receptors and triggers innappropropriate immune responses by these host cells, resulting in pathology. Because the toxin can diffuse throughout the host, the necrosis directly caused by the GAS toxins may be physically located in sites distant from the bacterial infection.
  • the final phase of GAS infection can occur long after the original bacteria have been cleared from the host system. At this stage, the host's previous immune response to the GAS bacteria due to cross reactivity between epitopes of a GAS surface protein, M, and host tissues, such as the heart.
  • M GAS surface protein
  • host tissues such as the heart.
  • an effective vaccine against GAS will preferably facilitate host elimination of the bacteria during the initial attachment and invasion stage.
  • IF C " lolaAI Ii$loip.A Ilreplii ⁇ ic ⁇ are historically classified according to the M surface protein described above.
  • the M protein is surface exposed trypsin-sensitive protein generally comprising two polypeptide chains complexed in an alpha helical formation.
  • the carboxyl terminus is anchored in the cytoplasmic membrane and is highly conserved among all group A streptococci.
  • the amino terminus, which extend through the cell wall to the cell surface, is responsible for the antigenic variability observed among the 80 or more serotypes of M proteins.
  • T-antigen A second layer of classification is based on a variable, trypsin-resistant surface antigen, commonly referred to as the T-antigen.
  • T-antigen a variable, trypsin-resistant surface antigen
  • Decades of epidemiology based on M and T serological typing have been central to studies on the biological diversity and disease causing potential of Group A Streptococci. While the M-protein component and its inherent variability have been extensively characterized, even after five decades of study, there is still very little known about the structure and variability of T-antigens.
  • Antisera to define T types is commercially available from several sources, including Sevapharma (http://www.sevapharma.cz/en).
  • T-antigen T-type 6
  • M6 strain of GAS M6 strain of GAS
  • FCT Fibronectin-binding, Collagen-binding T-antigen
  • the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used.
  • GAS AI sequences can be identified in numerous M types, Applicants have surprisingly discovered a correlation between the four main pilus subunits from the four different GAS AI types and specific T classifications. While other trypsin-resistant surface exposed proteins are likely also implicated in the T classification designations, the discovery of the role of the GAS adhesin islands (and the associated hyper-oligomeric pilus like structures) in T classification and GAS serotype variance has important implications for prevention and treatment of GAS infections. Applicants have identified protein components within each of the GAS adhesin islands which are associated with the pilus formation. These proteins are believed to be involved in the bacteria's initial adherence mechanisms. Immunological recognition of these proteins may allow the host immune response to slow or prevent the bacteria's transition into the more pathogenic later stages of infection.
  • GBS pili structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysaccharide matrix).
  • Biofilms are generally associated with bacterial resistance, as antibiotic treatments and host immune response are frequently unable to erradicate all of the bacteria components of the biofilm.
  • Direction of a host immune response against surface proteins exposed during the first steps of bacterial attachment is preferable.
  • the invention therefore provides for improved immunogenic compositions against GAS infection which may target GAS bacteria during their initial attachment efforts to the host epithelial cells and may provide protection against a wide range of GAS serotypes.
  • the immunogenic compositions of the invention include GAS AI surface proteins which may be formulated in an oligomeric, or hyperoligomeric (pilus) form.
  • the invention also includes combinations of GAS AI surface proteins. Combinations of GAS AI surface proteins may be selected from the same adhesin island or they may be selected from different GAS adhesin islands.
  • GAS AI sequences may be generally characterized as Type 1, Type 2, Type 3, and Type 4, depending on the number and type of sortase sequence within the island and the percentage identity of other proteins within the island.
  • Schematics of the GAS adhesin islands are set forth in FIGURE 51A and FIGURE 162.- In all strains identified so far, the adhesin island region is flanked by highly conserved open reading frames Ml_123 and Ml_136. Between three and five genes in each GAS adhesin island code for ECM binding adhesin proteins containing LPXTG motifs.
  • GAS AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-I proteins").
  • GAS AI-I preferably comprises surface proteins, a srtB sortase, and a rofA divergently transcribed transcriptional regulator.
  • lipidXTG sortase substrate motif such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with serine).
  • GAS AI-I includes open reading frames encoding for two or more (i.e., 2, 3, 4 or 5) of M6_SpyO157, M6_SpyO158, M6_SpyO159, M6_SpyO 160, M6_Spy0161.
  • a GAS AI-I may comprise a polynucleotide encoding any one of CDC SS 410_f ⁇ mbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
  • the hyper-oligomeric pilus structure of GAS AI-I appears to be responsible for the T-antigen type 6 classification, and GAS AI-I corresponds to the FCT region previously identified for tee ⁇ .
  • the tee ⁇ FCT region includes open reading frames encoding for a collagen adhesion protein (cpa, capsular polysaccharide adhesion) and a fibronectin binding protein (prtFl).
  • cpa collagen adhesion protein
  • prtFl fibronectin binding protein
  • Immunoblots with antiserum specific for Cpa also recognize a high molecular weight ladder structure, indicating Cpa involvement in the GAS AI-I pilus structure or formation.
  • Cpa antiserum reveals abundant staining on the surface of the bacteria and occasional gold particles extended from the surface of the bacteria.
  • immunoblots with antiserum specific for PrtFl recognize only a single molecular species with electrophoretic mobility corresponding to its predicted molecular mass, indicating that PrtFl may not be associated with the oligomeric pilus structure.
  • a preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • the oligomeric form is a hyperoligomer.
  • Another preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which has been isolated in an oligomeric (pilis) form.
  • the oligomer or hyperoligomeric pilus structures comprising the GAS AI-I surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
  • One or more of the GAS AI-I open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-I open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the GAS AI-I surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the LPXTG sortase substrate motif of a GAS AI surface protein may be generally represented by the formula XXXXG, wherein X at amino acid position 1 is an L, a V, an E, or a Q, wherein X at amino acid position 2 is a P if X at amino acid position 1 is an L, wherein X at amino acid position 2 is a V if X at amino acid position 1 is a E or a Q, wherein X at amino acid position 2 is V, wherein X at amino acid position 3 is any amino acid residue, wherein X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, or Q, and wherein X at amino acid position 4 is a T, S, or A if X at amino acid position 1 is an L
  • LPXTG motifs present in GAS AI surface proteins include LPSXG (SEQ ID NO: 134), VVXTG (SEQ ID NO: 135), EVXTG (SEQ ID NO: 136), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138), LPXAG (SEQ ID NO: 139), QVPTG (SEQ ID NO: 140), and FPXTG (SEQ ID NO: 141).
  • the GAS AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells.
  • AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer.
  • one or more GAS AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • GAS AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • GAS AI-I sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI-I may encode for at least one surface protein.
  • GAS AI-I may encode for at least two surface exposed proteins and at least one sortase.
  • GAS AI-I encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • GAS AI-I preferably includes a srtB sortase.
  • GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a GAS AI-I surface protein such as M6_Spy0157, M6_SpyO159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, or DSM2071_fimbrial.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the ⁇ oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligome ⁇ c, pilus-hke structures of the invention will preferably include a pilin motif.
  • the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more GAS Adhesin Island 1 ("GAS AI-I") proteins and one or more GAS Adhesin Island 2 ("GAS AI-2"), GAS Adhesin Island 3 (“GAS AI-3"), or GAS Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the GAS Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • GAS Adhesin Island 1 GAS Adhesin Island 1
  • GAS AI-3 GAS Adhesin Island 3
  • GAS Adhesin Island 4 GAS Adhesin Island 4
  • GAS AI-I may also include a divergently transcribed transcriptional regulator such as RofA ⁇ i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction).
  • GAS Adhesin Island 2 A second adhesin island, "GAS Adhesin Island 2" or "GAS AI-2" has also been identified in
  • GAS AI-2 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-2 proteins"). Specifically, GAS AI-2 includes open reading frames encoding for two or more ⁇ i.e., 2, 3, 4, 5, 6, 7, or 8) of GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, S ⁇ yO133, and GAS20.
  • a preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which may be formulated or purified in an oligomeric (pills) form.
  • the oligomeric form is a hyperoligomer.
  • Another preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which has been isolated in an oligomeric (pilis) form.
  • the oligomer or hyperoligomeric pilus structures comprising the GAS AI-2 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
  • One or more of the GAS AI-2 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-2 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the GAS AI-2 surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. AI surface proteins may also be able to bind to or associate with fibrinogen, f ⁇ bronectin, or collagen. H " " ! '-" predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI-2 may encode for at least one surface protein. Alternatively, GAS AI-2 may encode for at least two surface exposed proteins and at least one sortase. Preferably, GAS AI-2 encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as GAS 15, GAS 16, or GAS 18.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the oligomeric, pilus like structures may be used alone or in the combinations of the invention.
  • the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more GAS Adhesin Island 2 ("GAS AI-2") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I”), GAS Adhesin Island 3 (“GAS AI-3”), or GAS Adhesin Island 4 (“GAS AI-4”) proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • GAS AI-2 GAS Adhesin Island 2
  • GAS AI-I GAS Adhesin Island 1
  • GAS Adhesin Island 3 GAS Adhesin Island 3
  • GAS Adhesin Island 4 GAS Adhesin Island 4
  • GAS AI-2 may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction).
  • rofA a divergently transcribed transcriptional regulator
  • GAS Adhesin Island 3 A third adhesin island, "GAS Adhesin Island 3" or “GAS AI-3” has also been identified in several Group A Streptococcus serotypes and isolates.
  • GAS AI-3 comprises a series of approximately sdvefir ⁇ perf ' reMu ⁇ g mine's eh'c ⁇ ' ⁇ irig for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-3 proteins").
  • GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, SPsOlOO, orf78, orf79, orfSO, orf81, orf82, orf83, orf84, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, s ⁇ yM18_0131, spyM18_0132, SpyoM01000156, SpyoMO 1000155, SpyoMO 1000154, SpyoMO 1000153, SpyoMO 1000152, SpyoM01000151, Spyo
  • GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyM3_0098, SpyM3_0099, S ⁇ yM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104.
  • GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPsO104, SPs0105, and SPsOlOO.
  • GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of orf78, orf79, orfSO, orf ⁇ l, orf82, orf83, and orf84.
  • GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132.
  • GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyoMO 1000156, SpyoMO 1000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151, SpyoMO 1000150, and SpyoMO 1000149.
  • Applicants have also identified open reading frames encoding fimbrial structural subunits in other GAS bacteria harbouring an AI-3. These open reading frames encode fimbrial structural subunits ISS3040J ⁇ mbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
  • a GAS AI-3 may comprise a polynucleotide encoding any one of ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959j5mbrial.
  • GAS AI-3 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-3 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • a preferred immunogenic composition of the invention comprises a GAS AI-3 surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • the oligomeric form is a hyperoligomer.
  • Another preferred immunogenic composition of the invention comprises a GAS AI-3 surface protein which has been isolated in an oligomeric (pilis) form.
  • the oligomer or hyperoligomeric pilus structures comprising the GAS AI-3 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
  • One or more of the GAS AI-3 surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer.
  • one or more AI surface proteins are capable of binding to or otherwise associating with an I P , Ii Ij'" / Il H c; in Kj; ,. ⁇ ' p> "7 ⁇ $ "" -I! CI)I epitH'el ⁇ a ⁇ tfell 'Surface.
  • '"Al surface protein ' s may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • GAS AI-3 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI-3 may encode for at least one surface protein.
  • GAS AI-3 may encode for at least two surface exposed proteins and at least one sortase.
  • GAS AI-3 encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine or alanine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., ' Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPs0106, orf78, orfSO, orf82, orf84, spyM18_0126, spyM18_0128, spyM18_0130, spyM18_0132, SpyoM01000155, Sp'yoMO 1000153, SpyoMO 1000151, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
  • an AI surface protein such as SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPs0106, orf78, orfSO,
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyM3_0098, SpyM3_0100, S ⁇ yM3_0102, and SpyM3_0104.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SPsOlOO, SPs0102, SPs0104, and SPsOlOo.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as orf78, orf80, orf82, and orf84.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as spyM18_0126, spyM18_0128, s ⁇ yM18_0130, and s ⁇ yM18_0132.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyoMO 1000155, SpyoM01000153, S ⁇ yoM01000151, and SpyoM01000149.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric siibiitrits may%e''covaleritly as's ⁇ 'cia ' ⁇ ed'via an LPXTG motif, preferably, via the threonine amino acid residue.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the oligomeric, pilus like structures may be used alone or in the combinations of the invention.
  • the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more GAS Adhesin Island 3 ("GAS AI-3") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I”), GAS Adhesin Island 2 (“GAS AI-2”), or GAS Adhesin Island 4 (“GAS AI-4”) proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • GAS AI-3 GAS Adhesin Island 3
  • GAS AI-I GAS Adhesin Island 1
  • GAS Adhesin Island 2 GAS Adhesin Island 2
  • GAS Adhesin Island 4 GAS Adhesin Island 4
  • GAS AI-3 may also include a transcriptional regulator such as Nra.
  • GAS Adhesin Island 4 A fourth adhesin island, "GAS Adhesin Island 4" or "GAS AI-4" has also been identified in
  • GAS AI-4 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-4 proteins"). Specifically, GAS AI-4 includes open reading frames encoding for two or more ⁇ i.e., 2, 3, 4, 5, 6, 7, or 8) of 19224134, 19224135, 19223136, 19223137, 19224138, 19224139, 19224140, and 19224141.
  • a GAS AI-4 may comprise a polynucleotide encoding any one of 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
  • GAS AI-4 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the GAS AI-4 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • a preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • the oligomeric form is a hyperoligomer.
  • Another preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which has been isolated in an oligomeric (pilis) form.
  • the oligomer or hyperoligomeric pilus structures comprising the GAS AI-4 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
  • One or more of the GAS AI-4 surface protein sequences typically include an LPXTG motif
  • AI surface proteins of the ir ⁇ elttio ⁇ i ⁇ a ⁇ '4SJtlti : e ! ability ' 1 Of r ' tie :; GA ; ⁇ bacteria to adhere to and invade epithelial cells.
  • AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer.
  • one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • GAS AI-4 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • GAS AI-4 may encode for at least one surface protein.
  • GAS AI-4 may encode for at least two surface exposed proteins and at least one sortase.
  • GAS AI-4 encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid IL
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_f ⁇ mbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more GAS Adhesin Island 4 ("GAS AI-4") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I "), GAS Adhesin Island 2 (“GAS AI-2”), or GAS Adhesin Island 3 (“GAS AI-3”) proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • GAS AI-4 may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction).
  • rofA a divergently transcribed transcriptional regulator
  • the oligomeric, pilus-like structures of the invention may be combined with one or more additional GAS proteins.
  • the oligomeric, pilus-like structures comprise one or more AI surface proteins in combination with a second GAS protein.
  • the oligomeric, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an AI surface protein.
  • the invention therefore includes a method for manufacturing an oligomeric AI surface antigen comprising culturing a GAS bacterium that expresses the oligomeric AI protein and isolating the expressed oligomeric AI protein from the GAS bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed AI protein.
  • the AI protein is in a hyperoligomeric form.
  • the oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing an AI surface protein.
  • the invention therefore includes a method for manufacturing an oligomeric Adhesin Island surface antigen comprising culturing a GAS bacterium adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the GAS bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed
  • Adhesin Island protein Preferably, the Adhesin Island protein is in a hyperoligomeric form.
  • the GAS bacteria are preferably adapted to increase AI protein expression by at least two
  • GAS bacteria may be adapted to increase AI protein expression by any means known in the art, including methods of increasing gene dosage and methods of gene upregulation. Such means include, for example, transformation of the GAS bacteria with a plasmid encoding the AI protein.
  • the plasmid may include a strong promoter or it may include multiple copies of the sequence encoding the AI protein.
  • the sequence encoding the AI protein within the GAS bacterial genome may be deleted.
  • Island may be modified to increase expression.
  • the invention further includes GAS bacteria which have been adapted to produce increased levels of AI surface protein.
  • the invention includes GAS bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein.
  • the Gram positive bacteria of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the invention further includes GAS bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface.
  • the GAS bacteria may be adapted AI proteins on its surface by increasing expression levels of LepA polypeptide, or an equivalent signal peptidase, in the GAS bacteria.
  • Applicants have shown that deletion of LepA in strain SF370 bacteria, which harbour a GAS AI-2, abolishes surface exposure of M and pili proteins on the GAS, Increased levels of LepA expression in GAS are expected to result in increased exposure of M and pili proteins on the surface of GAS.
  • Increased expression of LepA in GAS may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation.
  • the GAS bacteria adapted to have increased levels of LepA expression may additionally be adapted to express increased levels of at least one pili protein.
  • the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes" Infection and Immunity (2004) 72(6):3444-3450).
  • non-pathogenic Gram positive bacteria refer to Gram positive bacteria which are compatible with a human host subject and are not associated with human pathogenisis.
  • the non-pathogenic bacteria are modified to express the AI surface protein in oligomeric, or hyper-oligomeric form. Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non-pathogenic Gram positive bacterial genome or inserted into a plasmid.
  • the non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the AI surface protein may be isolated or purified from a bacterial culture of the non-pathogenic Gram positive bacteria.
  • the AI surface protein may be isolated from cell extracts or culture supernatants.
  • the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria.
  • the non-pathogenic Gram positive bacteria may be used to express any of the GAS Adhesin Island proteins described herein.
  • the non-pathogenic Gram positive bacteria are transformed to express an Adhesin Island surface protein.
  • the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase.
  • the AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with pathogenic GAS.
  • L. lactis was transformed with pAM401 constructs encoding entire pili gene clusters of AI-I, AI-2, and AI-4 adhesin islands.
  • the ⁇ AM401 is a promoterless high-copy plasmid.
  • the entire pili gene clusters of anM6 (AI-I), Ml (AI-2), and M12 (AI-4) bacteria were inserted into the pAM401 construct.
  • the gene clusters were transcribed under the control their own (M6, Ml, or M12) promoter or the GBS promoter that successfully initiated expression of the GBS AI-I adhesin islands in Z.
  • Figure 172 provides a schematic depiction of GAS M6 (AI-I), ]yll 1 'VM-I);'ail!d'M ⁇ tA ⁇ -4) idh ⁇ sSfis ⁇ i ⁇ m ⁇ i and indicates the portions of the adhesin island sequences inserted in the pAM401 construct.
  • FIG. 173 A-C provide results of Western blot analysis of surface protein-enriched extracts of L. lactis transformed with M6 ( Figure 173 A), Ml ( Figure 173 B), or Ml 2 ( Figure 173 C) adhesin island gene clusters using antibodies that bind to the fimbrial structural subunit encoded by each cluster.
  • Figure 173 A at lanes 3 and 4 shows detection of high molecular structures in L.
  • FIG. 173B at lanes 3 and 4 shows detection of high molecular weight structures in L. lactis transformed with an adhesin island pilus gene cluster from an M 12 AI-4 using an antibody that binds to fimbrial structural subunit EftLSL.A.
  • Figure 173C at lane 3 shows detection of high molecular weight structures in L. lactis transformed with an adhesin island pilus gene cluster from an M6 AI- 1 using an antibody that binds to fimbrial structural subunit M6_Spy0160.
  • S. pneumoniae from TIGR4 Adhesin Island As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae from TIGR4.
  • the S. pneumoniae from TIGR4 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae from TIGR4 AI proteins includes open reading frames encoding for two or more ⁇ i.e., 2, 3, 4, 5, 6, or 7) of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, and SP0468.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae from TIGR4 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • the oligomeric form is a hyperoligomer.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae from TIGR4 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • the oligomer or hyperoligomer pilus structures comprising S. pneumoniae surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • I' "1 " ** TIGR4 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae from TIGR4 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae from TIGR4 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae from TIGR4 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells.
  • AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer.
  • one or more S. pneumoniae from TIGR4 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • S. pneumoniae from TIGR4 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • S. pneumoniae from TIGR4 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae from TIGR4 AI may encode for at least one surface protein.
  • S. pneumoniae from TIGR4 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae from TIGR4 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae from TIGR4 AI surface protein such as SP0462, SP0463, SP0464, or SP0465.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif B" ' Trie oTigomeric' j 'pilus ' lfke sMctures may be used alone or in the combinations of the invention.
  • the invention comprises a S. pneumoniae from TIGR4 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae from TIGR4 AI proteins and one or more S. pneumoniae strain 670 AI proteins, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae from TIGR4 AI may also include a transcriptional regulator.
  • S. pneumoniae strain 670 Adhesin Island As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 670.
  • the S. pneumoniae strain 670 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S.
  • pneumoniae strain 670 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of orfl_670, or£3_670, orf4_670, orf5_670, orf6_670, orf7_670, orf8_670.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 670 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 670 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 670 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 670 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 670 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 670 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 670 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 670 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • S. pneumoniae strain 670 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 670 AI may encode for at least one surface protein.
  • S. pneumoniae strain 670 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 670 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, prefeMbly M ' twee ' n lie thre ⁇ ineW glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 670 AI surface protein such as orf3_670, orf4_670, or orf5_670.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 670 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 670 AI proteins and one or more S. pneumoniae from TIGR4 AI proteins, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 670 AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 14 CSR 10 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae strain 14 CSR 10 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2J4CSR, ORF3_14CSR, ORF4J4CSR, ORF5_14CSR, ORF6_14CSR, ORF7_14CSR, ORF8J4CSR.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 14 CSR 10 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 14 CSR 10 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • ft"" " One oi” ⁇ SMjf't ⁇ e Erpi ⁇ e& ⁇ m ⁇ i strain 14 CSR 10 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 14 CSR 10 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 14 CSR 10 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 14 CSR 10 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 14 CSR 10 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 14 CSR 10 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • S. pneumoniae strain 14 CSR 10 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 14 CSR 10 AI may encode for at least one surface protein.
  • S. pneumoniae strain 14 CSR 10 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 14 CSR 10 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 14 CSR 10 AI surface protein such as orf3_CSR, orf4_CSR, or orf5_CSR.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif. in. ii T' / 11 1 iq; n iir , ⁇ " p> 7 1 ;p Ti; q ⁇
  • the invention comprises a S. pneumoniae strain 14 CSR 10 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 14 CSR 10 AI proteins, and one or more AI proteins of any of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 14 CSR 10AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 19A Hungary 6 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae strain 19A Hungary 6 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF6_19AH, ORF7_19AH, ORF8_19AH.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 19A Hungary 6 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 19A Hungary 6 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 19A Hungary 6 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 19A Hungary 6 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 19A Hungary 6 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 19A Hungary 6 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 19A Hungary 6 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 19A Vietnamese 6 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • the S. pneumoniae strain 19A Hungary 6 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 19A Hungary 6 AI may encode for at least one surface protein.
  • S. pneumoniae strain 19A may encode for at least one surface protein.
  • Hungary 6 AI may encode for at least two surface exposed proteins and at least one sortase.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 19A Hungary 6 AI surface protein such as or ⁇ _19AH, orf4_19AH, or orf5_19AH.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 19A Hungary 6 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 19A Hungary 6 AI proteins and one or more AI proteins from one of any one of S. pneumoniae from TIGR4, 670, 14 CSR 10, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI GR4 AI proteins, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 19A Hungary 6 AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 19F Taiwan 14 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 19F
  • Taiwan 14 AI proteins includes open reading frames encoding for two or more ⁇ i.e., 2, 3, 4, 5, 6, or 7) o
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 19F Taiwan 14 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a 5. pneumoniae strain 19F Taiwan 14 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 19F Taiwan 14 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORP.
  • one or more of the S. pneumoniae strain 19F Taiwan 14 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • Taiwan 14 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 19F Taiwan 14 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 19F Taiwan 14 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 19F Taiwan 14 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The JS. pneumoniae strain 19F Taiwan 14 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S.
  • pneumoniae strain 19F Taiwan 14 AI may encode for at least one surface protein.
  • S. pneumoniae strain 19F Taiwan 14 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 19F Taiwan 14 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an Al sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 19F Taiwan 14 AI surface protein such as orf3_19FTW, orf4_19FTW, or orf5_19FTW.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 3
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 19F Taiwan 14 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 19F Taiwan 14 AI proteins and one or more AI proteins of any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 14 CSR 10, 23F Taiwan 15, or 23F Poland 16, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 19F Taiwan 14 AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 23F Poland 16 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae strain 23F Tru 16 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_23FP, ORF3J23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, and ORF8_23FP.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Tru 16 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Tru 16 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 23F Tru 16 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 23F Poland 16 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 23F Tru 16 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 23F Tru 16 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells.
  • AI surface proteins may ⁇ [afffcf.'fHbJk ⁇ ilit ⁇ ⁇ S. '' pOevifiv&Mie li translocate through an epithelial cell layer.
  • one or more S. pneumoniae strain 23F Tru 16 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • S. pneumoniae strain 23 F Poland 16 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • pneumoniae strain 23 F Poland 16 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 23F ' Tru 16 AI may encode for at least one surface protein.
  • S. pneumoniae strain 23F Poland 16 Al may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 23F Poland 16 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface ' protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 23F Tru 16 AI surface protein such as or ⁇ _23FP, orf4_23FP, or orf5_23FP.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, ' respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 23F Tru 16 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 23F Tru 16 AI proteins and one or more AI proteins from any one or more S. pneumoniae strains of TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 14 CSR 10, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 23F Poland 16 AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 23F Taiwan 15 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae strain 23F Taiwan 15 AI proteins includes open reading frames encoding for two or more ⁇ i.e., 2, 3, 4, 5, 6, or 7) of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, ORF8_23FTW.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Taiwan 15 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Taiwan 15 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 23F Taiwan 15 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 23F Taiwan 15 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 23F Taiwan 15 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 23F Taiwan 15 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 23F Taiwan 15 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 23 F Taiwan 15 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • S. pneumoniae strain 23F Taiwan 15 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 23F Taiwan 15 AI may encode for at least one surface protein.
  • S. pneumoniae strain 23 F Taiwan 15 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 23F Taiwan 15 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the asl ⁇ yffiuli ⁇ Iibaiiipepiliiialtiin ⁇ ilactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 23F Taiwan 15 AI surface protein such as orG_23FTW, orf4_23FTW, or orf5_23FTW.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 23F Taiwan 15 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 23F Taiwan 15 AI proteins and one or more AI proteins from any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 14 CSR 10, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 23F Taiwan 15 AI may also include a transcriptional regulator. ⁇ !> ⁇ pneumoniae strain 6B Finland 12 Adhesin Island
  • the S. pneumoniae strain 6B Finland 12 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 6B
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Finland 12 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Finland 12 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 6B Finland 12 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • ⁇ e! ⁇ ! ⁇ yp ⁇ lJ ⁇ S ⁇ S ⁇ v ⁇ ' ⁇ BS ⁇ & ⁇ 3 ⁇ oniae strain 6B Finland 12 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 6B Finland 12 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the >S. pneumoniae strain 6B Finland 12 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 6B Finland 12 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 6B Finland 12 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • S. pneumoniae strain 6B Finland 12 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 6B Finland 12 AI may encode for at least one surface protein.
  • S. pneumoniae strain 6B Finland 12 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 6B Finland 12 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 6B Finland 12 AI surface protein such as orf3_6BF, orf4_6BF, or orf5_6BF.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif. ii ; '" C l ⁇ ?' ' °lil ⁇ iIli':iP, ⁇ i:lip' IMiMlS may be used alone or in the combinations of the invention.
  • the invention comprises a S. pneumoniae strain 6B Finland 12 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 6B Finland 12 AI proteins and one or more AI proteins of any one or more of S.
  • S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 6B Finland 12 AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 6B Spain 2 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae strain 6B Spain 2 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_6BSP, ORF3_6BSP, ORF4 6BSP, ORF5 6BSP, ORF6_6BSP, ORF7_6BSP, and ORF8_6BSP.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Spain 2 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Spain 2 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 6B Spain 2 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 6B Spain 2 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 6B Spain 2 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 6B Spain 2 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 6B Spain 2 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 6B Spain 2 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The S. pneumoniae strain 6B Spain 2 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S.
  • pneumoniae strain 6B Spain 2 AI may encode for at least one surface protein.
  • S. pneumoniae strain 6B Spain 2 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 6B Spain 2 AI surface protein such as or ⁇ _6BSP, orf4_6BSP, or orf5_6BSP.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 6B Spain 2 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 6B Spain 2 AI proteins and one or more AI proteins of any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 14 CSR 10, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 6B Spain 2 AI may also include a transcriptional regulator.
  • the S. pneumoniae strain 9V Spain 3 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases.
  • the S. pneumoniae strain 9V Spain 3 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP, and ORF8_9VSP.
  • a preferred immunogenic composition of the invention comprises a S. pneumoniae strain 9V Spain 3 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
  • Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 9V Spain 3 Al surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 9 V Spain 3 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF.
  • one or more of the S. pneumoniae strain 9V Spain 3 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the S. pneumoniae strain 9V Spain 3 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • the S. pneumoniae strain 9V Spain 3 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more & pneumoniae strain 9V Spain 3 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 9V Spain 3 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • the S. pneumoniae strain 9V Spain 3 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • S. pneumoniae strain 9 V Spain 3 AI may encode for at least one surface protein.
  • S. pneumoniae strain 9V Spain 3 AI may encode for at least two surface exposed proteins and at least one sortase.
  • S. pneumoniae strain 9V Spain 3 AI encodes for at least three surface exposed proteins and at least two sortases.
  • the AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 9V Spain 3 AI surface protein such as orf3_9VSP, orf4_9VSP, or orf5_9VSP.
  • the oligomeric, pilus-like structure may comprise numerous units of AI surface protein.
  • the oligomeric, pilus-like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,
  • oligomeric subunits wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
  • AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
  • the invention comprises a S. pneumoniae strain 9V Spain 3 AI protein in oligomeric form, preferably in a hyperoligomeric form.
  • the invention comprises a composition comprising one or more S. pneumoniae strain 9 V Spain 3 AI proteins and one or more AI proteins from any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
  • S. pneumoniae strain 9V Spain 3 AI may also include a transcriptional regulator.
  • the S. pneumoniae oligomeric, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an S. pneumoniae AI surface protein.
  • the invention therefore includes a method for manufacturing an oligomeric AI surface antigen comprising culturing a S. pneumoniae bacterium that expresses the oligomeric AI protein and isolating the expressed oligomeric AI protein from the S. pneumoniae bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed AI protein.
  • the AI protein is in a hyperoligomeric form.
  • the oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing an AI surface protein.
  • the invention therefore includes a method for manufacturing an S. pneumoniae oligomeric Adhesin Island surface antigen comprising culturing a S. pneumoniae bacterium adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the S. pneumoniae bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed Adhesin Island protein.
  • the Adhesin Island protein is in a hyperoligomeric form.
  • the S. pneumoniae bacteria are preferably adapted to increase AI protein expression by at least two ⁇ e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels.
  • S. pneumoniae bacteria may be adapted to increase AI protein expression by any means known in the art, including methods of increasing gene dosage and methods of gene upregulation.
  • Such means include, for example, transformation of the S. pneumoniae bacteria with a plasmid enco pdin igz t ' jhe , A/I u prostei ⁇ n. s Thye p ⁇ lasm ' zid ⁇ m3ayB inc,lud ,e a strong promoter or i .t may i .nc,lud,e mu ,lti.p,le copies of the sequence encoding the AI protein.
  • the sequence encoding the AI protein within the S. pneumoniae bacterial genome may be deleted.
  • the promoter regulating the S. pneumoniae Adhesin Island may be modified to increase expression.
  • the invention further includes S.
  • the invention includes S. pneumoniae bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein.
  • the S. pneumoniae of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the invention further includes S. pneumoniae bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface.
  • the S. pneumoniae bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide.
  • Increased levels of a local signal peptidase expression in Gram positive bacteria are expected to result in increased exposure of pili proteins on the surface of Gram positive bacteria.
  • Increased expression of a leader peptidase in S. pneumoniae may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation.
  • the S. pneumoniae bacteria adapted to have increased levels of leader peptidase may additionally be adapted to express increased levels of at least one pili protein.
  • the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes” Infection and Immunity (2004) 72(6):3444-3450).
  • Streptococus gordonii See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors
  • non-pathogenic Gram positive bacteria refer to Gram positive bacteria which are compatible with a human host subject and are not associated with human pathogenisis.
  • the non-pathogenic bacteria are modified to express the AI surface protein in oligomeric, or hyper-oligomeric form. Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non-pathogenic Gram positive bacterial genome or inserted into a plasmid.
  • the non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the AI surface protein may be isolated or purified from a bacterial culture of the non-pathogenic Gram positive bacteria.
  • the AI surface protein may be isolated from cell extracts or culture supernatants.
  • the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria.
  • the non-pathogenic Gram positive bacteria may be used to express any of the S. pneumoniae
  • the non-pathogenic Gram positive bacteria are transformed to e PxpCresTs anz A- UdheSsinO IsElaiIn 1 Zd sEurfyaceE prBoteQin.
  • the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase.
  • the AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with pathogenic S. pneumoniae.
  • Figures 190 A and B, and 193-195 provide examples of three methods successfully practiced by applicants to purify pili from S. pneumoniae TIGR4.
  • the Gram positive bacteria AI proteins described herein are useful in immunogenic compositions for the prevention or treatment of Gram positive bacterial infection.
  • the GBS AI surface proteins described herein are useful in immunogenic compositions for the prevention or treatment of GBS infection.
  • the GAS AI surface proteins described herein may be useful in immunogenic compositions for the prevention or treatment of GAS infection.
  • the S. pneumoniae AI surface proteins may be useful in immunogenic cojmpositions for the prevention or treatment of S. pneumoniae infection.
  • Gram positive bacteria AI surface proteins that can provide protection across more than one serotype or strain isolate may be used to increase immunogenic effectiveness.
  • a particular GBS AI surface protein having an amino acid sequence that is at least 50% ⁇ i.e., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous to the particular GBS AI surface protein of at least 2 (i.e., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) other GBS serotypes or strain isolates may be used to increase the effectiveness of such compositions.
  • fragments of Gram positive bacteria AI surface proteins that can provide protection across more than one serotype or strain isolate may be used to increase immunogenic effectiveness.
  • a fragment may be identified within a consensus sequence of a full length amino acid sequence of a Gram positive bacteria AI surface protein.
  • Such a fragment can be identified in the consensus sequence by its high degree of homology or identity across multiple (Le, at least 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram positive bacteria serotypes or strain isolates.
  • a high degree of ' homology is a degree of homology of at least 90% (Le., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) across Gram positive bacteria serotypes or strain isolates.
  • a high degree of identity is a degree of identity of at least 90% (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) across Gram positive bacteria serotypes or strain isolates.
  • a fragment of a Gram positive bacteria AI surface protein may be used in the immunogenic compositions.
  • AI surface protein oligomeric pilus structures may be formulated or purified for use in immunization. Isolated AI surface protein oligomeric pilus structures may also be used for immunization.
  • the invention includes an immunogenic composition comprising a first Gram positive bacteria AI protein and a second Gram positive bacterial AI protein.
  • j I" ' ,• ⁇ " ji Ii > ⁇ »::: if 1 Ii ii,::;; ,, ⁇ ::::n "7 ;:;:'n r, ⁇ ' iqi may be a surface protein Sudh" surface p rdteins may contain an LPXTG motif or other sortase substrate motif.
  • the first and second AI proteins may be from the same or different genus or species of Gram positive bacteria. If within the same species, the first and second AI proteins may be from the same or different AI subtypes. If two AIs are of the same subtype, the AIs have the same numerical designation. For example, all AIs designated as AI-I are of the same AI subtype. If two AIs are of a different subtype, the AIs have different numerical designations. For example, AI-I is of a different AI subtype from AI-2, AI-3, AI-4, etc. Likewise, AI-2 is of a different AI subtype from AI-I, AI-3, and AI-4, etc.
  • the invention includes an immunogenic composition comprising one or more
  • GBS AI-I proteins and one or more GBS AI-2 proteins.
  • One or more of the AI proteins may be a surface protein.
  • Such surface proteins may contain an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) and may bind fibrinogen, fibronectin, or collagen.
  • One or more of the AI proteins may be a sortase.
  • the GBS AI-I proteins may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648.
  • the GBS AI-I proteins include GBS 80 or GBS 104.
  • the GBS AI-2 proteins may be selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525.
  • the GBS AI-2 proteins are selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406.
  • the GBS AI-2 proteins may be selected from the group consisting of 01520, 01521, 01522, 01523, 01523, 01524 and 01525.
  • the GBS AI-2 protein includes GBS 59 or GBS 67.
  • the invention includes an immunogenic composition comprising one or more of any combination of GAS AI-I, GAS AI-2, GAS AI-3, or GAS AI-4 proteins.
  • GAS AI proteins may be a sortase.
  • the GAS AI-I proteins may be selected from the group consisting of M6_SpyO156, M6_SpyO157, M6_Spy0158, M6_SpyO159, M6_Spy0160, M6_SpyO161, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
  • the GAS AI-I proteins are selected from the group consisting of M6_SpyO157, M6_SpyO159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
  • the GAS AI-2 proteins may be selected from the group consisting of SpyO124, GAS15, SpyO127, GAS16, GAS17, GAS18, SpyOBl, SpyO133, and GAS20.
  • the GAS AI-2 proteins are selected from the group consisting of GAS 15, GAS 16, and GAS 18.
  • the GAS AI-3 proteins may be selected from the group consisting of S ⁇ yM3_0097, SpyM3_OO98, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SPs0099, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf77, orf78, orf79, orfSO, orf ⁇ l, orf82, orf83, orf84, spyM18_0125, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, s ⁇ yM18_0131, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, Sp
  • the GAS AI-3 proteins are selected from the group consisting of SPs0099, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, and SPs0106.
  • the GAS AI-3 proteins are selected from the group consisting of orf77, orf78, orf79, orf ⁇ O, orf ⁇ l, orf82, orf83, and orf84.
  • the GAS AI-3 proteins are selected from the group consisting of spyM18_0125, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132.
  • the GAS AI-3 proteins are selected from the group consisting of SpyoMO 1000156, SpyoMO 1000155, SpyoMO 1000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoMO 1000150, and SpyoM01000149.
  • the GAS AI-4 proteins may be selected from the group consisting of 19224133, 19224134, 19224135, 19224136, 19224137, 19224138, 19224139, 19224140, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
  • the GAS-AI4 proteins are selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
  • the invention includes an immunogenic composition comprising one or more of any combination of S. pneumonaie from TIGR4, S. pneumonaie strain 670, S. pneumonaie from 19A Hungary 6, S. pneumonaie from 6B Finland 12, S. pneumonaie from 6B Spain 2, S. pneumonaie from 9V Spain 3, S. pneumonaie from 14 CSR 10, S. pneumonaie from 19F Taiwan 14, S. pneumonaie from 23F Taiwan 15, or S. pneumonaie from 23F Poland 16 AI proteins.
  • One or more of the AI proteins may be a surface protein.
  • Such surface proteins may contain an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) and may bind fibrinogen, fibronectin, or collagen.
  • One or more of the AI proteins may be a sortase.
  • the S. pneumonaie from TIGR4 AI proteins may be selected from the group consisting of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, SP0468.
  • the S. pneumonaie from TIGR4 AI proteins include SP0462, SP0463, or SP0464.
  • the S. pneumonaie strain 670 AI proteins may be selected from the group consisting of Orfl_670, Orf3_670, Orf4_670, Orf5_670, Orf6_670, Orf7_670, and Orf8_670.
  • the S. pneumonaie strain 670 AI proteins include Orf3_670, Orf4_670, or Orf5_670.
  • the S. pneumonaie from 19A Hungary 6 AI proteins may be selected from the group consisting of ORF2_19AH, ORF3_19AH, ORF4J9AH, ORF5_19AH, ORF6_19AH, ORF7J9AH, or ORF8_19AH.
  • the S. pneumonaie from 6B Finland 12 AI proteins may be selected from the group consisting of ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF , or ORF8_6BF.
  • S3pa9in 2 AI proteins may be selected from the group consisting of ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP , or ORF8_6BSP.
  • the S. pneumonaie from 9V Spain 3 AI proteins may be selected from the group consisting of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP , or ORF8_9VSP.
  • the S. pneumonaie from 14 CSR 10 AI proteins may be selected from the group consisting of ORF2J4CSR, ORF3_14CSR, ORF4J4CSR, ORF5J4CSR, ORF6_14CSR, ORF7_14CSR , or ORF8_14CSR.
  • the S. pneumonaie from 19F Taiwan 14 AI proteins may be selected from the group consisting of ORF2_19FTW, ORF3J9FTW, ORF4_19FTW, ORF5J9FTW, ORF6_19FTW, ORF7_19FTW , or ORF8_19FTW.
  • the S. pneumonaie from 23F Taiwan 15 AI proteins may be selected from the group consisting of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, or ORF8_23FTW.
  • the S. pneumonaie from 23F Poland 16 AI proteins may be selected from the group consisting of ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP , or ORF8_23FP.
  • the Gram positive bacteria AI proteins included in the immunogenic compositions of the invention can provide protection across more than one serotype or strain isolate.
  • the immunogenic composition may comprise a first AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 91, 98, 99 or 100%) homologous to the amino acid sequence of a second AI protein, and wherein said first AI protein and said second AI protein are derived from the genomes of different serotypes of a Gram positive bacteria.
  • the first AI protein may also be homologous to the amino acid sequence of a third AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different serotypes of a Gram positive bacteria.
  • the first AI protein may also be homologous to the amino acid sequence of a fourth AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different serotypes of a Gram positive bacteria.
  • the GBS AI proteins included in the immunogenic compositions of the invention can provide protection across more than one GBS serotype or strain isolate.
  • the immunogenic composition may comprise a first GBS AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e.,, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) homologous to the amino acid sequence of a second GBS AI protein, and wherein said first AI protein and said second AI protein are derived from the genomes of different GBS serotypes.
  • the first GBS AI protein may also be homologous to the amino acid sequence of a third GBS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of P C T/ Ii EIi O 5 / ⁇ 723 «3 different GBS serotypes.
  • the first AI protein may also be homologous to the amino acid sequence of a fourth GBS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different GBS serotypes.
  • the first AI protein may be selected from an AI-I protein or an AI-2 protein.
  • the first AI protein may be a GBS AI-I surface protein such as GBS 80.
  • the amino acid sequence of GBS 80 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 80 amino acid sequence from GBS serotype III, strain isolates NEM316 and COHl and the GBS 80 amino acid sequence from GBS serotype Ia, strain isolate A909.
  • the first AI protein may be GBS 104.
  • the amino acid sequence of GBS 104 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 104 amino acid sequence from GBS serotype III, strain isolates NEM316 and COHl, the GBS 104 amino acid sequence from GBS serotype Ia, strain isolate A909, and the GBS 104 amino acid sequence serotype II, strain isolate 18RS21.
  • Table 12 provides the amino acid sequence identity of GBS 80 and GBS 104 across GBS serotypes Ia, Ib, II, III, V, and VIII.
  • the GBS strains in which genes encoding GBS 80 and GBS 104 were identified share, on average, 99.88 and 99.96 amino acid sequence identity, respectively. This high degree of amino acid identity indicates that an immunogenic composition comprising a first protein of GBS 80 or GBS 104 may provide protection across more than one GBS serotype or strain isolate.
  • the first AI protein may be an AI-2 protein such as GBS 67.
  • the amino acid sequence of GBS 61 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 67 amino acid sequence from GBS serotype III, strain isolate NEM316, the GBS 67 amino acid sequence from GBS serotype Ib, strain isolate H36B, and the GBS 67 amino acid sequence from GBS serotype II, strain isolate 17RS21.
  • the first AI protein may be an AI-2 protein such as spbl .
  • the amino acid sequence of spbl from GBS serotype III, strain isolate COHl is greater than 90% homologous to the spbl amino acid sequence from GBS serotype Ia, strain isolate A909.
  • the first AI protein may be an AI-2 protein such as GBS 59.
  • the amino acid sequence of GBS 59 from GBS serotype II, strain isolate 18RS21 is 100% homologous to the GBS 59 amino acid sequence from GBS serotype V, strain isolate 2603.
  • compositions of the invention may also be designed to include Gram positive AI proteins from divergent serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of serotypes or strain isolates of a Gram positive bacteria and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.
  • the invention may include an immunogenic composition comprising a first and second Gram positive bacteria AI protein, wherein a polynucleotide sequence encoding for the full length sequence of the first AI protein is not present in a similar Gram positive bacterial genome comprising a polynucleotide sequence encoding for the second AI protein.
  • compositions of the invention may also be designed to include AI proteins from divergent GBS serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of GBS serotypes or strain isolates and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.
  • the invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein a polynucleotide sequence encoding for the full length sequence of the first GBS Al protein is not present in a genome comprising a polynucleotide sequence encoding for the second GBS AI protein.
  • the first AI protein could be GBS 80 (such as the GBS 80 sequence from GBS serotype V, strain isolate 2603).
  • the sequence for GBS 80 in GBS sertoype II, strain isolate 18RS21 is disrupted.
  • the second AI protein could be GBS 104 or GBS 67 (sequences selected from the GBS serotype II, strain isolate 18RS21).
  • PC IV ⁇ J S O 5 / E 7 E .3 1 O
  • the invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein the first GBS AI protein has detectable surface exposure on a first GBS strain or serotype but not a second GBS strain or serotype and the second GBS AI protein has detectable surface exposure on a second GBS strain or serotype but not a first GBS strain or serotype.
  • the first AI protein could be GBS 80 and the second AI protein could be GBS 67.
  • Table 15 there are some GBS serotypes and strains that have surface exposed GBS 80 but that do not have surface exposed GBS 67 and vice versa.
  • An immunogenic composition comprising a GBS 80 and a GBS 67 protein may provide protection across a wider group of GBS strains and serotypes.
  • the invention may include an immunogenic composition comprising a first and second Gram positive bacteria AI protein, wherein the polynucleotide sequence encoding the sequence of the first AI protein is less than 90 % (i.e., less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second AI protein.
  • the invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein the polynucleotide sequence encoding the sequence of the first GBS AI protein is less than 90 % (i.e., less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second GBS AI protein.
  • the first GBS AI protein could be GBS 67 (such as the GBS 67 sequence from GBS serotype Ib, strain isolate H36B).
  • the GBS 67 sequence for this strain is less than 90% homologous (87%) to the corresponding GBS 67 sequence in GBS serotype V, strain isolate 2603.
  • the second GBS AI protein could then be the GBS 80 sequence from GBS serotype V, strain isolate 2603.
  • An example immunogenic composition of the invention may comprise adhesin island proteins GBS 80, GBS 104, GBS 67, and GBS 59, and non-AI protein GBS 322.
  • FACS analysis of different GBS strains demonstrates that at least one of these five proteins is always found to be expressed on the surface of GBS bacteria.
  • Figure 227 provides the FACS data obtained for surface exposure of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on each of 37 GBS strains.
  • Figure 228 provides the FACS data obtained for surface exposure of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on each of 41 GBS strains obtained from the CDC.
  • each GBS strain had surface expression of at least one of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59.
  • the surface exposure of at least one of these proteins on each bacterial strain indicates that an immunogenic composition coinprising these proteins will provide wide protection across GBS strains and serotypes.
  • the surface exposed GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 proteins are also present at high levels as determined by FACS.
  • Table 49 summarizes the FACS results for the initial 70 GBS strains examined for GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 surface expression.
  • a protein was designated as having high levels of surface expression of a protein if a five-fold shift in fluorescence was observed when using antibodies for the protein relative to preimmune control serum.
  • Table 49 Exposure Levels of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on GBS Strains
  • Table 50 details which of the surface proteins is highly expressed on the different GBS serotype. Table 50: High Levels of Surface Protein Expression on GBS Serotypes
  • the immunogenic composition of the invention may include GBS 80, GBS 104,
  • an immunogenic composition containing GBS 80, GBS 104, GBS 67, GBS 59 and GBS 322 will provide protection for 89% of GBS strains and serotypes, the same percentage as an immunogenic composition containing GBS 80, GBS 104, GBS 67, and GBS 322 proteins. See Figure 229. However, it may be preferable to include GBS 59 in the composition to increase its immunogenic strength.
  • GBS 59 is highly expressed on the surface two-thirds of GBS bacteria examined by FACS analysis, unlike GBS 80, GBS 104, and GBS 322, which are highly expressed in less than half of GBS bacteria examined.
  • GBS 59 opsonophagocytic activity is also comparable to that of a mix of GBS 322, GBS 104, GBS 67, and GBS 80 proteins. See Figure 230.
  • the GAS AI proteins included in the immunogenic compositions of the invention can provide protection across more than one GAS serotype or strain isolate.
  • the immunogenic composition may comprise a first GAS AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) homologous to the amino acid sequence of a second GAS AI protein, and wherein said first AI protein and said second AI protein are derived from the genomes of different GAS serotypes.
  • the first GAS AI protein may also be homologous to the amino acid sequence of a third GAS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different GAS serotypes.
  • the first AI protein may also be homologous to the amino acid sequence of a fourth GAS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different GAS serotypes.
  • compositions of the invention may also be designed to include GAS AI proteins from divergent serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of serotypes or strain isolates of a GAS bacteria and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.
  • the first AI protein could be a ⁇ rtF2 protein (such as the 19224141 protein from GAS serotype M12, strain isolate A735).
  • the sequence for a prtF2 protein is not present in GAS AI types 1 or 2.
  • the second AI protein could be collagen binding protein M6_SpyO159 (from M6 isolate (MGAS10394), which comprises an AI-I) or GAS 15 (from Ml isolate (SF370), which comprises an AI-2).
  • the invention may include an immunogenic composition comprising a first and second GAS AI protein, wherein the first GAS AI protein has detectable surface exposure on a first GAS strain or serotype but not a second GAS strain or serotype and the second GAS AI protein has detectable surface exposure on a second GAS strain or serotype but not a first GAS strain or serotype.
  • the invention may include an immunogenic composition comprising a first and second GAS AI protein, wherein the polynucleotide sequence encoding the sequence of the first GAS AI protein is less than 90 % (i.e., less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second GAS AI protein.
  • the first and second GAS AI proteins are subunits of the pilus.
  • the first and second GAS AI proteins are selected from the major pilus forming proteins (i.e., M6_S ⁇ y0160 from M6 strain 10394, SPyO128 from Ml strain SF370, SpyM3_0100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM18_0128 from M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from M12 strain A735, fimbrial structural subunit from M77 strain ISS4959, fimbrial structural subunit from M44 strain ISS3776, fimbrial structural subunit from M50 strain ISS3776 ISS 4538, fimbrial structural subunit from M12strain CDC SS635, fimbrial structural subunit from M23 strain DSM2071, fimbrial structural subunit from M6 strain CDC SS410).
  • M6_S ⁇ y0160 from M6 strain 10394 SPyO128 from M
  • Table 45 provides the percent identity between the amino acidic sequences of each of the main pilus forming subunits from GAS AI-I, AI-2, AI-3, and AI-4 representative strains (i.e., M6_Spy0160 from M6 strain 10394, SPyO128 from Ml strain SF370, S ⁇ yM3_0100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM18_0128 from M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from M12 strain A735, Fimbrial structural subunit from M77 strain ISS4959, fimbrial structural subunit from M44 strain ISS3776, fimbrial structural subunit from M50 strain ISS3776 ISS 4538, fimbrial structural subunit from M12strain CDC SS635, fimbrial structural subunit from M23 strain DSM2071, fimbrial structural subunit from M6 strain CDC
  • the first main pilus subunit may be selected from bacteria of GAS serotype M6 strain 10394 and the second main pilus subunit may be selected from bacteria of GAS serotype Ml strain 370.
  • the main pilus subunits encoded by these strains of bacteria share only 23% nucleotide identity.
  • An immunogenic composition comprising pilus main subunits from each of these strains of bacteria is expected to provide protection across a wider group of GAS strains and serotypes.
  • main pilus subunits that can be used in combination to provide increased protection across a wider range of GAS strains and serotypes include proteins encoded by GAS serotype M5 Manfredo isolate and serotype M6 strain 10394, which share 23% sequence identity, GAS serotype M18 strain 8232 and serotype Ml strain 370, which share 38% sequence identity, GAS serotype M3 strain 315 and serotype M12 strain A735, which share 61% sequence identity, and GAS serotype M3 strain 315 and serotype M6 strain 10394 which share 25% sequence identity.
  • Figures 198-201 provide further tables comparing the percent identity of adhesin island-encoded surface exposed proteins for different GAS serotypes relative to other GAS serotypes harbouring an adhesin island of the same or a different subtype (GAS AI-I, GAS AI-2, GAS AI-3, and GAS AI-4). See also further discussion below.
  • Applicants have discovered that surface exposure of GBS 104 is dependent on the concurrent expression of GBS 80.
  • reverse transcriptase PCR analysis of AI-I shows that all of the AI genes are co-transcribed as an operon.
  • Applicants constructed a series of mutant GBS containing in frame deletions of various AI-I genes. (A schematic of the GBS mutants is presented in Figure 7). FACS analysis of the various mutants comparing mean shift values using anti-GBS 80 versus anti-GBS 104 antibodies is presented in Figure 8. Removal of the GBS 80 operon prevented surface exposure of GBS 104; removal of the 80. While not being limited to a specific theory, it is thought that GBS 80 is involved in the transport or localization of GBS 104 to the surface of the bacteria. The two proteins may be oligomerized or otherwise associated. It is possible that this association involves a conformational change in GBS 104 that facilitates its transition to the surface of the GBS bacteria.
  • PiIi structures that comprise GBS 104 appear to be of a lower molecular weight than pili structures lacking GBS 104.
  • Figure 68 shows that polyclonal anti-GBS 104 antibodies (see lane marked ⁇ -104 POLIC.) cross-hybridize with smaller structures than do polyclonal anti-GBS 80 antibodies (see lane marked ⁇ -GBS 80 POLIC).
  • Applicants have shown that removal of GBS 80 can cause attenuation, further suggesting the protein contributes to virulence.
  • the LD 50 's for the ⁇ 80 mutant and the ⁇ 80, ⁇ 104 double mutant were reduced by an order of magnitude compared to wildtype and ⁇ 104 mutant.
  • sortases within the adhesin island also appear to play a role in localization and presentation of the surface proteins.
  • FACS analysis of various sortase deletion mutants showed that removal of sortase SAG0648 prevented GBS 104 from reaching the surface and slightly reduced the surface exposure of GBS 80.
  • sortase SAG0647 and sortase SAG0648 were both knocked out, neither GBS 80 nor GBS 104 were surface exposed. Expression of either sortase alone was sufficient for GBS 80 to arrive at the bacterial surface. Expression of SAG0648, however, was required for GBS 104 surface localization.
  • compositions of the invention may include two or more AI proteins, wherein the AI proteins are physically or chemically associated.
  • the two AI proteins may form an oligomer.
  • the associated proteins are two AI surface proteins, such as GBS 80 and GBS 104.
  • the associated proteins may be AI surface proteins from different adhesin islands, including host cell adhesin island proteins if the AI surface proteins are expressed in a recombinant system.
  • the associated proteins may be GBS 80 and GBS 67. Adhesin Island proteins from other Gram positive bacteria
  • Adhesin Island or “AI” refers to a series of open reading frames within a bacterial genome that encode for a collection of surface proteins and sortases.
  • An Adhesin Island may encode for amino acid sequences comprising at least one surface protein.
  • the Adhesin Island may encode at least one surface protein.
  • an Adhesin Island may encode for at least two surface proteins and at least one sortase.
  • an Adhesin Island encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • surface protein ' s may participate in me formation of a pilus structure on the surface of the Gram positive bacteria.
  • Gram positive adhesin islands of the invention preferably include a divergently transcribed transcriptional regulator.
  • the transcriptional regulator may regulate the expression of the AI operon.
  • the invention includes a composition comprising one or more Gram positive bacteria AI surface proteins. Such AI surface proteins may be associated in an oligomeric or hyperoligomeric structure.
  • Preferred Gram positive adhesin island proteins for use in the invention may be derived from Staphylococcus (such as S. aureus), Streptococcus (such as S. agalactiae (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutans), Enterococcus (such as E.faecalis and E.faecium), Clostridium (such as C. difficile), Listeria (such as L. monocytogenes) and Corynebacterium (such as C. diphtheria).
  • Staphylococcus such as S. aureus
  • Streptococcus such as S. agalactiae (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutans
  • Enterococcus such as E.faecalis and E.faecium
  • Clostridium such as C. difficile
  • Listeria such as L.
  • Gram positive AI surface protein sequences typically include an LPXTG motif or other sortase substrate motif.
  • Gram positive AI surface proteins of the invention may affect the ability of the Gram positive bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of Gram positive bacteria to translocate through an epithelial cell layer.
  • one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
  • Gram positive AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • Gram positive AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins.
  • a Gram positive bacteria AI may encode for at least one surface exposed protein.
  • the Adhesin Island may encode at least one surface protein.
  • a Gram positive bacteria AI may encode for at least two surface exposed proteins and at least one sortase.
  • a Gram positive AI encodes for at least three surface exposed proteins and at least two sortases.
  • Gram positive AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase.
  • the sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif.
  • the sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II.
  • the precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
  • Gram positive bacteria AI surface proteins of the invention will contain an N-terminal leader or secretion signal to facilitate translocation of the surface protein across the bacterial membrane.
  • Gram positive bacteria AI surface proteins of the invention may affect the ability of the Gram positive bacteria to adhere to and invade target host cells, such as epithelial cells.
  • Gram positive bacteria AI surface proteins may also affect the ability of the gram positive bacteria to translocate through an epithelial cell layer.
  • one or more of the Gram positive AI surface proteins are P C T/ U . ⁇ O 5 / 1 E! 7 E! B 1 Qi capable of binding to or other associating with an epithelial cell surface.
  • one or more Gram positive AI surface proteins may bind to fibrinogen, fibronectin, or collagen protein.
  • the invention includes a composition comprising oligomeric, pilus-like structures comprising a Gram positive bacteria AI surface protein.
  • the oligomeric, pilus-like structure may comprise numerous units of the AI surface protein.
  • the oligomeric, pilus- like structures comprise two or more AI surface proteins.
  • the oligomeric, pilus- like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof.
  • the oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif.
  • the oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
  • Gram positive bacteria AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include one or both of a pilin motif comprising a conserved lysine residue and an E box motif comprising a conserved glutamic acid residue.
  • the oligomeric, pilus like structures may be used alone or in the combinations of the invention.
  • the invention comprises a Gram positive bacteria Adhesin Island in oligomeric form, preferably in a hyperoligomeric form.
  • the oligomeric, pilus-like structures of the invention may be combined with one or more additional Gram positive AI proteins (from the same or a different Gram positive species or genus).
  • the oligomeric, pilus-like structures comprise one or more Gram positive bacteria AI surface proteins in combination with a second Gram positive bacteria protein.
  • the second Gram positive bacteria protein may be a known antigen, and need not normally be associated with an AI protein.
  • the oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing a Gram positive bacteria AI surface protein.
  • the invention therefore includes a method for manufacturing an oligomeric Adhesin Island surface antigen comprising culturing a Gram positive bacteria adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the Gram positive bacteria.
  • the AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface.
  • the method may further comprise purification of the expressed Adhesin Island protein.
  • the Adhesin Island protein is in a hyperoligomeric form.
  • Gram positive bacteria are preferably adapted to increase AI protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels.
  • Gram positive bacteria may be adapted to increase AI protein expression by means known in the art, including methods of increasing gene dosage and methods of gene upregulation. Such means .
  • PC T/ Il J S O Eii ./ ⁇ 7' i ⁇ ! 39 include, for example, transformation of the Gram positive bacteria with a plasmid encoding the AI protein.
  • the plasmid may include a strong promoter or it may include multiple copies of the sequence encoding the AI protein.
  • the sequence encoding the AI protein within the Gram positive bacterial genome may be deleted.
  • the promoter regulating the Gram positive Adhesin Island may be modified to increase expression.
  • the invention further includes Gram positive bacteria which have been adapted to produce increased levels of AI surface protein.
  • the invention includes Gram positive bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein.
  • the Gram positive bacteria of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
  • the invention further includes Gram positive bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface.
  • the Gram positive bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide.
  • Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pili proteins on the surface of Gram positive bacteria.
  • Increased expression of a leader peptidase in Gram positive may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation.
  • the Gram positive bacteria adapted to have increased levels of leader peptidase may additionally be adapted to express increased levels of at least one pili protein.
  • the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal VaccineMade from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes” Infection and Immunity (2004) 72(6):3444-3450). It has already been demonstrated, above, that L. lactis expresses GBS and GAS AI polypeptides in oligomeric form and on its surface.
  • the oligomeric, pilus-like structures may be produced recombinantly.
  • the Gram positive bacteria AI surface protein will preferably be expressed in coordination with the expression of one or more of the AI sortases of the invention.
  • AI sortases will facilitate oligomeric or hyperoligomeric formation of the AI surface protein subunits.
  • Gram positive AI Sortases of the invention will typically have a signal peptide sequence within the first 70 amino acid residues. They may also include a transmembrane sequence within 50 amino acid residues of the C terminus. The sortases may also include at least one basic amino acid residue within the last 8 amino acids. Preferably, the sortases have one or more active site residues, such as a catalytic cysteine and histidine. Ihesinlslai ⁇ d'siirfa'e'e' protein's from two or more Gram positive bacterial genus or species may be combined to provide an immunogenic composition for prophylactic or therapeutic treatment of disease or infection of two more Gram positive bacterial genus or species.
  • the adhesin island surface proteins may be associated together in an oligomeric or hyperoligomeric structure.
  • the invention comprises an adhesin island surface proteins from two or more Streptococcus species.
  • the invention includes a composition comprising a GBS AI surface protein and a GAS adhesin island surface protein.
  • the invention includes a composition comprising a GAS adhesin island surface protein and a S. pneumoniae adhesin island surface protein.
  • the invention comprises an adhesin island surface protein from two or more Gram positive bacterial genus.
  • the invention includes a composition comprising a Streptococcus adhesin island protein and a Corymb acterium adhesin island protein.
  • adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus.
  • Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fasciitis.
  • post-streptococcal autoimmune responses are still a major cause of cardiac pathology in children.
  • Group A Streptococcal infection of its human host can generally occur in three phases.
  • the first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces. Generally this attachment phase begins in the throat or the skin. The deeper the tissue level infected, the more severe the damage that can be caused.
  • the bacteria secrete a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. This toxin binds to susceptible host cell receptors and triggers innappropropriate immune responses by these host cells, resulting in pathology. Because the toxin can diffuse throughout the host, the necrosis directly caused by the GAS toxins may be physically located in sites distant from the bacterial infection.
  • the final phase of GAS infection can occur long after the original bacteria have been cleared from the host system. At this stage, the host's previous immune response to the GAS bacteria due to cross reactivity between epitopes of a GAS surface protein, M, and host tissues, such as the heart.
  • a general review of GAS infection can be found in Principles of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
  • an effective vaccine against GAS will preferably facilitate host elimination of the bacteria during the initial attachment and invasion stage.
  • M protein is surface exposed trypsin-sensitive protein generally comprising two polypeptide chains complexed in an alpha helical formation.
  • the carboxyl terminus is anchored in the cytoplasmic membrane and is highly conserved among all group A streptococci.
  • the amino terminus which extends through the cell wall to the cell surface, is responsible for the antigenic variability observed among the 80 or more serotypes of M proteins.
  • T-antigen A second layer of classification is based on a variable, trypsin-resistant surface antigen, commonly referred to as the T-antigen.
  • T-antigen a variable, trypsin-resistant surface antigen
  • Decades of epidemiology based on M and T serological typing have been central to studies on the biological diversity and disease causing potential of Group A Streptococci. While the M-protein component and its inherent variability have been extensively characterized, even after five decades of study, there is still very little known about the structure and variability of T-antigens.
  • Antisera to define T types are commercially available from several sources, including Sevapharma (http://www.sevapharma.cz/en).
  • T-antigen T-type 6
  • M6 strain of GAS M6 strain of GAS
  • FCT Fibronectin-binding, Collagen-binding T-antigen
  • the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the tee ⁇ gene product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used. See Figure 163 A, last lane labeled "M6_Tee6.”
  • the FCT region in M6JSS3650 contains two other genes (prtFl and cpa) predicted to code for surface exposed proteins; these proteins are characterized as containing the cell wall attachment motif LPXTG.
  • Western blot analysis using antiserum specific for PrtFl detected a single molecular species with electrophoretic mobility corresponding to the predicted molecular mass of the protein and one smaller band of unknown origin.
  • Western blot analysis using antisera specific for Cpa recognized a high molecular weight covalently linked ladder (Fig 163 A, second lane).
  • Immunogold labelling of Cpa with specific antiserum followed by transmission electron microscopy detected an abundance of Cpa at the cell surface and only occasional structures extending from the cell surface (Fig. 163J).
  • FCT region Four classes of FCT region can be discerned by the types and order of the genes contained within the region.
  • the FCT region of strains of types M3, M5, M 18 and M49 have a similar organization whereas those of M6, Ml and M12 differ. See Figure 164.
  • these four FCT regions correlate to four GAS Adhesin Island types (AI-I, AI-2, AI-3 and AI-4).
  • Ml strain SF370 there are three predicted surface proteins (Cpa (also referred to as Ml_126 and GAS 15), Ml_128 (a f ⁇ mbrial protein also referred to as SpyO128 and GAS 16), and Ml_130 (also referred to as Spy0130 and GAS 18)) (GAS AI-2).
  • Cpa also referred to as Ml_126 and GAS 15
  • Ml_128 a f ⁇ mbrial protein also referred to as SpyO128 and GAS 16
  • Ml_130 also referred to as Spy0130 and GAS 18
  • Antisera specific for each surface protein reacted with a ladder of high molecular weight material (Fig. 163B).
  • Immunogold staining of Ml strain SF370 with antiserum specific for Ml_128 revealed pili structures similar to those seen when M6 strain ISS3650 was immunogold stained with antiserum specific for tee6 (See Fig 1163K).
  • the Ml_128 protein appears to be necessary for polymerization of Cpa and Ml_130 proteins. If the Ml_128 gene in Ml_SF370 was deleted, Western blot analysis using antibodies that hybridize to Cpa and Ml_130 no longer detected high molecular weight ladders comprising the Cpa and Ml_130 proteins (Fig. 163 E).
  • Figures 177 A-C which provide the results of Western blot analysis of the Ml_128 ( ⁇ 128) deleted bacteria using anti-Ml_130 antiserum ( Figure 177 A), anti-Ml_128 antiserum ( Figure 177 B), and anti-Ml_126 antiserum ( Figure 177 C ⁇ " nigh 'molecular weighH'adders;i ⁇ d ⁇ cative of pilus formation on the surface of Ml strain SF370, could not be detected by any of the three antisera in ⁇ 128 bacteria. If the ⁇ l28 bacteria were transformed with a plasmid containing the gene for Ml_128, Western blot analysis using antisera specific for Cpa and Ml_130 again detected high molecular weight ladders ( Figure 163 H).
  • FIG. 177 A-C provide Western blot analysis results of the Ml_130 deleted ( ⁇ 130) strain SF370 bacteria using anti-Ml_130 ( Figure 177 A), anti-Ml_128 ( Figure 177 B), and anti-Ml_126 antiserum ( Figure 177 C).
  • the anti-Ml_128 and anti-Ml_126 antiserum both detected the presence of high molecular weight ladders in the ⁇ 130 strain SF370 bacteria, indicating that the ⁇ 130 bacteria form pili that comprise Ml_126 and Ml_128 polypeptides in the absence of Ml_130.
  • the Western blot probed with antiserum immunoreactive with Ml_130 did not detect any proteins for the ⁇ 130 bacteria( Figure 177A).
  • composition of the pili in GAS resembles that previously described for both C. diphtheria (7, 8) and S. agalactiae (described above) (9) in that each pilus is formed by a backbone component which abundantly stains the pili in EM and is essential for the incorporation of the other components.
  • LepA signal peptidase SpyO127
  • LepA deletion mutants ( ⁇ LepA) of strain SF370 fail to assemble pili on the cell surface. Not only are the ⁇ LepA mutants unable to assemble pili, they are also deficient at cell surface Ml expression. See Figure 180, which provides a FACS analysis of the wildtype (A) and ⁇ LepA mutant (B) SF370 bacteria using Ml antisera. No shift in fluorescence is observed for the ⁇ LepA mutant bacteria in the presence of Ml immune serum.
  • deletion mutants of LepA will be useful for detecting non-M, non-pili, surface exposed antigens on the surface of GAS, or any Gram positive bacteria. These antigens may also be useful in immunogenic compositions. Pili were also observed in M5 strain ISS4882 and M12 strain 20010296. The M5 strain
  • ISS4882 contains genes for four predicted surface exposed proteins (GAS ⁇ I-3). Antisera against three of the four products of the FCT region (GAS AI-3) of M5JSS4883 (Cpa, M5_orf80, M5_orf82) stained high molecular weight ladders in Western blot analysis ( Figure 163 C). Long pili were visible when antisera against M5_orf80 was used in immunogold staining followed by electron microscopy ( Figure 163L).
  • the M12 strain 20010296 contains genes for five predicted surface exposed proteins.
  • GAS AI-4 Antisera against three of the five products of the FCT region (GAS AI-4) of Ml 2_20010296 (Cpa, EftLSL.A, Orf2) stained high molecular weight ladders in Westen blot analysis ( Figure 163 D). Long pili were visible when antisera against EftLSL.A were used (Fig. 163 M).
  • the major pilus forming proteins identified in the four strains studied by applicants share between 23% and 65% amino acid identity in any pairwise comparison, indicating that each pilus may represent a different Lancef ⁇ eld T-antigen.
  • Each pilus is part of a trypsin resistant structure on the GAS bacteria surface, as is the case for the Lancefield T antigens. See Figure 165, which provides a FACS analysis of bacteria harboring each of the FCT types that had or had not been treated with trypsin ( ⁇ 5).
  • Applicants have identified at least four different Group A Streptococcus Adhesin Islands. While these GAS AI sequences can be identified in numerous M types, Applicants have surprisingly discovered a correlation between the four main pilus subunits from the four different GAS AI types and specific T classifications. While other trypsin-resistant surface exposed proteins are likely also implicated in the T classification designations, the discovery of the role of the GAS adhesin islands (and the associated hyper-oligomeric pilus like structures) in T classification and GAS serotype variance has important implications for prevention and treatment of GAS infections. Applicants have identified protein components within each of the GAS adhesin islands which are associated with the pilus formation. These proteins are believed to be involved in the bacteria's initial adherence mechanisms.
  • GAS pili may be involved in formation of biofilms.
  • the GBS pili structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysaccharide matrix).
  • Biofilms are generally associated with bacterial resistance, as antibiotic treatments and host immune response are frequently un ' ail'e to 6i ⁇ a ⁇ ic;'aie"air" ⁇ f the'bactefia components of the biofilm.
  • Direction of a host immune response against surface proteins exposed during the first steps of bacterial attachment i.e., before complete biofilm formation) is preferable.
  • the invention therefore provides for improved immunogenic compositions against GAS infection which may target GAS bacteria during their initial attachment efforts to the host epithelial cells and may provide protection against a wide range of GAS serotypes.
  • the immunogenic compositions of the invention include GAS AI surface proteins which may be formulated in an oligomeric, or hyperoligomeric (pilus) form.
  • the invention also includes combinations of GAS AI surface proteins. Combinations of GAS AI surface proteins may be selected from the same adhesin island or they may be selected from different GAS adhesin islands.
  • the invention comprises compositions comprising a first GAS AI protein and a second GAS AI protein wherein the first and second GAS Al proteins are derived from different GAS adhesin islands.
  • the invention includes a composition comprising at least two GAS AI proteins wherein the GAS AI proteins are encoded by the adhesin islands selected from the group consisting of GAS AI-I and AI-2; GAS AI-I and GAS AI-3; GAS AI-I and GAS AI-4; GAS AI-2 and GAS AI-3; GAS AI-2 and GAS AI-4; and GAS AI-3 and GAS AI-4.
  • the two GAS AI proteins are derived from different T-types.
  • FIGURE 162 A schematic arrangement of GAS Adhesin Island sequences is set forth in FIGURE 162.
  • the AI region is flanked by the highly conserved open reading frames Ml_123 and Ml- 136. Between three and five genes in each locus code for surface proteins containing LPXTG motifs. These surface proteins also all belong to the family of genes coding for ECM binding adhesins.
  • GAS Adhesin island sequences can be identified in numerous M types of Group A Streptococcus. Examples of AI sequences within Ml, M6, M3, M5, M12, M18, and M49 serotypes are discussed below.
  • GAS Adhesin Islands generally include a series of open reading frames within a GAS genome that encode for a collection of surface proteins and sortases.
  • a GAS Adhesin Island may encode for amino acid sequences comprising at least one surface protein.
  • a GAS Adhesin Island may encode for at least two surface proteins and at least one sortase.
  • a GAS Adhesin Island encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • One or more GAS AI surface proteins may participate in the formation of a pilus structure on the surface of the Gram positive bacteria.
  • GAS Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator.
  • the transcriptional regulator may regulate the expression of the GAS AI operon. Examples of transcriptional regulators found in GAS AI sequences include RofA and Nra.
  • the GAS AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen.
  • One or more of the GAS AI surface proteins may comprise a fimbrial structural subunit.
  • " " Orie of 1 more of me GAS * Al ' surface proteins may include an LPXTG motif or other sortase substrate motif.
  • the LPXTG motif may be followed by a hydrophobic region and a charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. See Barnett, et al., J. Bacteriology (2004) 186 (17): 5865-5875.
  • GAS AI sequences may be generally categorized as Type 1, Type 2, Type 3, or Type 4, depending on the number and type of sortase sequences within the island and the percentage identity of other proteins (with the exception of RofA and cpa) within the island.
  • Figure 167 provides a chart indicating the number and type of sortase sequences identified within the adhesin islands of various strains and serotypes of GAS.
  • GAS Adhesin Island within M6 serotype (MGAS 10394) is outlined in Table 4 below.
  • This GAS adhesin island 1 (“GAS AI-I") comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator.
  • GAS AI-I surface proteins include S ⁇ yO157 (a fibronectin binding protein), SpyO159 (a collagen adhesion protein) and Spy0160 (a fimbrial structural subunit).
  • each of these GAS AM surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with serine).
  • GAS AI-I includes a srtB type sortase.
  • GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
  • M6_S ⁇ y0160 appears to be present on the surface of GAS as part of oligomeric (pilus) structures.
  • Figures 127-132 present electron micrographs of GAS serotype M6 strain 3650 immunogold stained for M6_SpyO16O using anti-M6_S ⁇ y0160 antiserum.
  • Oligomeric or hyperoligme ⁇ c structures labelled witn gold particles can be seen extending from the surtace of the GAS in each of these figures, indicating the presence of multiple M6_Spy0160 polypeptides in the oligomeric or hyperoligomeric structures.
  • Figure 176 A-F present electron micrographs of GAS M6 strain 2724 immunogold stained for M6_Spy0160 using anti-M6_Spy0160 antiserum ( Figures 176 A- E) or immunogold stained for M6_Spy0159 using anti-M6_SpyO159 antiserum ( Figure 176 F). Oligomeric or hyperoligomeric structures labelled with gold particles can again be seen extending from the surface of the M6 strain 2724 GAS bacteria immunogold stained for M6_Spy0160. M6_SpyO159 is also detected on the surface of the M6 strain 2724 GAS.
  • Figure 73 provides the results of FACS analysis for surface expression of spyM6_0159 on each of GAS serotypes M6 2724, M6 3650, and M62894. A shift in fluorescence is observed for each GAS serotype when anti-spyM6_0159 antiserum is present, demonstrating cell surface expression.
  • Table 18 quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-spyM6_0159 antiserum, and the difference in fluorescence value between the pre-immune and anti-spyM6_0159 antiserum.
  • Figure 74 provides the results of FACS analysis for surface expression of spyM6_0160 on each of GAS serotypes M6 2724, M6 3650, and M6 2894.
  • anti-spyM6_0160 antiserum In the presence of of anti-spyM6_0160 antiserum, a shift in fluorescence is observed for each GAS serotype, which demonstrates its cell surface expression.
  • Table 19, below quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-spyM6_0160 antiserum, and the change in fluorescence value between the pre-immune and anti-s ⁇ yM6_0160 antiserum.
  • FIG. 98 shows that while pre-immune sera (P ⁇ -0159) does not detect expression of M6_S ⁇ yO159 in GAS serotype M6, anti-M6_SpyO159 immune sera (I ⁇ - 0159) is able to detect M6_S ⁇ yO159 protein in both total GAS M6 extracts (M6 tot) and GAS M6 fractions enriched for cell surface proteins (M6 surf prot).
  • M6_SpyO159 proteins detected in the t ⁇ tal"GAS"M6 ' extracts " bf the " G ⁇ ' Mo extracts enriched for surface proteins are also present as high molecular weight structures, indicating that M6_SpyO159 may be in an oligomeric (pilus) form.
  • Figure 112 shows that while preimmune sera (Preimmune Anti 106) does not detect expression of M6_Spy0160 in GAS serotype M6 strain 2724, anti-M6_Spy0160 immune sera (Anti 160) does in both total GAS M6 strain 2724 extracts (M6 2724 tot) and GAS M6 strain 2724 fractions enriched for surface proteins.
  • the M6_Spy0160 proteins detected in the total GAS M6 strain 2724 extracts or the GAS M6 strain 2724 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that M6_SpyO16O may be in an oligomeric (pilus) form.
  • Figures 110 and 111 both further verify the presence of M6_S ⁇ yO 159 and M6_Spy0160 in higher molecular weight structures on the surface of GAS.
  • Figure 110 provides a Western blot performed to detect M6_SpyO159 and M6_Spy0160 in GAS M6 strain 2724 extracts enriched for surface proteins. Antiserum raised against either M6_Spy0159 (Anti- 159) or M6_S ⁇ yO16O (Anti- 160) cross-hybridizes with high molecular weight structures (pili) in these extracts.
  • Figure 111 provides a similar Western blot that verifies the presence of M6_ S ⁇ yO159 and M6_Spy0160 in high molecular weight structures in GAS M6 strain 3650 extracts enriched for surface proteins.
  • SpyM6_0157 (a fibronectin-binding protein) may also be expressed on the surface of GAS serotype M6 bacteria.
  • Figure 174 shows the results of FACS analysis for surface expression of spyM6_0157 on M6 strain 3650. A slight shift in fluorescence is observed, which demonstrates that some spyM6_0157 may be expressed on the GAS cell surface.
  • Adhesin Island sequence within M6 GAS Adhesin Island 2 ("GAS AI-2")
  • GAS Adhesin Island within Ml serotype is outlined in Table 5 below.
  • This GAS adhesin island 2 (“GAS AI-2") comprises surface proteins, a SrtB sortase, a SrtCl sortase and a RofA divergently transcribed transcriptional regulator.
  • GAS AI-2 surface proteins include GAS 15 (Cpa), SpyO128 (thought to be a fimbrial protein) and Spy0130 (a hypothetical protein).
  • each of these GAS AI-2 surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), WXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
  • GAS AI-2 includes a srtB type sortase and a srtCl sortase.
  • GAS SrtB sortases may preferably anchor surface proteins with an LPSTG (SEQ ID NO: 166) motif, particularly where the motif is followed by a serine.
  • GAS SrtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO: 167) motif.
  • GAS SrtCl may be differentially regulated by RofA.
  • GAS AI-2 may also include a LepA putative signal peptidase I protein.
  • Table 5 GAS AI-2 sequence from Ml isolate (SF370)
  • GAS 15, GAS 16, and GAS 18 appear to be present on the surface of GAS as part of oligomeric (pilus) structures.
  • Figures 113-115 present electron micrographs of GAS serotype Ml strain SF370 immunogold stained for GAS 15 using anti-GAS 15 antiserum.
  • Figures 116-121 provide electron micrographs of GAS serotype Ml strain SF370 immunogold stained for GAS 16 using anti- GAS 16 antiserum.
  • Figures 122-125 present electron micrograph of GAS serotype Ml strain SF370 immunogold stained for GAS 18 using anti-GAS 18 antiserum. Oligomers of these proteins can be seen on the surface of SF370 bacteria in the immuno-gold stained micrographs.
  • Figure 126 reveals a hyperoligomer on the surface of a GAS serotype Ml strain SF370 bacterium immunogold stained for GAS 18. This long hyperoliogmeric structure comprising GAS 18 stretches far out into the supernatant from the surface of the bacteria.
  • FIG. 75 provides the results of FACS analysis for surface expression of GAS 15 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348. A shift in fluorescence is observed for each GAS serotype when anti-GAS 15 antiserum is present, demonstrating cell surface expression.
  • Table 20, below quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-GAS 15 antiserum, and the difference in fluorescence value between the pre- immune and anti-GAS 15 antiserum.
  • Figures 76 and 79 provide the results of FACS analysis for surface expression of GAS 16 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348.
  • the FACS data in Figure 76 was obtained using antisera was raised against full length GAS 16.
  • the FACS data in Figure 79 was obtained using antisera was raised against a truncated GAS 16, which is encoded by SEQ ID NO: 179, shown below.
  • Table 22 Summary of FACS values for surface expression of GAS 16 using a second antisera
  • Table 24 Summary of FACS values for surface expression of GAS 18 using a second antisera
  • FIG. 89 shows that while pre-immune sera does not detect GAS Ml expression of GAS 15, anti-GAS 15 immune sera is able to detect GAS 15 protein in both total GAS Ml extracts and GAS Ml proteins enriched for cell surface proteins.
  • the GAS 15 proteins detected in the Ml extracts enriched for surface proteins are also present as high molecular weight structures, indicating that GAS 15 may be in an oligomeric (pilus) form.
  • Figure 90 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 15 antisera.
  • Figure 91 provides an additional Western blot identical to that of Figure 90, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
  • Figure 92 provides a Western blot that was probed for GAS 16 protein. While pre-immune sera does not detect GAS Ml expression of GAS 16, anti-GAS 16 immune sera is able to detect GAS eV ⁇ c ⁇ ecl for cell surface proteins. The GAS 16 proteins detected in the Ml extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 16 may be in an oligomeric (pilus) form.
  • Figure 93 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 16 antisera.
  • the lanes that contain total GAS Ml protein (Ml tot new and Ml tot old) and the lane that contains GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 16.
  • Figure 94 provides an additional Western blot identical to that of Figure 93, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
  • Figure 95 provides a Western blot that was probed for GAS 18 protein. While pre-immune sera does not detect GAS Ml expression of GAS 18, anti-GAS 18 immune sera is able to detect GAS 18 protein in GAS Ml extracts enriched for cell surface proteins.
  • the GAS 18 proteins detected in the Ml extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 18 may be in an oligomeric (pilus) form.
  • Figure 96 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 18 antisera.
  • the lane that contains GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 18.
  • Figure 97 provides an additional Western blot identical to that of Figure 96, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
  • Figures 102-106 provide additional Western blots to verify the presence of GAS 15, GAS 16, and GAS 18 in high molecular weight structures in GAS.
  • Each Western blot was performed using proteins from a different GAS Ml strain, 2580, 2913, 3280, 3348, and 2719.
  • Each Western blot was probed with antisera raised against each of GAS 15, GAS 16, and GAS 18.
  • Figure 107 provides a similar Western blot performed to detect GAS 15, GAS 16, and GAS 18 proteins in a GAS serotype Ml strain SF370 protein fraction enriched for surface proteins. This Western blot also shows detection of GAS 15 (Anti-15), GAS 16 (Anti-16), and GAS 18 (Anti-18) as high molecular weight structures.
  • GAS Adhesin Island sequence within M3. M5. and M18 GAS Adhesin Island 3 (“GAS AI-3") GAS Adhesin Island sequences within M3, M5, and M18 serotypes are outlined in Tables 6 — 8 and 10 below.
  • This GAS adhesin island 3 (“GAS AI-3") comprises surface proteins, a SrtC2 sortase, and a Negative transcriptional regulator (Nra) divergently transcribed transcriptional regulator.
  • GAS AI-3 surface proteins within include a collagen binding protein, a fimbrial protein, a F2 like fibronectin-binding protein.
  • GAS AI-3 surface proteins may also include a hypothetical surface l surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
  • GAS AI-3 includes a SrtC2 type sortase.
  • GAS SrtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail.
  • GAS SrtC2 may be differentially regulated by Nra.
  • GAS AI-3 may also include a LepA putative signal peptidase I protein.
  • GAS AI-3 may also include a putative multiple sugar metabolism regulator.
  • FIG 51A A schematic of AI-3 serotypes M3, M5, M18, and M49 is shown in Figure 51A. Each contains an open reading frame encoding a SrtC2-type sortase of nearly identical amino acid sequence. See Figure 52B for an amino acid sequence alignment for each of the SrtC2 amino acid sequences.
  • the protein F2-like fibronectin-binding protein of each these type 3 adhesin islands contains a pilin motif and an E-box.
  • Figure 60 indicates the amino acid sequence of the pilin motif and E-box of each of GAS AI-3 serotype M3 MGAS315 (SpyM3_0104/21909640), GAS AI-3 serotype M3 SSI (SpsO 106/28895018), GAS AI-3 serotype M18 (SpyM18_0132/19745307), and GASAI-3 serotype
  • FIG. 80 provides the results of FACS analysis for surface expression of S ⁇ yM3_0098 on each of GAS ⁇ t ⁇ i. S l ⁇ iS ⁇ n fluorescence is observed for each GAS serotype when anti-SpyM3_0098 antiserum is present, demonstrating cell surface expression.
  • Table 25 quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0098 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0098 antiserum.
  • Figure 81 provides the results of FACS analysis for surface expression of SpyM3_0100 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0100 antiserum is present, demonstrating cell surface expression.
  • Table 26, below quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0100 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0100 antiserum.
  • Figure 82 provides the results of FACS analysis for surface expression of SpyM3_0102 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-S ⁇ yM3_0102 antiserum is present, demonstrating cell surface expression.
  • Table 27, below quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0102 antiserum.
  • Figure 82 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM3_0102 identified in a different GAS serotype, M6.
  • FACS analysis conducted with the SpyM3_0102 antisera was able to detect surface expression of the homologous SpyM3_0102 antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894.
  • Table 28 quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0102 antiserum, and the difference in fluorescence value between the pre-immune and anti-S ⁇ yM3_0102 antiserum.
  • Table 28 Summary of FACS values for surface expression of SpyM3_0102 in M6 serotypes
  • SpyM3_0102 is also homologous to pilin antigen 19224139 of GAS serotype M12. Antisera raised against SpyM3_0102 is able to detect high molecular weight structures in GAS serotype M12 strain 2728 protein fractions enriched for surface proteins, which would contain the 19224139 antigen. See Figure 109 at the lane labelled M12 2728 surf prot.
  • Figure 83 provides the results of FACS analysis for surface expression of SpyM3_0104 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0104 antiserum is present, demonstrating cell surface expression.
  • Table 29, below quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-SpyM3_0104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0104 antiserum.
  • Figure 83 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM3_0104 identified in a different GAS serotype, Ml 2.
  • FACS analysis conducted with the SpyM3_0104 antisera was able to detect surface expression of the homologous SpyM3_0104 antigen on GAS serotype M12 2728.
  • Table 30, below quantitatively summarizes the FACS fluorescence values obtained for this GAS serotype in the presence of pre-immune antiserum, anti-S ⁇ yM3_0104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0104 antiserum.
  • Table 30 Summary of FACS values for surface expression of SpyM3_0104 in an M12 serotype
  • Figure 84 provides the results of FACS analysis for surface expression of SPs_0106 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SPs_0106 antiserum is present, demonstrating cell surface expression.
  • Table 31, below quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SPs_0106 antiserum, and the difference in fluorescence value between the pre-immune and anti-SPs_0106 antiserum.
  • Figure 84 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SPs_0106 identified in a different GAS serotype, M12. FACS analysis able to detect surface expression of the homologous SPs_0106 antigen on GAS serotype M12 2728.
  • Table 32 below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SPs_0106 antiserum, and the difference in fluorescence value between the pre-immune and anti- SPs_0106 antiserum.
  • Table 32 Summary of FACS values for surface expression of SPs_0106 in an M12 serotype
  • GAS Adhesin Island 4 GAS Adhesin Island 4
  • GAS Adhesin Island sequences within M12 serotype are outlined in Table 11 below.
  • This GAS adhesin island 4 (“GAS AI-4") comprises surface proteins, a SrtC2 sortase, and a RofA regulatory protein.
  • GAS AI-4 surface proteins within may include a fimbrial protein, an F or F2 like fibronectin- binding protein, and a capsular polysaccharide adhesion protein (Cpa). GAS AI-4 surface proteins may also include a hypothetical surface protein in an open reading frame (orf). Preferably, each of these GAS AI-4 surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
  • LPXTG sortase substrate motif such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
  • GAS AI-4 includes a SrtC2 type sortase.
  • GAS SrtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail.
  • GAS AI-4 may also include a LepA putative signal peptidase I protein and a MsmRL protein. Table 11; GAS AI-4 se uences from M12 isolate A735
  • a schematic of AI-4 serotype M12 is shown in Figure 51A.
  • One of the open reading frames encodes a SrtC2-type sortase having an amino acid sequence nearly identical to the amino acid sequence of the SrtC2-type sortase of the AI-3 serotypes described atfdV&r sWFlflufe 1 sifffof afr ⁇ ' amin ⁇ a ' ⁇ icF'sequence alignment for each of the SrtC2 amino acid sequences.
  • FIG. 52 is an amino acid alignment of the capsular polysaccharide adhesion protein (cpa) of AI-4 serotype M12 (19224135), GAS AI-3 serotype M5 (ORF78), S. pyogenes strain MGAS315 serotype M3 (21909634), S. pyogenes SSI-I serotype M3 (28810257), S.
  • cpa capsular polysaccharide adhesion protein
  • Figure 54 is an amino acid sequence alignment that illustrates that the F2-like fibronectin- binding protein of AI-4 serotype Ml 2 (19224141) shares homology with the F2-like fibronectin- binding protein of S. pyogenes strain MGAS8232 serotype M3 (19745307), GAS AI-3 serotype M5 (ORF84), S. pyogenes strain SSI serotype M3 (28810263), and S. pyogenes strain MGAS315 serotype M3 (21909640).
  • Figure 55 is an amino acid sequence alignment that illustrates that the fimbrial protein of AI-4 serotype ' M12 (19224137) shares homology with the fimbrial protein of GAS AI-3 serotype M5 (ORF80), and the hypothetical protein of S. pyogenes strain MGAS315 serotype M3 (21909636), S. pyogenes strain SSI serotype M3 (28810259), S. pyogenes strain MGAS8732 serotype M3 (19745303), and S. pyogenes strain Ml GAS serotype Ml (13621428).
  • Figure 56 is an amino acid sequence alignment that illustrates that the hypothetical protein of GAS AI-4 serotype M12 (19224139) shares homology with the hypothetical protein of S. pyogenes strain MGAS315 serotype M3 (21909638), S. pyogenes strain SSI-I serotype M3 (28810261), GAS AI-3 serotype M5 (ORF82), and S. pyogenes strain MGAS8232 serotype M3 (19745305).
  • the protein F2-like fibronectin-binding protein of the type 4 adhesin island also contains a highly conserved pilin motif and an E-box.
  • Figure 60 indicates the amino acid sequence of the pilin motif and E-box in AI-4 serotype Ml 2. FACS analysis has confirmed that the GAS AI-4 surface proteins 19224134, 19224135,
  • Figure 85 provides the results of FACS analysis for surface expression of 19224134 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224134 antiserum is present, demonstrating cell surface expression.
  • Table 33 below, quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M12 2728 in the presence of pre-immune antiserum, anti-19224134 antiserum, and the difference in fluorescence value between the pre-immune and anti-19224134 antiserum.
  • Table 33 Summary of FACS values for surface expression of 19224134 in an M12 serotype
  • Figxire 85 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to 19224134 identified in a different GAS serotype, M6.
  • FACS analysis conducted with the 19224134 antisera was able to detect surface expression of the homologous 19224134 antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894.
  • Table 34 quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-19224134 antiserum, and the difference in fluorescence value between the pre-immune and anti-19224134 antiserum.
  • Figure 86 provides the results of FACS analysis for surface expression of 19224135 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224135 antiserum is present, demonstrating cell surface expression.
  • Table 35 quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M12 2728 in the presence of pre-immune antiserum, anti-19224135 antiserum, and the difference in fluorescence value between the pre-immune and anti- 19224135 antiserum.
  • Table 35 Summary of FACS values for surface expression of 19224135 in an M12 serotype
  • Figure 87 provides the results of FACS analysis for surface expression of 19224137 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224137 antiserum is present, demonstrating cell surface expression.
  • Table 36 quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M12 2728 in the presence of pre-immune antiserum, anti-19224137 antiserum, and the difference in fluorescence value between the pre-immune and anti- 19224137 antiserum.
  • Table 36 Summary of FACS values for surface expression of 19224137 in an M12 serotype
  • Figure 88 provides the results of FACS analysis for surface expression of 19224141 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224141 antiserum is present, demonstrating cell surface expression.
  • Table 37 quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M 12 2728 in the presence of pre-immune antiserum, anti-19224141 antiserum, and the difference in fluorescence value between the pre-immune and anti- 19224141 antiserum.
  • 19224139 (designated as orf2) may also be expressed on the surface of GAS serotype M12 bacteria.
  • Figure 175 shows the results of FACS analysis for surface expression of 19224139 on M12 strain 2728. A slight shift in fluorescence is observed, which demonstrates that some 19224139 may be expressed on the GAS cell surface.
  • FIG. 99 shows that while pre-immune sera (P ⁇ -4135) does not detect GAS M12 expression of 19224135, anti-19224135 immune sera (I ⁇ -4135) is able to detect 19224135 protein in both total GAS M12 extracts (M12 tot) and GAS M12 fractions enriched for cell surface proteins (M12 surf prot).
  • the 19224135 proteins detected in the total GAS M12 extracts or the GAS M12 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that 19224135 may be in an oligomeric (pilus) form.
  • Figure 108 provides a further Western blot showing that anti-19224135 antiserum (Anti-19224135) imrnunoreacts with high molecular weight structures in GAS M12 strain 2728 protein extracts enriched for surface proteins.
  • Surface expression of 19224137 on M12 serotype GAS has also been confirmed by Western blot analysis.
  • Figure 100 shows that while pre-immune sera (P ⁇ -4137) does not detect GAS M 12 expression of 19224137, anti-19224137 immune sera (I ⁇ -4137) is able to detect 19224137 protein in both total GAS M 12 extracts (M 12 tot) and GAS M12 fractions enriched for cell surface proteins (M12 surf prot).
  • the 19224137 proteins detected in the total GAS M12 extracts or the GAS M12 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that 19224137 may be in an oligomeric (pilus) form. See also Figure 108, which provides a further Western blot showing that anti-19224137 antiserum (Anti-19224137) immunoreacts with high molecular weight structures in GAS M12 strain 2728 protein extracts enriched for surface proteins.
  • Streptococcus pneumoniae Adhesin island sequences can be identified in Streptococcus pneumoniae genomes. Several of these genomes include the publicly available Streptococcus pneumoniae TIGR4 genome or Streptococcus pneumoniae strain 670 genome. Examples of these S. pneumoniae AI sequence are discussed below.
  • S. pneumoniae Adhesin Islands generally include a series of open reading frames within a S. pneumoniae genome that encode for a collection of surface proteins and sortases.
  • a S. pneumoniae Adhesin Island may encode for amino acid sequences comprising at least one surface protein.
  • an S. pneumoniae Adhesin Island may encode for at least two surface proteins and at least one sortase.
  • a S. pneumoniae Adhesin Island encodes for at least three surface proteins and at least two sortases.
  • One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
  • One or more S. pneumoniae AI i C surface pro ' te: i ⁇ Mbiia ⁇ ; ii ⁇ ;i ation of a pilus structure on the surface of the S. pneumoniae bacteria.
  • S. pneumoniae Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator.
  • the transcriptional regulator may regulate the expression of the S. pneumoniae AI operon.
  • the S. pneumoniae AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen.
  • FIG 137 A schematic of the organization of a S. pneumoniae AI locus is provided in Figure 137.
  • the locus comprises open reading frames encoding a transcriptional regulator (rlrA), cell wall surface proteins (rrgA, rrgB, rrgC), and sortases (srtB, srtC, srtD).
  • Figure 137 also indicates the S. pneumoniae strain TIGR4 gene name corresponding to each of these open reading reading frames.
  • Tables 9 and 38 identify the genomic location of each of these open reading frames in S. pneumoniae strains TIGR.4 and 670, respectively.
  • Table 9 S. pneumoniae AI sequences from TIGR4
  • Genomic Location Strand Length PID Synonym (AI Sequence Functional description Identifier)
  • the full-length nucleotide sequence of the S. pneumoniae strain 670 AI is also shown in Figure 101, as is its translated amino acid sequence.
  • At least eight other S. pneumoniae strains contain an adhesin island locus described by the locus depicted in Figure 137. These strains were identified by an amplification analysis. The genomes of different S. pneumoniae strains were amplified with eleven separate sets of primers. The sequence of each of these primers is provided below in Table 41.
  • Figure 138 which is a schematic of the location where each of these primers hybridizes to the S. pneumoniae AI locus.
  • Figure 139A provides the set of amplicons obtained from amplification of the AI locus in S. pneumoniae strain TIGR4.
  • Figure 139B provides the length, in base pairs, of each amplicon in S. pneumoniae strain TIGR4.
  • Amplification of the genome of S. pneumoniae strains 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9 V Spain 3, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, and 23F Poland 16 produced a set of eleven amplicons for the eleven primer pairs, indicating that each of these strains also contained the S. pneumoniae AI locus.
  • the S. pneumoniae strains were also identified as containing the AI locus by comparative genome hybridization (CGH) analysis.
  • CGH comparative genome hybridization
  • the genomes of sixteen S. pneumoniae strains were interrogated for the presence of the AI locus by comparison to unique open reading frames of strain TIGR4.
  • the AI locus was detected by this method in strains 19A Hungary 6 (19AHUN), 6B Finland 12 (6BFIN12), 6B Spain 2 (6BSP2), 14CSR10 (14 CSRlO), 9V Spain 3 (9VSP3), 19F Taiwan 14 (19FTW14), 23F Taiwan 15 (19FTW15), and 23F Poland 16 (23FP16). See Figure 140.
  • the AI locus has been sequenced for each of these strains and the nucleotide and encoded amino acid seqeunce for each orf has been determined.
  • An alignment of the complete nucleotide sequence of the adhesin island present in each of the ten strains is provided in Figure 196. Aligning the amino acid sequences encoded by the orfs reveals conservation of many of the AI polypeptide amino acid sequences. For example, Table 39 provides a comparison of the percent identities of the polypeptides encoded within the S. pneumoniae strain 670 and TIGR4 adhesin islands.
  • Figures 141-147 each provide a multiple sequence alignment for the polypeptides encoded by one of the open reading frames in all ten AI-positive S. pneumoniae strains.
  • light shading indicates an LPXTG motif
  • dark shading indicates the presence of an E- box motif with the conserved glutamic acid residue of the E-box motif in bold.
  • polypeptides encoded by most of the open reading frames may be divided into two groups of homology, S. pneumoniae AI-a and AI-b.
  • S. pneumoniae strains that comprise AI-a include 14 CSR 10, 19A Hungary 6, 23F Tru 15, 670, 6B Finland 12, and 6B Spain 2.
  • S. pneumoniae strains that comprise AI-b include 19F Taiwan 14, 9V Spain 3, 23F Taiwan 15, and TIGR4.
  • An immunogenic composition of the invention may comprise one or more polypeptides from within each of S. pneumoniae AI-a and AI-b.
  • polypeptide RrgB encoded by open reading frame 4
  • One group contains the RrgB sequences of six S.
  • a second group contains the RrgB sequences of four S. pneumoniae strains. While the amino acid sequence of the strains within each individual group is 99-100 percent identical, the amino acid sequence identity of the strains in the first relative to the second group is only 48%. Table 41 provides the identity comparisons of the amino acid sequences encoded by each open reading frame for the ten S. pneumoniae strains.
  • the division of homology between the RrgB polypeptide in the S. pneumoniae strains is due a lack of amino acid sequence identity in the central amino acid residues.
  • Amino acid residues 1-30 and 617-665 are identical for each of the ten S. pneumoniae strains. However, amino acid residues 31-616 share between 42 and 100 percent identity between strains. See Figure 149.
  • the shared N- and C-terminal regions of identity in the RrgB polypeptides may be preferred portions of the RrgB polypeptide for use in an immunogenic composition.
  • shared regions of identity in any of the polypeptides encoded by the S. pneumoniae AI locus may be preferable for use in immunogenic compositions.
  • One of skill in the art using the amino acid alignments provided in Figures 141-147, would readily be able to determine these regions of identity.
  • the S. pneumoniae comprising these AI loci do, in fact, express high molecular weight polymers on their surface, indicating the presence of pili.
  • Figure 182 which shows detection of high molecular weight structures expressed by S. pneumoniae strains that comprise the adhesin island lqcli£SepGtelll:FipiFiia7iplhSyitiilnsi ! are indicated as rlrA+. Confirming these findings, electron microscopy and negative staining detects the presence of pili extending from the surface of S. pneumoniae. See Figure 185. To demonstrate that the adhesin island locus was responsible for the pili, the rrgA-srtD region of TIGR 4 were deleted.
  • S. pneumoniae TIGR4 that lack the pilus operon have significantly diminished ability to adhere to A549 alveolar cells in vitro. See Figure 184.
  • the SpO463 (S. pneumoniae TIGR4 rrgB) adhesion island polypeptide is expressed in oligomeric form.
  • Whole cell extracts were analyzed by Western blot using a SpO463 antiserum.
  • the antiserum cross-hybridized with high molecular weight SpO463 polymers. See Figure 156.
  • the antiserum did not cross-hybridize with polypeptides from D39 or R6 strains of S. pneumoniae, which do not contain the AI locus depicted in Figure 137.
  • Immunogold labelling of S. pneumoniae TIGR 4 using RrgB antiserum confirms the presence of RrgB in pili.
  • Figure 189 shows double-labeling of S.
  • the RrgA protein appears to be present in and necessary for formation of high molecular weight structures on the surface of 5. pneumoniae TIGR4. See Figure 181 which provides the results of Western blot analysis of TIGR4 S. pneumoniae lacking the gene encoding RrgA. No high molecular weight structures are detected in S. pneumoniae that do not express RrgA using antiserum raised against RrgB. See also Figure 183.
  • FIG. 148 A detailed diagram of the amino acid sequence comparions of the RrgA protein in the ten 5.. pneumoniae strains is shown in Figure 148. The diagram reveals the division of the individual S. pneumoniae strains into the two different homology groups.
  • a polyacrylamide gel showing successful recombinant expression of RrgA is provided in Figure 190A. Detection of the RrgA protein, which is expressed in pET21b with a histidine tag, is also shown by Western blot analysis in Figure 190B, using an anti-histidine tag antibody.
  • Non-AI polypeptide may be genetically manipulated to additionally contain AI , polypeptide sequences, e.g., a sortase substrate, pilin, or E-box motif, which may cause expression of the non-AI polypeptide as an AI polypeptide.
  • non-AI polypeptide may be genetically manipulated to replace an amino acid sequence within the non-AI polypeptide for AI polypeptide sequences, e.g., a sortase substrate, pilin, or E-box motif, which may cause expression of the non-AI polypeptide as an AI polypeptide.
  • Any number of amino acid residues may be added to the non-AI polypeptide or may be replaced within the non-AI polypeptide to cause its expression as an AI polypeptide. At least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 75, 100, 150, 200, or 250 amino acid residues may be replaced or added to the non-AI polypeptide amino acid sequence.
  • GBS 322 may be one such non-AI polypeptide that may be expressed as an AI polypeptide.
  • the GBS AI polypeptides of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from GBS, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form.
  • the GBS AI proteins of the invention may include polypeptide sequences having sequence identity to the identified GBS proteins.
  • the degree of sequence identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more).
  • the GBS adhesin island polynucleotide sequences may include polynucleotide sequences having sequence identity to the identified GBS adhesin island polynucleotide sequences.
  • the degree of sequence identity may vary depending on the polynucleotide sequpnce in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9.7%, 98%, 99%, 99.5% or more).
  • the GBS adhesin island polynucleotide sequences of the invention may include polynucleotide fragments of the identified adhesin island sequences.
  • the length of the fragment may vify't ⁇ epending' ⁇ iii! fee-pblyn ⁇ el ⁇ fia&lfcq ⁇ lence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
  • the GBS adhesin island amino acid sequences of the invention may include polypeptide fragments of the identified GBS proteins.
  • the length of the fragment may vary depending on the amino acid sequence of the specific GBS antigen, but the fragment is preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
  • the fragment comprises one or more epitopes from the sequence.
  • Other preferred fragments include (1) the N-terminal signal peptides of each identified GBS protein, (2) the identified GBS protein without their N-terminal signal peptides, and (3) each identified GBS protein wherein up to 10 amino acid residues (e.g.
  • N- tenninus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more are deleted from the N- tenninus and/or the C-terminus e.g. the N-terminal amino acid residue may be deleted.
  • Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
  • GBS 80 fragments are discussed below.
  • Polynucleotide and polypeptide sequences of GBS 80 from a variety of GBS serotypes and strain isolates are set forth in Figures 18 and 22.
  • the polynucleotide and polypeptide sequences for GBS 80 from GBS serotype V, strain isolate 2603 are also included below as SEQ ID NOS 1 and 2: SEQ ID NO. 1

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Abstract

The invention relates to the identification of a new adhesin islands within the genomes of several Group A and Group B Streptococcus serotypes and isolates. The adhesin islands are thought to encode surface proteins which are important in the bacteria's virulence. Thus, the adhesin island proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against GAS or GBS infection. For example, the invention may include an immunogenic composition comprising one or more of the discovered adhesin island proteins.

Description

IMMUWΩ<δErøeCQMPΘSlTIiQNS FOR GRAM POSITIVE BACTERIA SUCH AS
STREPTOCOCCUS A GALA CTIAE
FIELD OF THE INVENTION The invention relates to the identification of adhesin islands within the genome Streptococcus agalactiae ("GBS") and the use of adhesin island amino acid sequences encoded by these adhesin islands in compositions for the treatment or prevention of GBS infection. Similar sequences have been identified in other Gram positive bacteria. The invention further includes immunogenic compositions comprising adhesin island amino acid sequences of Gram positive bacteria for the treatment or prevention of infection of Gram positive bacteria. Preferred immunogenic compositions of the invention include an adhesin island surface protein which may be formulated or purified in an oligomeric or pilus form. BACKGROUND OF THE INVENTION
GBS has emerged in the last 20 years as the major cause of neonatal sepsis and meningitis that affects 0.5 - 3 per 1000 live births, and an important cause of morbidity among older age groups affecting 5 — 8 per 100,000 of the population. Current disease management strategies rely on intrapartum antibiotics and neonatal monitoring which have reduced neonatal case mortality from >50% in the 1970's to less than 10% in the 1990's. Nevertheless, there is still considerable morbidity and mortality and the management is expensive. 15 — 35% of pregnant women are asymptomatic carriers and at high risk of transmitting the disease to their babies. Risk of neonatal infection is associated with low serotype specific maternal antibodies and high titers are believed to be protective. In addition, invasive GBS disease is increasingly recognized in elderly adults with underlying disease such as diabetes and cancer.
The "B" in "GBS" refers to the Lancefϊeld classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate, designated A to O, that could be serologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains can be divided into at least 9 serotypes (Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII) based on the structure of their polysaccharide capsule. In the past, serotypes Ia, Ib, II, and III were equally prevalent in normal vaginal carriage and early onset sepsis in newborns. Type V GBS has emerged as an important cause of GBS infection in the USA, however, and strains of types VI and VIII have become prevalent among Japanese women.
The genome sequence of a serotype V strain 2603 V/R has been published (See Tettelin et al. (2002) Proc. Natl. Acad. ScL USA, 10.1073/pnas.l82380799) and various polypeptides for use a vaccine antigens have been identified (WO 02/34771). The vaccines currently in clinical trials, however, are based primarily on polysaccharide antigens. These suffer from serotype-specifϊcity and poor immunogenicity, and so there is a need for effective vaccines against S.agalactiae infection. „,.,. „ &..αsα/αG|ώαιeii*'Θlassifiedιιas-ιa"gra-iι positive bacterium, a collection of about 21 genera of
I-- II 1 / obub/1 ii::::; / ir,,:f,S ":«* bacteria that colonize humans, have a generally spherical shape, a positive Gram stain reaction and lack endospores. Gram positive bacteria are frequent human pathogens and include Staphylococcus (such as S. aureus), Streptococcus (such as S. pyogenes (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutatis), Enterococcus (such as E.faecalis and E. faeciuin), Clostridium (such as C. difficile), Listeria (such as L. monocytogenes) and Cory neb act erium (such as C. diphtheria).
It is an object of the invention to provide further and improved compositions for providing immunity against disease and/or infection of Gram positive bacteria. The compositions are based on the identification of adhesin islands within Streptococcal genomes and the use of amino acid sequences encoded by these islands in therapeutic or prophylactic compositions. The invention further includes compositions comprising immunogenic adhesin island proteins within other Gram positive bacteria in therapeutic or prophylactic compositions. SUMMARY OF THE INVENTION
Applicants have identified a new adhesin island, "GBS Adhesin Island 1", "AI-I" or "GBS AI-I", within the genomes of several Group B Streptococcus serotypes and isolates. This adhesin island is thought to encode surface proteins which are important in the bacteria's virulence. In addition, Applicants have discovered that surface proteins within GBS Adhesin Islands form a previously unseen pilus structure on the surface of GBS bacteria. Amino acid sequences encoded by such GBS Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of GBS infection.
A preferred immunogenic composition of the invention comprises an AI-I surface protein, such as GBS 80, which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Electron micrographs depicting some of the first visualizations of this pilus structure in a wild type GBS strain are shown in Figures 16, 17, 49, and 50. In addition, Applicants have transformed a GBS strain with a plasmid comprising the AI surface protein GBS 80 which resulted in increased production of that AI surface protein. The electron micrographs of this mutant GBS strain in Figures 13 - 15 reveal long, hyper-oligomeric structures comprising GBS 80 which appear to cover portions of the surface of the bacteria and stretch far out into the supernatant. These hyper-oligomeric pilus structures comprising a GBS AI surface protein may be purified or otherwise formulated for use in immunogenic compositions.
GBS AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("AI-I proteins"). Specifically, AI-I includes polynucleotide sequences encoding for two or more of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. One or more of the AI-I polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the AI-I open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF. f'"!i Ir1111
Figure imgf000004_0001
ff£,sides;;pn;san;,japjrjDKimately 16.1 kb transposon-like element frequently inserted into the open reading frame for trmA. One or more of the AI-I surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. The AI surface proteins of the invention may affect the ability of the GBS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. AI-I may encode at least one surface protein. Alternatively, AI-I may encode at least two surface proteins and at least one sortase. Preferably, AI-I encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif or other sortase substrate motif.
The GBS AI-I protein of the composition may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. GBS AI-I surface proteins GBS 80 and GBS 104 are preferred for use in the immunogenic compositions of the invention.
In addition to the open reading frames encoding the AI-I proteins, AI-I may also include a divergently transcribed transcriptional regulator such as araC {i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). It is believed that araC may regulate the expression of the GBS AI operon. (See Korbel et al., Nature Biotechnology (2004) 22(7): 911 - 917 for a discussion of divergently transcribed regulators in E. colϊ).
A second adhesin island, "Adhesin Island-2", "AI-2" or "GBS AI-2", has also been identified in numerous GBS serotypes. Amino acid sequences encoded by the open reading frames of AI-2 may also be used in immunogenic compositions for the treatment or prevention of GBS infection.
GBS AI-2 comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, AI-2 includes open reading frames encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525. The GBS AI-2 sequences may be divided into two subgroups. In one embodiment, AI-2 includes open reading frames encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406. This collection of open reading frames may be generally referred to as GBS AI-2 subgroup 1. Alternatively, AI-2 may include open reading frames encoding for two or more of 01520, 01521, 01522, 01523, 01523, 01524 and 01525. This collection of open reading frames may be generally referred to as GBS AI-2 subgroup 2. One or more of the AI-2 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the AI-2 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF. ,ril ,, Qne,.or typically include an LPXTG motif (such as LPXTG
Figure imgf000005_0001
(SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. AI-2 may encode for at least one surface protein. Alternatively, AI-2 may encode for at least two surface proteins and at least one sortase. Preferably, AI-2 encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif.
The AI-2 protein of the composition may be selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525. AI- 2 surface proteins GBS 67, GBS 59, and 01524 are preferred AI-2 proteins for use in the immunogenic compositions of the invention. GBS 67 or GBS 59 is particularly preferred.
GBS AI-2 may also include a divergently transcribed transcriptional regulator such as a RofA like protein (for example rogB). As in AI-I, rogB is thought to regulate the expression of the AI-2 operon.
The GBS AI proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against GBS infection. For example, the invention may include an immunogenic composition comprising one or more GBS AI-I proteins and one or more GBS AI-2 proteins.
The immunogenic compositions may also be selected to provide protection against an increased range of GBS serotypes and strain isolates. For example, the immunogenic composition may comprise a first and second GBS AI protein, wherein a full length polynucleotide sequence encoding for the first GBS AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second GBS AI protein. In addition, each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple GBS serotypes and strain isolates. Preferably, each antigen is presnt in the genomes of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) GBS strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5 or more) GBS serotypes.
Within GBS AI-I, Applicants have found that Group B Streptococcus surface exposure of GBS 104 is dependent on the concurrent expression of GBS 80. It is thought that GBS 80 is involved in the transport or localization of GBS 104 to the surface of the bacteria. The two proteins may be oligomerized or otherwise chemically or physically associated. It is possible that this association involves a conformational change in GBS 104 that facilitates its transition to the surface of the GBS bacteria. In addition, one or more AI sortases may also be involved in this surface localization and chemical or physical association. Similar relationships are thought to exist within GBS AI-2. The compositions of the invention may therefore include at least two AI proteins, wherein the two AI proteins are physically or chemically associated. Preferably, the two AI proteins form an oligomer. Preferably, one or more of the AI proteins are in a hyper-oligomeric form. In one embodiment, the associated AI proteins may be purified or isolated from a GBS bacteria or recombinant host cell. It1Is .alsp.ao.pbiect Qflhαjraftsntiαn to provide further and improved compositions for p Ii il / U !!:::iι ILIi H:::iι / c:; ./ n:;;:: „3 h» providing prophylactic or therapeutic protection against disease and/or infection of Gram positive bacteria. The compositions are based on the identification of adhesin islands within Streptococcal genomes and the use of amino acid sequences encoded by these islands in therapeutic or prophylactic compositions. The invention further includes compositions comprising immunogenic adhesin island proteins within other Gram positive bacteria in therapeutic or prophylactic compositions. Preferred Gram positive adhesin island proteins for use in the invention may be derived from Staphylococcus (such as S. aureus), Streptococcus (such as S. agalactiae (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutans), Enterococcus (such as E.faecalis and E. faecium), Clostridium (such as C. difficile), Listeria (such as L. monocytogenes) and Corynebacterium (such as C. diphtheria). Preferably, the Gram positive adhesin island surface proteins are in oligomeric or hyperologimeric form.
For example, Applicants have identified adhesin islands within the genomes of several Group A Streptococcus serotypes and isolates. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus.
Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fasciitis. In addition, poststreptococcal autoimmune responses are still a major cause of cardiac pathology in children.
Group A Streptococcal infection of its human host can generally occur in three phases. The first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces. Generally this attachment phase begins in the throat or the skin. The deeper the tissue level infected, the more severe the damage that can be caused. In the second stage of infection, the bacteria secretes a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. This toxin binds to susceptible host cell receptors and triggers innappropropriate immune responses by these host cells, resulting in pathology. Because the toxin can diffuse throughout the host, the necrosis directly caused by the GAS toxins may be physically located in sites distant from the bacterial infection. The final phase of GAS infection can occur long after the original bacteria have been cleared from the host system. At this stage, the host's previous immune response to the GAS bacteria due to cross reactivity between epitopes of a GAS surface protein, M, and host tissues, such as the heart. A general review of GAS infection can be found in Principles of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
In order to prevent the pathogenic effects associated with the later stages of GAS infection, an effective vaccine against GAS will preferably facilitate host elimination of the bacteria during the initial attachment and invasion stage.
Isolates of Group A Streptococcus are historically classified according to the M surface protein described aboγe. The M protein is surface exposed trypsin-sensitive protein generally capitis!!' ^..^βSlffli^ H^WS^M^^ in an alPha helical formation. The carboxyl terminus is anchored in the cytoplasmic membrane and is highly conserved among all group A streptococci. The amino terminus, which extend through the cell wall to the cell surface, is responsible for the antigenic variability observed among the 80 or more serotypes of M proteins. A second layer of classification is based on a variable, trypsin-resistant surface antigen, commonly referred to as the T-antigen. Decades of epidemiology based on M and T serological typing have been central to studies on the biological diversity and disease causing potential of Group A Streptococci. While the M-protein component and its inherent variability have been extensively characterized, even after five decades of study, there is still very little known about the structure and variability of T-antigens. Antisera to define T types is commercially available from several sources, including Sevapharma (http://www.sevapharma.cz/en).
The gene coding for one form of T-antigen, T-type 6, from an M 6 strain of GAS (D741) has been cloned and characterized and maps to an approximately 11 kb highly variable pathogenicity island. Schneewind et al., J Bacteriol. (1990) 172(6):3310 - 3317. This island is known as the Fibronectin-binding, Collagen-binding T-antigen (FCT) region because it contains, in addition to the T6 coding gene (teeό), members of a family of genes coding for Extra Cellular Matrix (ECM) binding proteins. Bessen et al., Infection & Immunity (2002) 70(3): 1159-1167. Several of the protein products of this gene family have been shown to directly bind either fibronectin and/or collagen. See Hanski et al., Infection & Immunity (1992) 60(12):5119-5125; Talay et al., Infection & Immunity (1992( 60(9):3837-3844; Jaffe et al. (1996) 21(2):373-384; Rocha et al., Adv Exp Med Biol. (1997) 418:737-739; Kreikemeyer et al., J Biol Chem (2004) 279(16):15850-15859; Podbielski et al., MoI. Microbiol. (1999) 31(4): 1051-64; and Kreikemeyer et al., Int. J. Med Microbiol (2004) 294(2-3):177- 88. In some cases direct evidence for a role of these proteins in adhesion and invasion has been obtained. Applicants raised antiserum against a recombinant product of the teeό gene and used it to explore the expression of T6 in M6 strain 2724. In immunoblot of mutanolysin extracts of this strain, the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used. This pattern of high molecular weight products is similar to that observed in immunoblots of the protein components of the pili identified in Streptococcus agalactiae (described above) and previously in Corynebacterium diphtheriae. Electron microscropy of strain M6_2724 with antisera specific for the product of teeό revealed abundant surface staining and long pilus like structures extending up to 700 nanometers from the bacterial surface, revealing that the T6 protein, one of the antigens recognized in the original Lancefiled sero typing system, is located within a GAS Adhesin Island (GAS AI-I) and forms long covalently linked pilus structures.
Applicants have identified at least four different Group A Streptococcus Adhesin Islands.
While these GAS AI sequences can be identified in numerous M types, Applicants have surprisingly
Figure imgf000008_0001
pilus subunits from the four different GAS AI types and specific T classifications. While other trypsin-resistant surface exposed proteins are likely also implicated in the T classification designations, the discovery of the role of the GAS adhesin islands (and the associated hyper-oligomeric pilus like structures) in T classification and GAS serotype variance has important implications for prevention and treatment of GAS infections. Applicants have identified protein components within each of the GAS adhesin islands which are associated with the pilus formation. These proteins are believed to be involved in the bacteria's initial adherence mechanisms. Immunological recognition of these proteins may allow the host immune response to slow or prevent the bacteria's transition into the more pathogenic later stages of infection. In addition, Applicants have discovered that the GBS pili structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysaccharide matrix). Biofilms are generally associated with bacterial resistance, as antibiotic treatments and host immune response are frequently unable to erradicate all of the bacteria components of the biofilm. Direction of a host immune response against surface proteins exposed during the first steps of bacterial attachment (i.e., before complete biofilm formation) is preferable.
The invention therefore provides for improved immunogenic compositions against GAS infection which may target GAS bacteria during their initial attachment efforts to the host epithelial cells and may provide protection against a wide range of GAS serotypes. The immunogenic compositions of the invention include GAS AI surface proteins which may be formulated in an oligomeric, or hyperoligomeric (pilus) form. The immunogenic compositions of the invention may include one or more GAS AI surface proteins. The invention also includes combinations of GAS AI surface proteins. Combinations of GAS AI surface proteins may be selected from the same adhesin island or they may be selected from different GAS adhesin islands.
Amino acid sequence encoded by such GAS Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of GAS infection. Preferred immunogenic compositions of the invention comprise a GAS AI surface protein which has been formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric foπn is a hyperoligomer.
GAS Adhesin Islands generally include a series of open reading frames within a GAS genome that encode for a collection of surface proteins and sortases. A GAS Adhesin Island may encode for an amino acid sequence comprising at least one surface protein. The Adhesin Island, therefore, may encode at least one surface protein. Alternatively, a GAS Adhesin Island may encode for at least two surface proteins and at least one sortase. Preferably, a GAS Adhesin Island encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or more GAS AI surface proteins may participate in the formation of a pilus structure on the surface of the Gram positive bacteria. B-I, ji-.. 'GAS- Adfeesjm Islanfepfiitheiiinipption preferably include a divergently transcribed transcriptional regulator. The transcriptional regulator may regulate the expression of the GAS AI operon. Examples of transcriptional regulators found in GAS AI sequences include RofA and Nra.
The GAS AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen. One or more of the GAS AI surface proteins may comprise a fϊmbrial structural subunit. One or more of the GAS AI surface proteins may include an LPXTG motif or other sortase substrate motif. The LPXTG motif may be followed by a hydrophobic region and a charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. See Barnett, et al, J. Bacteriology (2004) 186 (17): 5865-5875. GAS AI sequences may be generally categorized as Type 1, Type 2, Type 3, or Type 4, depending on the number and type of sortase sequences within the island and the percentage identity of other proteins (with the exception of RofA and cpa) within the island. Schematics of the GAS adhesin islands are set forth in FIGURE 51A and FIGURE 162. "GAS Adhesin Island-1 or "GAS AI- 1" comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-I proteins"). GAS AI-I preferably comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator. GAS AI-I surface proteins may include a fibronectin binding protein, a collagen adhesion protein and a fϊmbrial structural subunit. The fϊmbrial structural subunit (also known as teeό) is thought to form the shaft portion of the pilus like structure, while the collagen adhesion protein (Cpa) is thought to act as an accessory protein facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule.
Specifically, GAS AI-I includes polynucleotide sequences encoding for two or more of M6_SpyO157, M6_SpyO158, M6_SpyO159, M6_SpyO16O, M6_SpyO161. The GAS AI-I may also include polynucleotide sequences encoding for any one of CDC SS 410_fϊmbrial, ISS3650_fimbrial, DSM2071Jϊmbrial
A preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. The immunogenic composition of the invention may alternatively comprise an isolated GAS AI-I surface protein in oligomeric (pilus) form. The oligomer or hyperoHgomeric pilus structures comprising GAS AI-I surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
One or more of the GAS AI-I polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-I open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
One or more of the GAS AI-I surface proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI- 1 may enojOide
Figure imgf000010_0001
GAS AI-I may encode for at least two surface proteins and at least one sortase. Preferably, GAS AI-I encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif.
GAS AI-I preferably includes a srtB sortase. GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
The GAS AI-I protein of the composition may be selected from the group consisting of M6_SpyO157, M6_SpyO158, M6_SpyO159, M6_Spy0160 M6_Spy0161, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fϊmbrial. GAS AI-I surface proteins M6_SρyO157 (a fibronectin binding protein), M6_SρyO159 (a collagen adhesion protein, Cpa), M6_Spy0160 (a fimbrial structural subunit, teeό), CDC SS 410_fimbrial (a fimbrial structural subunit), ISS3650_fϊmbrial (a fimbrial structural subunit), and DSM2071_fimbrial (a fimbrial structural subunit) are preferred GAS AI-I proteins for use in the immunogenic compositions of the invention. The fimbrial structural subunit tee6 and the collagen adhesion protein Cpa are preferred GAS AI -1 surface proteins. Preferably, each of these GAS AI-I surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with serine).
In addition to the open reading frames encoding the GAS AI-I proteins, GAS AI-I may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the GAS AI protein open reading frames, but it transcribed in the opposite direction).
The GAS AI-I surface proteins may be used alone, in combination with other GAS AI-I surface proteins or in combination with other GAS AI surface proteins. Preferably, the immunogenic compositions of the invention include the GAS AI-I fimbrial structural subunit (teeό) and the GAS AI-I collagen binding protein. Still more preferably, the immunogenic compositions of the invention include the GAS AI-I fimbrial structural subunit (teeό).
A second GAS adhesion island, "GAS Adhesin Island-2" or "GAS AI-2," has also been identified in GAS serotypes. Amino acid sequences encoded by the open reading frames of GAS AI- 2 may also be used in immunogenic compositions for the treatment or prevention of GAS infection. A preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-2 surface protein in oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising GAS AI-2 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
GAS AI-2 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-2 proteins"). Gjf iSfAT-p pϊ-φU?©d|pip'ri|s;;ilif|kc:| ©steins, a srtB sortase, a srtCl sortase and a rofA divergently transcribed transcriptional regulator.
Specifically, GAS AI-2 includes polynucleotide sequences encoding for two or more of GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SpyO133, and GAS20. One or more of the GAS AI-2 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-2 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
One or more of the GAS AI-2 surface proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-2 may encode for at least one surface protein. Alternatively, GAS AI-2 may encode for at least two surface proteins and at least one sortase. Preferably, GAS AI-2 encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif. GAS AI-2 preferably includes a srtB sortase and a srtCl sortase. As discussed above, GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine. GAS srtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO: 167) motif. GAS srtCl may be differentially regulated by rofA. The GAS AI-2 protein of the composition may be selected from the group consisting of
GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SpyO133, and GAS20. GAS AI-2 surface proteins GAS15 (Cpa), GAS16 (thought to be a fimbrial protein, Ml_128), GAS18 (Ml_Spy0130), and GAS20 are preferred for use in the immunogenic compositions of the invention. GAS 16 is thought to form the shaft portion of the pilus like structure, while GAS 15 (the collagen adhesion protein Cpa) and GAS 18 are thought to act as accessory proteins facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule. Preferably, each of these GAS AI-2 surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VVXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
In addition to the open reading frames encoding the GAS AI-2 proteins, GAS AI-2 may also include a divergently transcribed transcriptional regulator such as rofA {i.e., the transcriptional regulator is located near or adjacent to the GAS AI protein open reading frames, but it transcribed in the opposite direction). The GAS AI-2 surface proteins may be used alone, in combination with other GAS AI-2 surface proteins or in combination with other GAS AI surface proteins. Preferably, the immunogenic compositions of the invention include the GAS AI-2 fimbrial protein (GAS 16), the GAS AI-2 collagen binding protein (GAS 15) and GAS 18 (Ml_SpyO13O). More preferably, the immunogenic compositions of the invention include the GAS AI-2 fimbrial protein (GAS 16). A third GAS adhesion island, "GAS Adhesin Island-3" or "GAS AI-3," has also been identified in numerous GAS serotypes. Amino acid sequences encoded by the open reading frames of
Figure imgf000012_0001
compositions for the treatment or prevention of GAS infection.
A preferred immunogenic composition of the invention comprises a GAS AI-3 surface protein which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-3 surface protein in oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising GAS AI-3 surface proteins may be purified or otherwise formulated for use in immunogenic compositions. GAS AI-3 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-3 proteins"). GAS AI-3 preferably comprises surface proteins, a srtC2 sortase, and a Negative transcriptional regulator (Nra) divergently transcribed transcriptional regulator. GAS AI-3 surface proteins may include a collagen binding protein, a fϊmbrial protein, and a F2 like fibronectin-binding protein. GAS AI-3 surface proteins may also include a hypothetical surface protein. The fϊmbrial protein is thought to form the shaft portion of the pilus like structure, while the collagen adhesion protein (Cpa) and the hypothetical surface protein are thought to act as accessory proteins facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule. Preferred AI-3 surface proteins include the fimbrial proein, the collagen binding protein and the hypothetical protein. Preferably, each of these GAS AI-3 surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
Specifically, GAS AI-3 includes polynucleotide sequences encoding for two or more of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SpsOlOO, SpsOlOl, Sps0102, Sps0103, Sps0104, Sps0105, SpsO106, orf78, orf79, orf80, orfSl, orf82, orf83, orf84, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_O13O, spyM18_0131, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoMO 1000152, SpyoM01000151, SpyoMO 1000150, SpyoMO 1000149, ISS3040_fimbrial, ISS3776_fϊmbrial, and ISS4959_fimbrial. In one embodiment, GAS AI-3 may include open reading frames encoding for two or more of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of SpsOlOO, SpsOlOl, Sps01O2, Sps0103, Sps0104, Sps0105, and SpsOlOό. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of orf78, orf79, orfSO, orfδl, orf82, orf83, and orf84. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of sρyM18_0126, spyM18_0127, sρyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and SpyoM01000149. Alternatively, GAS AI-I may also include polynucleotide sequences encoding for any one of ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. |j',"ϋ £* "prje" §i)|i@it|'|!c|fj|t:l)£i'<iΑ'^i''4,;Ef1;;ϊ'δI!|τiucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-3 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF. One or more of the GAS AI-3 surface proteins typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-3 may encode for at least one surface protein. Alternatively, GAS AI-3 may encode for at least two surface proteins and at least one sortase. Preferably, GAS AI-3 encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif.
GAS AI-3 preferably includes a srtC2 type sortase. GAS srtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail. GAS SrtC2 may be differentially regulated by Nra. The GAS AI-3 protein of the composition may be selected from the group consisting of
SpyM3_O098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SpsOlOO, SpsOlOl, Sps0102, Sps0103, Sps0104, Sps0105, Sps0106, orf78, orf79, orf80, orfδl, orf82, orf83, orf84, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_O130, spyM18_0131, spyM18_0132, SpyoM01000156, SpyoMO 1000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151, SpyoMO 1000150, SpyoMO 1000149, ISS3040_fimbrial, ISS3776_fϊmbrial, and ISS4959_fimbrial. GAS AI-3 surface proteins SpyM3_O098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPsOlOo, orf78, orf80, orf82, orf84, spyM18_0126, spyM18_0128, spyM18J)130, spyM18_0132, SpyoM01000155, SpyoMO 1000153, SpyoMO 1000151, SpyoMO 1000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial are preferred GAS AI-3 proteins for use in the immunogenic compositions of the invention.
In addition to the open reading frames encoding the GAS AI-3 proteins, GAS AI-3 may also include a transcriptional regulator such as Nra.
GAS AI-3 may also include a LepA putative signal peptidase I protein. The GAS AI-3 surface proteins may be used alone, in combination with other GAS AI-3 surface proteins or in combination with other GAS AI surface proteins. Preferably, the immunogenic compositions of the invention include the GAS AI-3 fimbrial protein, the GAS AI-3 collagen binding protein, the GAS AI-3 surface protein (such as SρyM3_0102, M3_Sps0104, M5_orf82, or spyM18_O130), and fibronectin binding protein PrtF2. More preferably, the immunogenic compositions of the invention include the GAS AI-3 fimbrial protein, the GAS AI-3 collagen binding protein, and the GAS AI-3 surface protein. Still more preferably, the immunogenic compositions of the invention include the GAS AI-3 fimbrial protein. p £ '"p:§firybSWlM::feώliJ§§'tf!tB l&S AI-3 fimbrial protein include SpyM3_0100, M3_Sps0102, M5_orf80, spyM18_128, SpyoM01000153, ISS3040_fimbrial, ISS3776_fimbrial, ISS4959Jimbrial.
Representative examples of the GAS AI-3 collagen binding protein include SpyM3_0098, M3_Sρs0100, M5_orf 78, sρyM18_0126, and SpyoMO 1000155.
Representative examples of the GAS AI-3 fibronectin binding protein PrtF2 include SpyM3_0104, M3_Sps0106, M5_orf84 and spyM18_0132, and SpyoMO 1000149.
A fourth GAS adhesion island, "GAS Adhesin Island-4" or "GAS AI-4," has also been identified in GAS serotypes. Amino acid sequences encoded by the open reading frames of GAS AI- 4 may also be used in immunogenic compositions for the treatment or prevention of GAS infection.
A preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-4 surface protein in oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising GAS AI-3 surface proteins may be purified or otherwise formulated for use in immunogenic compositions. The oligomeric or hyperoligomeric pilus structures comprising GAS AI-4 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
GAS AI-4 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-4 proteins"). This GAS adhesin island 4 ("GAS AI-4") comprises surface proteins, a srtC2 sortase, and a RofA regulatory protein. GAS AI-4 surface proteins within may include a fimbrial protein, Fl and F2 like fibronectin-binding proteins, and a capsular polysaccharide adhesion protein (cpa). GAS AI-4 surface proteins may also include a hypothetical surface protein in an open reading frame (orf). The fϊmbral protein (EftLSL) is thought to form the shaft portion of the pilus like structure, while the collagen adhesion protein (Cpa) and the hypothetical protein are thought to act as accessory proteins facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule. Preferably, each of these GAS AI-4 surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
Specifically, GAS AI-4 includes polynucleotide sequences encoding for two or more of 19224134, 19224135, 19224136, 19224137, 19224138, 19224139, 19224140, and 19224141. A GAS AI-4 polynucleotide may also include polynucleotide sequences encoding for any one of 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, ISS4538_fimbrial. One or more of the GAS AI-4 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-4 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF. p C 'Ψ≠'M&EύB1≠'<®W4M$M∞e proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-4 may encode for at least one surface protein. Alternatively, GAS AI-4 may encode for at least two surface proteins and at least one sortase. Preferably, GAS AI-4 encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif.
GAS AI-4 includes a SrtC2 type sortase. GAS SrtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail. The GAS AI-4 protein of the composition may be selected from the group consisting of
19224134, 19224135, 19224136, 19224137, 19224138, 19224139, 19224140, 19224141, 20010296_fϊmbrial, 20020069 jSmbrial, CDC SS 635_fimbrial, ISS4883_fϊmbrial, and ISS4538_fimbrial. GAS AI-4 surface proteins 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069__fimbrial, CDC SS 635_fϊmbrial, ISS4883_fnnbrial, ISS4538_fimbrial are preferred proteins for use in the immunogenic compositions of the invention.
In addition to the open reading frames encoding the GAS AI-4 proteins, GAS AI-4 may also include a divergently transcribed transcriptional regulator such as RofA {i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction. GAS AI-4 may also include a LepA putative signal peptidase I protein and a MsmRL protein.
The GAS AI-4 surface proteins may be used alone, in combination with other GAS AI-4 surface proteins or in combination with other GAS AI surface proteins. Preferably, the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein (EftLSL or 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, or ISS4538_fimbrial), the GAS AI-4 collagen binding protein, the GAS AI-4 surface protein (such as M12 isolate A735 orf 2), and fibronectin binding protein PrtFl and PrtF2. More preferably, the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein, the GAS AI-4 collagen binding protein, and the GAS AI-4 surface protein. Still more preferably, the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein. The GAS AI proteins of the invention maybe used in immunogenic compositions for prophylactic or therapeutic immunization against GAS infection. For example, the invention may include an immunogenic composition comprising one or more GAS Al- 1 proteins and one or more of any of GAS AI-2, GAS AI-3, or GAS AI-4 proteins. For example, the invention includes an immunogenic composition comprising at least two GAS AI proteins where each protein is selected from a different GAS adhesin island. The two GAS AI proteins may be selected from one of the following GAS AI combinations: GAS AM and GAS AI-2; GAS AI-I and GAS AI-3; GAS AI-I and GAS AI-4; GAS AI-2 and GAS AI-3; GAS AI-2 and GAS AI-4; and GAS AI 3 and GAS AI-4.
Preferably the combination includes fimbrial proteins from one or more GAS adhesin islands. F" C 'T^'ϊSΛMdySiie'ciβϊni&33iαϊisi'iiay also be selected to provide protection against an increased range of GAS serotypes and strain isolates. For example, the immunogenic composition may comprise a first and second GAS AI protein, wherein a full length polynucleotide sequence encoding for the first GAS AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second GAS AI protein. In addition, each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple GAS serotypes and strain isolates. Preferably, each antigen is present in the genomes of at least two (i.e., 3,
4, 5, 6, 7, 8, 9, 10, or more) GAS strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5, or more) GAS serotypes. Applicants have also identified adhesin islands within the genome of Streptococcus pneumoniae. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence. Amino acid sequence encoded by such S. pneumoniae Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of S. pneumoniae infection. Preferred immunogenic compositions of the invention comprise a S. pneumoniae AI surface protein which has been formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. A preferred immunogenic composition of the invention alternatively comprises an isolated S. pneumoniae surface protein in oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising S. pneumoniae surface proteins may be purified or otherwise formulated for use in immunogenic compositions. The S. pneumoniae Adhesin Islands generally include a series of open reading frames within a
5. pneumoniae genome that encode for a collection of surface proteins and sortases. A S. pneumoniae Adhesin Island may encode for an amino acid sequence comprising at least one surface protein. Alternatively, the S. pneumoniae Adhesin Island may encode for at least two surface proteins and at least one sortase. Preferably, a S. pneumoniae Adhesin Island encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPTXG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or more S. pneumoniae AI surface proteins may participate in the formation of a pilus structure on the surface of the S. pneumoniae bacteria.
The S. pneumoniae Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator. The transcriptional regulator may regulate the expression of the S. pneumonaie AI operon. An example of a transcriptional regulator found in S. pneumoniae AI sequences is HrA.
A schematic of the organization of a S. pneumoniae AI locus is provided in Figure 137. The locus comprises open reading frames encoding a transcriptional regulator (rlrA), cell wall surface proteins (rrgA, rrgB, rrgC) and sortases (srt B, srtC, srtD).
S. pneumoniae AI sequences may be generally divided into two groups of homology, S. pneuamoniae AI-a and AI-b. S. pneumoniae strains that comprise AI-a include 14 CSR 10, 19A 12, and 6B Spain 2. S. pneumoniae AI strains that
Figure imgf000017_0001
comprise AI-b include 19F Taiwan 14, 9V Spain 3, 23F Taiwan 15 and TIGR 4.
S. pneumoniae AI from TIGR4 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S1. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from TIGR4 includes polynucleotide sequences encoding for two or more of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, and SP0468.
One or more of the & pneumoniae AI from TIGR4 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the iS1. pneumoniae AI from TIGR4 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
S. pneumoniae strain 670 AI comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("_?. pneumoniae AI proteins"). Specifically, S. pneumoniae strain 670 AI includes polynucleotide sequences encoding for two or more of orfl_670, orβ_670, orf4__670, orf5_670, orf6_670, orf7_670, and orf8_670.
One or more of the 5. pneumoniae strain 670 AI polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the iS. pneumoniae strain 670 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
S. pneumoniae AI from 14 CSRlO comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 14 CSRlO includes polynucleotide sequences encoding for two or more of ORF2 14CSR, ORF3J4CSR, ORF4_14CSR, ORF5__14CSR, ORF6J4CSR, ORF7_14CSR, and ORF8_14CSR.
One or more of the S. pneumoniae AI from 14 CSRlO polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 14 CSRlO open reading frames may be replaced by a sequence having sequence homology to the replaced ORF. S. pneumoniae AI from 19A Hungary 6 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 19A Hungary 6 includes polynucleotide sequences encoding for two or more of ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5J9AH, ORF6_19AH, ORF7_19AH, and ORF8_19AH. One or more of the S. pneumoniae AI from 19A Hungary 6 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 19A Hungary 6 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF. if"" C Υ-
Figure imgf000018_0001
14 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (US. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 19F Taiwan 14 includes polynucleotide sequences encoding for two or more of ORF2_19FTW, ORF3J9FTW, ORF4_19FTW, ORF5J9FTW, ORF6J9FTW, ORF7_19FTW, and ORF8J9FTW.
One or more of the S. pneumoniae AI from 19F Taiwan 14 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 19F Taiwan 14 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF. S. pneumoniae AI from 23F Poland 16 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 23F Poland 16 includes polynucleotide sequences encoding for two or more of ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, and ORF8_23FP. One or more of the S. pneumoniae AI from 23F Poland 16 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 23F Poland 16 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
S. pneumoniae AI from 23F Taiwan 15 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, 61. pneumoniae AI from 23F Taiwan 15 includes polynucleotide sequences encoding for two or more of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORP6_23FTW, ORF7_23FTW, and ORF8_23FTW.
One or more of the S. pneumoniae AI from 23F Taiwan 15 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 23F Taiwan 15 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
S. pneumoniae AI from 6B Finland 12 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 6B Finland 12 includes polynucleotide sequences encoding for two or more of ORF2 6BF, ORF3_6BF, ORF4 6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, and ORF8_6BF.
One or more of the S. pneumoniae AI from 6B Finland 12 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 6B Finland 12 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
S. pneumoniae AI from 6B Spain 2 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ('|S ©ei^Hye!iHiSeM^a.';;SispSdiily, S. pneumoniae AI from 6B Spain 2 includes polynucleotide sequences encoding for two or more of ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP, and ORF8_6BSP.
One or more of the S. pneumoniae AI from 6B Spain 2 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 6B Spain 2 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
S. pneumoniae Al from 9V Spain 3 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("iS1. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from 9V Spain 3 includes polynucleotide sequences encoding for two or more of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP, and ORF8_9VSP.
One or more of the S. pneumoniae AI from 9V Spain 3 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 9V Spain 3 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae AI surface proteins typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae AI may encode for at least one surface protein. The Adhesin Island, may encode at least one surface protein. Alternatively, S. pneumoniae AI may encode for at least two surface proteins and at least one sortase. Preferably, S. pneumoniae AI encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif.
The S. pneumoniae AI protein of the composition may be selected from the group consisting of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, SP0468, orfl_670, orf3_670, orf4_670, orf5_670, orf6_670, orf7_670, orf8_670, ORF2_14CSR, ORF3 14CSR, ORF4_14CSR, ORF5J4CSR, ORF6J4CSR, ORF7J4CSR, ORF8_14CSR, ORF2_19AH, ORF3_19AH, ORF4J9AH, ORF5_19AH, ORF6J9AH, ORF7_19AH, ORF8J9AH, ORF2_19FTW, ORF3J9FTW, ORF4_19FTW, ORF5J9FTW, ORF6J9FTW, ORF7_19FTW, ORF8J9FTW, ORF2 23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, ORF8_23FP, ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, ORF8_23FTW, ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, ORF8_6BF, ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP, ORF8_6BSP, ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP and, ORF8_9VSP.
S. pneumoniae AI surface proteins are preferred proteins for use in the immunogenic compositions of the invention. In one embodiment, the compositions of the invention comprise combinations of two or more S pneumoniae AI surface proteins. Preferably such combinations are sftefddFrp{H!t^i;€lffiφ'biffiel'gi;;6ij,pcflhsisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF3_19AH, ORF4_19AH, ORF5J9AH, ORF3J9FTW, ORF4_19FTW, ORF5_19FTW, ORF3_23FP, ORF4 23FP, ORF5_23FP, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF3_6BF, ORF4_6BF, ORF5J5BF, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF3_9VSP, ORF4_9VSP, and ORF5_9VSP.
In addition to the open reading frames encoding the S. pneumoniae AI proteins, S. pneumoniae AI may also include a transcriptional regulator.
The S. pneumoniae AI proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against S. pneumoniae infection. For example, the invention may include an immunogenic composition comprising one or more S. pneumoniae from TIGR4 AI proteins and one or more S. pneumoniae strain 670 proteins. The immunogenic composition may comprise one or more AI proteins from any one or more of S. pneumoniae strains TIGR4, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, 23F Poland 16, and 670. The immunogenic compositions may also be selected to provide protection against an increased range of S. pneumoniae serotypes and strain isolates. For example, the immunogenic composition may comprise a first and second S. pneumoniae AI protein, wherein a full length polynucleotide sequence encoding for the first S. pneumoniae AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second S. pneumoniae AI protein. In addition, each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple S. pneumoniae serotypes and strain isolates. Preferably, each antigen is present in the genomes of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) S. pneumoniae strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5, or more) S. pneumoniae serotypes. The immunogenic compositions may also be selected to provide protection against an increased range of serotypes and strain isolates of a Gram positive bacteria. For example, the immunogenic composition may comprise a first and second Gram positive bacteria AI protein, wherein a full length polynucleotide sequence encoding for the first Gram positive bacteria AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second Gram positive bacteria AI protein. In addition, each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple serotypes and strain isolates of the Gram positive bacteria. Preferably, each antigen is present in the genomes of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram positive bacteria strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3, 4, 5, or more) Gram positive bacteria serotypes.One or both of the first and second AI proteins may preferably be in oligomeric or hyperoligomeric form. Adhesin island surface proteins from two or more Gram positive bacterial genus or species may be combined to provide an immunogenic composition for prophylactic or therapeutic treatment ofecjji|easp"o,r'i|ifee|i)f| i&|t^qj;ito'^ej::©faKri:^ositive bacterial genus or species. Optionally, the adhesin island surface proteins may be associated together in an oligomeric or hyperoligomeric structure.
In one embodiment, the invention comprises adhesin island surface proteins from two or more Streptococcus species. For example, the invention includes a composition comprising a GBS AI surface protein and a GAS adhesin island surface protein. As another example, the invention includes a composition comprising a GAS adhesin island surface protein and a S. pneumoniae adhesin island surface protein. One or both of the GAS AI surface protein and the S. pneumoniae AI surface protein may be in oligomeric or hyperoligomeric form. As a further example, the invention includes a composition comprising a GBS adhesin island surface protein and a S. pneumoniae adhesin island surface protein.
In one embodiment, the invention comprises an adhesin island surface protein from two or more Gram positive bacterial genus. For example, the invention includes a composition comprising a Streptococcus adhesin island protein and a Corynebacterium adhesin island protein. One or more of the Gram positive bacteria AI surface proteins may be in an oligomeric or hyperoligomeric form. In addition, the AI polynucleotides and amino acid sequences of the invention may also be used in diagnostics to identify the presence or absence of GBS (or a Gram positive bacteria) in a biological sample. They may be used to generate antibodies which can be used to identify the presence of absence of an AI protein in a biological sample or in a prophylactic or therapeutic treatment for GBS (or a Gram positive bacterial) infection. Further, the AI polynucleotides and amino acid sequences of the invention may also be used to identify small molecule compounds which inhibit or decrease the virulence associated activity of the AI.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 presents a schematic depiction of Adhesin Island 1 ("AI-I") comprising open reading frames for GBS 80, GBS 52, SAG0647, SAG0648 and GBS 104. FIGURE 2 illustrates the identification of AI-I sequences in several GBS serotypes and strain isolates (GBS serotype V, strain isolate 2603; GBS serotype III, strain isolate nem316; GBS serotype II, strain isolate 18RS21; GBS serotype V, strain isolate CJBl I l; GBS serotype III, strain isolate COHl and GBS serotype Ia, strain isolate A909). (An AI-I was not identified in GBS serotype Ib, strain isolate H36B or GBS serotype Ia, strain isolate 515). FIGURE 3 presents a schematic depiction of the correlation between AI-I and the Adhesin
Island 2 ("AI-2") within the GBS serotype V, strain isolate 2603 genome. (This AI-2 comprises open reading frames for GBS 67, GBS 59, SAG1406, SAG1405 and GBS 150).
FIGURE 4 illustrates the identification of AI-2 comprising open reading frames encoding for GBS 67, GBS 59, SAG1406, SAG1404 and GBS 150 (or sequences having sequence homology thereto) in several GBS serotypes and strain isolates (GBS serotype V, strain isolate 2603; GBS serotype III, strain isolate NEM316; GBS serotype Ib, strain isolate H36B; GBS serotype V, strain isolate CJBl 11; GBS serotype II, strain isolate 18RS21; and GBS serotype Ia, strain isolate 515).
Figure 4 also illustrates the identification of AI-2 comprising open reading frames encoding for 01520 (f SC^VO ΪJ2B OSS2.,(aior:tasi|,i|r©3 (spbl), 01524 and 01525 (or sequences having sequence homology thereto).
FIGURE 5 presents data showing that GBS 80 binds to fibronectin and fibrinogen in ELISA.
FIGURE 6 illustrates that all genes in AI-I are co-transcribed as an operon. FIGURE 7 presents schematic depictions of in-frame deletion mutations within AI-I .
FIGURE 8 presents FACS data showing that GBS 80 is required for surface localization of GBS 104.
FIGURE 9 presents FACS data showing that sortases SAG0647 and SAG0648 play a semi- redundant role in surface exposure of GBS 80 and GBS 104. FIGURE 10 presents Western Blots of the in-frame deletion mutants probed with anti-GBS80 and anti-GBS 104 antisera.
FIGURE 11: Electron micrograph of surface exposed pili structures in Streptococcus agalactiae containing GBS 80.
FIGURE 12: PHD predicted secondary structure of GBS 067. FIGURE 13, 14 and 15: Electron micrograph of surface exposed pili structures of strain isolate COHl of Streptococcus agalactiae containing a plasmid insert encoding GBS 80.
FIGURE 16 and 17 : Electron micrograph of surface exposed pili structure of wild type strain isolate COHl of Streptococcus agalactiae.
FIGURE 18: Alignment of polynucleotide sequences of Al-I from serotype V, strain isolates 2603 and CJB 111; serotype II, strain isolate 18RS21 ; serotype III, strain isolates COHl and NEM316; and serotype Ia, strain isolate A909.
FIGURE 19: Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolates 2603 and CJBl 11; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype Ia, strain isolate 515. FIGURE 20: Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolate
2603 and serotype III, strain isolate NEM316.
FIGURE 21: Alignment of polynucleotide sequences of AI-2 from serotype III, strain isolate COHl and serotype Ia, strain isolate A909.
FIGURE 22: Alignment of amino acid sequences of AI-I surface protein GBS 80 from serotype V, strain isolates 2603 and CJBl 11; serotype Ia, strain isolate A909; serotype III, strain isolates COHl and NEM316.
FIGURE 23: Alignment of amino acid sequences of AI-I surface protein GBS 104 from serotype V, strain isolates 2603 and CJB 111; serotype III, strain isolates COHl and NEM316; and serotype II, strain isolate 18RS21. FIGURE 24: Alignment of amino acid sequences of AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJBl 11; serotype Ia, strain isolate 515; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype III, strain isolate NEM316. IJ;;;:! £;, "f ipillfljl:]!
Figure imgf000023_0001
GjpSjclosely associates with tight junctions and cross the monolayer of MEl 80 cervical epithelial cells by a paracellular route.
FIGURE 26: Illustrates GBS infection of MEl 80 cells.
FIGURE 27: Illustrates that GBS 80 recombinant protein does not bind to epithelial cells. FIGURE 28: Illustrates that deletion of GBS 80 does not effect the capacity of GBS strain
2603 V/R to adhere and invade MEl 80 cervical epithelial cells.
FIGURE 29: Illustrates binding of recombinant GBS 104 protein to epithelial cells.
FIGURE 30: Illustrates that deletion of GBS 104 in the GBS strain COHl, reduces the capacity of GBS to adhere to MEl 80 cervical epithelial cells. FIGURE 31 : Illustrates that GBS 80 knockout mutant strain partially loses the ability to translocate through an epithelial cell monolayer.
FIGURE 32: Illustrates that deletion of GBS 104, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cell line.
FIGURE 33: Illustrates that GBS 104 knockout mutant strain translocates through an epithelial monolayer less efficiently than the isogenic wild type.
FIGURE 34: Negative stained electron micrographs of GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80.
FIGURE 35: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles).
FIGURE 36: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles).
FIGURE 37: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 n m gold particles).
FIGURE 38: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 104 antibodies or preimmune sera (visualized with 10 nm gold particles). FIGURE 39: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles) and anti-GBS 104 antibodies (visualized with 10 nm gold particles).
FIGURE 40: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles) and anti-GBS 104 antibodies (visualized with 10 nm gold particles). p C "f 1,0XlMIQ Sϊxjst'rgK:rø <3rø8O is necessary for polymer formation and GBS 104 and sortase SAG0648 are necessary for efficient assembly of pili.
FIGURE 42: Illustrates that GBS 67 is part of a second pilus and that GBS 80 is polymerized in strain 515. FIGURE 43: Illustrates that two macro-molecules are visible in Cohl, one of which is the
GBS 80 pilin.
FIGURE 44: Illustrates pilin assembly.
FIGURE 45: Illustrates that GBS 52 is a minor component of the GBS pilus. FIGURE 46: Illustrates that the pilus is found in the supernatant of a bacterial culture. FIGURE 47: Illustrates that the pilus is found in the supernatant of bacterial cultures in all phases.
FIGURE 48: Illustrates that in Cohl, only the GBS 80 protein and one sortase (sagO647 or sagO648) is required for polymerization.
FIGURE 49: IEM image of GBS 80 staining of a GBS serotype VIII strain JM9030013 that express pili.
FIGURE 50: IEM image of GBS 104 staining of a GBS serotype VIII strain JM9030013 that express pili.
FIGURE 5 IA: Schematic depiction of open reading frames comprising a GAS AI-2 serotype Ml isolate, GAS AI-3 serotype M3, M5, M18, and M49 isolates, a GAS AI-4 serotype M12 isolate, and an GAS AI-I serotype M6 isolate.
FIGURE 51B: Amino acid alignment of SrtCl-type sortase of a GAS AI-2 serotype Ml isolate, SrtC2-type sortases of serotype M3, M5, M18, and M49 isolates, and a SrtC2-type sortase of a GAS AI-4 serotype M12 isolate.
FIGURE 52: Amino acid alignment of the capsular polysaccharide adhesion proteins of GAS AI-4 serotype M12 (A735), GAS AI-3 serotype M5 (Manfredo), S. pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI-I serotype M3, S. pyogenes strain MGAS8232 serotype M3, and GAS AI-2 serotype Ml.
FIGURE 53: Amino acid alignment of F-like fibronectin-binding proteins of GAS AI-4 serotype M12 (A735) and S. pyogenes strain MGAS10394 serotype M6. FIGURE 54: Amino acid alignment of F2-like fibronectin-binding proteins of GAS AI-4 serotype M12 (A735), S. pyogenes strain MGAS8232 serotype M3, GAS AI-3 strain M5 (Manfredo), S. pyogenes strain SSI serotype M3, and S. pyogenes strain MGAS315 serotype M3.
FIGURE 55: Amino acid alignment of fimbrial proteins of GAS AI-4 serotype M12 (A735), GAS AI-3 serotype M5 (Manfredo), S. pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI serotype M3, S. pyogenes strain MGAS 8232 serotype M3, and S. pyogenes Ml GAS serotype Ml. jp. I1;1;; "|I91UB!03Cllliti.ill(S<iU;a gS#>t of hypothetical proteins of GAS AI-4 serotype Ml 2 (A735), S. pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI-I serotype M3, GAS AI-3 serotype M5 (Manfredo), and S. pyogenes strain MGAS8232 serotype M3.
FIGURE 57: Results of FASTA homology search for amino acid sequences that align with the collagen adhesion protein of GAS AI- 1 serotype M6 (MGAS 10394) .
FIGURE 58: Results of FASTA homology search for amino acid sequences that align with the fimbrial structural subunit of GAS AI-I serotype M6 (MGAS 10394).
FIGURE 59: Results of FASTA homology search for amino acid sequences that align with the hypothetical protein of GAS AI-2 serotype Ml (SF370). FIGURE 60: Specifies pilin and E box motifs present in GAS type 3 and 4 adhesin islands.
FIGURE 61: Illustrates that surface expression of GBS 80 protein on GBS strains COH and JM9130013 correlates with formation of pili structures. Surface expression of GBS 80 was determined by FACS analysis using an antibody that cross-hybridizes with GBS 80. Formation of pili structures was determined by immunogold electron microscopy using gold-labelled anti-GBS 80 antibody.
FIGURE 62: Illustrates that surface exposure is capsule-dependent for GBS 322 but not for GBS 80.
FIGURE 63: Illustrates the amino acid sequence identity of GBS 59 proteins in GBS strains. FIGURE 64: Western blotting of whole GBS cell extracts with anti-GBS 59 antibodies. FIGURE 65: Western blotting of purified GBS 59 and whole GBS cell extracts with anti-
GBS 59 antibodies.
FIGURE 66: FACS analysis of GBS strains CJBl 11, 7357B, 515 using GBS 59 antiserum. FIGURE 67: Illustrates that anti-GBS 59 antibodies are opsonic for CJBl 11 GBS strain serotype V. FIGURE 68: Western blotting of GBS strain JM9130013 total extracts.
FIGURE 69: Western blotting of GBS stain 515 total extracts shows that GBS 67 and GBS 150 are parts of a pilus.
FIGURE 70: Western blotting of GBS strain 515 knocked out for GBS 67 expression FIGURE 71: FACS analysis of GBS strain 515 and GBS strain 515 knocked out for GBS 67 expression using GBS 67 and GBS 59 antiserum.
FIGURE 72: Illustrates complementation of GBS 515 knocked out for GBS 67 expression with a construct overexpressing GBS 80.
FIGURE 73: FACS analysis of GAS serotype M6 for spyM6_0159 surface expression. FIGURE 74: FACS analysis of GAS serotype M6 for spyM6_0160 surface expression. FIGURE 75: FACS analysis of GAS serotype Ml for GAS 15 surface expression.
FIGURE 76: FACS analysis of GAS serotype Ml for GAS 16 surface expression using a first anti-GAS 16 antiserum. P C ''Hø'ϋDMMl
Figure imgf000026_0001
serotype Ml for GAS 18 surface expression using a first anti-GAS 18 antiserum.
FIGURE 78: FACS analysis of GAS serotype Ml for GAS 18 surface expression using a second anti-GAS 18 antiserum. FIGURE 79: FACS analysis of GAS serotype Ml for GAS 16 surface expression using a second anti-GAS 16 antisera.
FIGURE 80: FACS analysis of GAS serotype M3 for spyM3_0098 surface expression. FIGURE 81: FACS analysis of GAS serotype M3 for spyM3_0100 surface expression. FIGURE 82: FACS analysis of GAS serotype M3 for spyM3_0102 surface expression. FIGURE 83: FACS analysis of GAS serotype M3 for spyM3_0104 surface expression.
FIGURE 84: FACS analysis of GAS serotype M3 for spyM3_0106 surface expression. .FIGURE 85: FACS analysis of GAS serotype M12 for 19224134 surface expression. FIGURE 86: FACS analysis of GAS serotype M12 for 19224135 surface expression. FIGURE 87: FACS analysis of GAS serotype M12 for 19224137 surface expression. FIGURE 88: FACS analysis of GAS serotype M12 for 19224141 surface expression.
FIGURE 89: Western blot analysis of GAS 15 expression on GAS Ml bacteria. FIGURE 90: Western blot analysis of GAS 15 expression using GAS 15 immune sera. FIGURE 91: Western blot analysis of GAS 15 expression using GAS 15 pre-immune sera. FIGURE 92: Western blot analysis of GAS 16 expression on GAS Ml bacteria. FIGURE 93 : Western blot analysis of GAS 16 expression using GAS 16 immune sera.
FIGURE 94: Western blot analysis of GAS 16 expression using GAS 16 pre-immune sera. FIGURE 95: Western blot analysis of GAS 18 on GAS Ml bacteria. FIGURE 96: Western blot analysis of GAS 18 using GAS 18 immune sera. FIGURE 97: Western blot analysis of GAS 18 using GAS 18 pre-immune sera. FIGURE 98: Western blot analysis of M6_SρyO159 expression on GAS bacteria.
FIGURE 99: Western blot analysis of 19224135 expression on M12 GAS bacteria. FIGURE 100: Western blot analysis of 19224137 expression on M12 GAS bacteria. FIGURE 101 : Full length nucleotide sequence of an S. pneumoniae strain 670 AL FIGURE 102: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 2580.
FIGURE 103: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 2913.
FIGURE 104: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 3280. FIGURE 105: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain
3348.
FIGURE 106: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain
2719. p C "T I<3t|M;il©:!:;KesIK;BQ Srøsis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain SF370.
FIGURE 108: Western blot analysis of 19224135 and 19224137 in GAS M12 strain 2728. FIGURE 109: Western blot analysis of 19224139 in GAS M12 strain 2728 using antisera raised against SpyM3_0102.
FIGURE 110: Western blot analysis of M6_SpyO159 and M6_Spy0160 in GAS M6 strain 2724.
FIGURE 111: Western blot analysis of M6_SpyO 159 and M6_SpyO 160 in GAS M6 strain SF370. FIGURE 112: Western blot analysis of M6_Spyl60 in GAS M6 strain 2724.
FIGURES 113-115: Electron micrographs of surface exposed GAS 15 on GAS Ml strain SF370.
FIGURES 116-121: Electron micrographs of surface exposed GAS 16 on GAS Ml strain SF370. FIGURES 122-125: Electron micrographs of surface exposed GAS 18 on GAS Ml strain
SF370 detected using anti-GAS 18 antisera.
FIGURE 126: IEM image of a hyperoligomer on GAS Ml strain SF370 detected using anti- GAS 18 antisera.
FIGURES 127-132: IEM images of oligomeric and hyperoligomeric structures containing M6_Spy0160 extending from the surface of GAS serotype M6 3650.
FIGURE 133A and B: Western blot analysis of L. lactis transformed to express GBS 80 with anti-GBS 80 antiserum.
FIGURES 134: Western blot analyses of L. lactis transformed to express GBS AI-I with anti-GBS 80 antiserum. FIGURE 135: Ponceau staining of same acrylamide gel as used in Figure 134.
FIGURE 136A: Western blot analysis of sonicated pellets and supematants of cultured L. lactis transformed to express GBS AI-I polypeptides using anti-GBS 80 antiserum.
FIGURE 136B: Polyacrylamide gel electrophoresis of sonicated pellets and supematants of cultured L. lactis transformed to express GBS AI polypeptides. FIGURE 137: Depiction of an example S. pneumoniae AI locus.
FIGURE 138: Schematic of primer hybridization sites within the S. pneumoniae AI locus of FIGURE 137.
FIGURE 139A: The set of amplicons produced from the S. pneumoniae strain TIGR4 AI locus. FIGURE 139B: Base pair lengths of amplicons produced from FIGURE 139A primers in S. pneumoniae strain TIGR4.
FIGURE 140: CGH analysis of S. pneumoniae strains for the AI locus. 1"I' C;;1 W^iBS€lt ilSpifi i$ϊ©elrøice alignment of polypeptides encoded by AI orf 2 in S. pneumoniae AI-positive strains.
FIGURE 142: Amino acid sequence alignment of polypeptides encoded by AI orf 3 in S. pneumoniae AI-positive strains. FIGURE 143: Amino acid sequence alignment of polypeptides encoded by AI orf 4 in S. pneumoniae AI-positive strains.
FIGURE 144: Amino acid sequence alignment of polypeptides encoded by AI orf 5 in S. pneumoniae AI-positive strains.
FIGURE 145: Amino acid sequence alignment of polypeptides encoded by AI orf 6 in S. pneumoniae AI-positive strains.
FIGURE 146: Amino acid sequence alignment of polypeptides encoded by AI orf 7 in S. pneumoniae AI-positive strains.
FIGURE 147: Amino acid sequence alignment of polypeptides encoded by AI orf 8 in S. pneumoniae AI-positive strains. FIGURE 148: Diagram comparing amino acid sequences of RrgA in S. pneumoniae strains.
FIGURE 149: Amino acid sequence comparison of RrgB S. pneumoniae strains. FIGURE 150A: SpO462 amino acid sequence.
FIGURE 150B: Primers used to produce a clone encoding the SpO462 polypeptide. FIGURE 15 IA: Schematic depiction of recombinant SpO462 polypeptide. FIGURE 151 B : Schematic depiction of full-length SpO462 polypeptide.
FIGURE 152A: Western blot probed with serum obtained from S. pneumoniae- infected patients for SpO462.
FIGURE 152B: Western blot probed with GBS 80 serum for SρO462. FIGURE 153 A: SpO463 amino acid sequence. FIGURE 153B: Primers used to produce a clone encoding the SpO463 polypeptide.
FIGURE 154A: Schematic depiction of recombinant SpO463 polypeptide. FIGURE 154B: Schematic depiction of full-length SρO463 polypeptide. FIGURE 155: Western blot detection of recombinant SpO463 polypeptide. FIGURE 156: Western blot detection of high molecular weight SpO463 polymers. FIGURE 157A: SpO464 amino acid sequence.
FIGURE 157B: Primers used to produce a clone encoding the SρO464 polypeptide. FIGURE 158 A: Schematic depiction of recombinant SpO464 polypeptide. FIGURE 158B: Schematic depiction of full-length SpO464 polypeptide. FIGURE 159: Western blot detection of recombinant SpO464 polypeptide. FIGURE 160: Amplification products prepared for production of SρO462, SpO463, and
SpO464 clones.
FIGURE 161 : Opsonic killing by anti-sera raised against L. lactis expressing GBS AI 1P C TtelMl::iO:iSolieiaaicftplcQg GAS adhesin islands GAS AI-I, GAS AI-2, GAS AI-3 and GAS AI-4.
FIGURES 163 A-D: Immunoblots of cell-wall fractions of GAS strains with antisera specific for LPXTG proteins of M6_ ISS3650 (A), Ml_SF370 (B)1 M5JSS4883 (C) and M12_20010296 (D). FIGURES 163 E-H: Immunoblots of cell-wall fractions of deletion mutants Ml_SF370Δ128
(E) Ml_SF370Δ130 (F) Ml_SF370ΔSrtCl (G) and the Ml_128 deletion strain complemented with plasmid pAM:: 128 which contains the Ml_128 gene (H) with antisera specific for the pilin components of M1J3F370.
FIGURES 163 I-N: Immunogold labelling and transmission electron microscopy of: T6 (I) and Cpa (J) in M6JSS3650; M1J28 in Ml_SF370 (K) and deletion strain Ml_SF370Δ128 (N); M5_prf8O in M5JSS4883 (L); M12_EftLSL.A in M12_20010296 (M). The strains used are indicated below the panels. Bars=200nm.
FIGURE 164: Schematic representation of the FCT region from 7 GAS strains FIGURES 165 A-H: Flow cytometry of GAS bacteria treated or not with trypsin and stained with sera specific for the major pilus component. Preimmune staining; black lines, untreated bacteria; green lines and trypsin treated bacteria; blue lines. M6_ISS3650 stained with sera which recognize the M6 protein (A) or anti-M6_T6 (B), Ml_SF370 stained with anti-Mi (C) or anti-Ml_128 (D), M5JSS4883 stained with anti-PrtF (E) or anti-M5_orf80 (F) and M12_20010296 with anti-M12 (G) or anti-EftLSL.A (H) FIGURES 166 A-C: Immunoblots of recombinant pilin components with polyvalent
Lancefield T-typing sera. The recombinant proteins are shown above the blot and the sera pool used is shown below the blot.
FIGURES 166 D-G: Immunoblots of pilin proteins with monovalent T-typing sera. The recombinant proteins are shown below the blot and the sera used above the blot. Figure 166 H and I Flow cytometry analysis of strain Ml_SF370 (H) and the deletion strain
Ml_SF370Δ128 (I) with T-typing antisera pool T.
FIGURE 167: Chart describing the number and type of sortase sequences identified within GAS AIs.
FIGURE 168 A: Immunogold-electronmicroscopy of L. lactis lacking an expression construct for GBS AI-I using anti-GBS 80 antibodies.
FIGURE 168 B and C: Immunogold-electronmicroscopy detects GBS 80 in oligomeric (pilus) structures on surface of L. lactis transformed to express GBS AI-I
FIGURE 169: FACS analysis detects expression of GBS 80 and GBS 104 on the surface of L. lactis transformed to express GBS AI-I. FIGURE 170: Phase contrast microscopy and immuno-electronmicroscopy shows that expression of GBS AI-I in L. lactis induces L. lactis aggregation.
FIGURE 171 : Purification of GBS pili from L. lactis transformed to express GBS AI-I . P C 'fløtlESiiiϊfirliiiSoώaialciiiiiepiyan of GAS M6 (AI-I), Ml (AI-2), and M12 (AI-4) adhesin islands and portions of the adhesin islands inserted in the pAM401 construct for expression in L. lactis.
FIGURE 173 A-C: Western blot analysis showing assembly of GAS pili in L. lactis expressing GAS AI-2 (Ml) (A), GAS AI-4 (M12) (B), and GAS AI-I (M6) (C).
FIGURE 174: FACS analysis of GAS serotype M6 for M6_SpyO157 surface expression. FIGURE 175: FACS analysis of GAS serotype M12 for 19224139 surface expression. FIGURE 176 A-E: Immunogold electron microscopy using antibodies against M6_SpyO16O detects pili on the surface of M6 strain 2724. FIGURE 176 F: Immunogold electron microscopy using antibodies against M6_SpyO159 detects M6_SpyO159 surface expression on M6 strain 2724.
FIGURE 177 A-C: Western blot analysis of Ml strain SF370 GAS bacteria individually deleted for Ml_130, SrtCl, or Ml_128 using anti-Ml_130 serum (A), anti-Ml_128 serum (B), and anti-Ml_126 serum (C). FIGURE 178 A-C: Immunogold electron microscopy using antibodies against Ml_128 to detect surface expression on wildtype strain SF370 bacteria (A), Ml_128 deleted SF370 bacteria (B), and SrtCl deleted SF370 bacteria (C).
FIGURE 179 A-C: FACS analysis to detect expression of Ml_126 (A), Ml_128 (B), and Ml_130 (C) on the surface of wildtype SF370 GAS bacteria. FIGURE 179 D-F: FACS analysis to detect expression of Ml_126 (D), Ml_128 (E), and
Ml_130 (F) on the surface of Ml_128 deleted SF370 GAS bacteria.
FIGURE 179 G-I: FACS analysis to detect expression of Ml_126 (G), Ml_128 (H), and Ml_130 (I) on the surface of SrtCl deleted SF370 GAS bacteria.
FIGURE 180 A and B: FACS analysis of wildtype (A) and LepA deletion mutant (B) strains of SF370 bacteria for Ml surface expression.
FIGURE 181: Western blot analysis detects high molecular weight polymers in S. pneumoniae TIGR4 using anti-RrgB antisera.
FIGURE 182: Detection of high molecular weight polymers in S. pnuemoniae rlrA positive strains. FIGURE 183: Detection of high molecular weight polymers in S. pneumoniae TIGR4 by silver staining and Western blot analysis using anti-RrgB antisera.
FIGURE 184: Deletion of S. pneumoniae TIGR4 adhesin island sequences interferes with the ability of S. pneumoniae to adhere to A549 alveolar cells.
FIGURE 185: Negative staining of S. pneumoniae strain TIGR4 showing abundant pili on the bacterial surface.
FIGURE 186: Negative staining of strain TIGR4 deleted for ixgA-srtD adhesin island sequences showing no pili on the bacterial surface I"1' C llGUffilO:ii:::K.egaHΫe1' sEi3rfiiof the TIGR4 mgrA mutant showing abundant pili on the bacterial surface.
FIGURE 188: Negative staining of the negative control TIGR4 mgrA mutant deleted for adhesin island sequences ixgA-srtD showing no pili on the bacterial surface. FIGURE 189: Immuno-gold labelling of S. pneumoniae strain TIGR4 grown on blood agar solid medium using α-RrgB (5nm) and α-RrgC (lOnm). Bar represents 200nm.
FIGURE 190 A and B: Detection of expression and purification of S. pneumoniae RrgA protein by SDS-PAGE (A) and Western blot analysis (B).
FIGURE 191: Detection of RrgB by antibodies produced in mice. FIGURE 192: Detection of RrgC by antibodies produced in mice.
FIGURE 193: Purification of S. pneumoniae TIGR 4 pili by a cultivation and digestion method and detection of the purified TIGR4 pili.
FIGURE 194: Purification of S. pneumoniae TIGR 4 pili by a sucrose gradient centrifugation method and detection of the purified TIGR4 pili. FIGURE 195: Purification of S. pneumoniae TIGR 4 pili by a gel filtration method and detection of the purified TIGR4 pili.
FIGURE 196: Alignment of full length S. pneumoniae adhesin island sequences from ten S. pneumoniae strains.
FIGURE 197 A: Schematic of GBS AI-I coding sequences. FIGURE 197 B: Nucleotide sequence of intergenic region between AraC and GBS 80 (SEQ
ID NO: 273.
FIGURE 197 C: FACS analysis results for GBS 80 expression in GBS strains having different length polyA tracts in the intergenic region between AraC and GBS 80.
FIGURE 198: Table comparing the percent identity of surface proteins encoded by a serotype M6 (harbouring a GAS AI-I) adhesin island relative to other GAS serotypes harbouring an adhesin island.
FIGURE 199: Table comparing the percent identity of surface proteins encoded by a serotype Ml (harbouring a GAS AI-2) adhesin island relative to other GAS serotypes harbouring an adhesin island. FIGURE 200: Table comparing the percent identity of surface proteins encoded by serotypes
M3, M18, M5, and M49 (harbouring GAS AI-3) adhesin islands relative to other GAS serotypes harbouring an adhesin island.
FIGURE 201: Table comparing the percent identity of surface proteins encoded by a serotype M 12 (harbouring a GAS AI-I) adhesin island- relative to other GAS serotypes harbouring an adhesin island.
FIGURE 202: GBS 80 recombinant protein does not bind to epithelial cells.
FIGURE 203: Deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade ME 180 cervical epithelial cells. P C 'ΪIGtMHiiMliii'irBδiisbl'ϊfflsIb^βitracellular matrix proteins.
FIGURE 205: Deletion of GBS 104 protein, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cells
FIGURE 206: GBS 104 knockout mutant strains of bacteria translocate through an epithelial monolayer less efficiently that the isogenic wild type strain.
FIGURE 207: GBS 80 knockout mutant strains of bacteria partially lose the ability to translocate through an epithelial monolayer.
FIGURE 208: GBS adherence to HUVEC endothelial cells.
FIGURE 209: Strain growth rate of wildtype, GBS 80-deleted, or GBS 104 deleted COHl GBS.
FIGURE 210: Binding of recombinant GBS 104 protein to epithelial cells by FACS analysis. FIGURE 211: Deletion of GBS 104 proteinin the" GBS strain COHl reduces the ability of GBS to adhere to MEl 80 cervical epithelial cells.
FIGURE 212: COHl strain GBS overexpressing GBS 80 protein has an impaired capacity to translocate through an epithelial monolayer.
FIGURE 213: Scanning electron microscopy shows that overexpression of GBS 80 protein on COHl strain GBS enhances the capacity of the COHl bacteria to form microcolonies on epithelial cells.
FIGURE 214: Confocal imaging shows that overexpression of GBS 80 proteins on COHl strain GBS enhances the capacity of the COHl bacteria to form microcolonies on epithelial cells.
FIGURE 215: Detection of GBS 59 on the surface of GBS strain 515 by immuno-electron microscopy.
FIGURE 216: Detection of GBS 67 on the surface of GBS strain 515 by immuno-electron microscopy. FIGURE 217: GBS 67 binds to fibronectin.
FIGURE 218: Western blot analysis shows that deletion of both GBS AI-2 sortase genes abolishes assembly of the pilus.
FIGURE 219: FACS analysis shows that deletion of both GBS AI-2 sortase genes abolishes assembly of the pilus. FIGURE 220 A-C: Western blot analysis shows that GBS 59, GBS 67, and GBS 150 form high molecular weight complexes.
FIGURE 221 A-C: Western blot analysis shows that GBS 59 is required for polymer formation of GBS 67 and GBS 150.
FIGURE 222: FACS analysis shows that GBS 59 is required for surface exposure of GBS 67. FIGURE 223: Summary Western blots for detection of GBS 59, GBS 67, or GBS 150 in
GBS 515 and GBS 515 mutant strain.
FIGURE 224: Description of GBS 59 allelic variants. P
Figure imgf000033_0001
only against a strain of GBS expressing a homologous GBS 59.
FIGURE 226 A and B: Results of FACS analysis for surface expression of GBS 59 using antibodies specific for different GBS 59isoforms. FIGURE 227 A and B: Results of FACS analysis for surface expression of GBS 80, GBS
104, GBS 322, GBS 67, and GBS 59 on 41 various strains of GBS bacteria.
FIGURE 228: Results of FACS analysis for surface expression of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 on 41 strains of GBS bacteria obtained from the CDC.
FIGURE 229: Expected immunogenicity coverage of different combinations of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 across strains of GBS bacteria.
FIGURE 230: GBS 59 opsonophagocytic activity is comparable to that of a mixture of GBS 80, GBS 104, GBS 322 and GBS 67.
FIGURE 231 A-C: Schematic presentation of example hybrid GBS AIs. FIGURE 232: Schematic presentation of an example hybrid GBS AI. FIGURE 233 A and B: Western blot and FACS analysis detect expression of GBS 80 and
GBS 67 on the surface of L. lactis transformed with a hybrid GBS AL FIGURE 234 A-E Hybrid GBS AI cloning strategy.
FIGURE 235: High magnification of S. pneumoniae strain TIGR4 pili double labeled with α- RrgB (5nm) and α-RrgC (lOnm). Bar represents lOOnm. FIGURE 236: Immuno-gold labeling of the S. pneumoniae TIGR4 rrgA-srtD deletion mutant with no visible pili on the surface detectable by α-RrgB- and α-RrgC. Bar represents 200nm.
FIGURE 237: Variability in GBS 67 amino acid sequences between strains 2603 and H36B. FIGURE 238: Strain variability in GBS 67 amino acid sequences of allele I (2603). FIGURE 239: Stran variability in GBS 67 amino acid sequence of allele II (H36B).
BRIEF DESCRIPTION OF THE TABLES
TABLE 1 : Active Maternal Immunization Assay for fragments of GBS 80 TABLE 2: Passive Maternal Immunization Assay for fragments of GBS 80 TABLE 3: Lethal dose 50% of AI-I mutants from GBS strain isolate 2603. TABLE 4: GAS AI-I sequences from M6 isolate (MGAS10394).
TABLE 5: GAS AI-2 sequences from Ml isolate (SF370). TABLE 6: GAS AI-3 sequences from M3 isolate (MGAS315). TABLE 7: GAS AI-3 sequences from M3 isolate (SSI-I). TABLE 8: GAS AI-3 sequences from M18 isolate (MGAS8232). TABLE 9: S. pneumoniae AI sequences from TIGR4 sequence.
TABLE 10: GAS AI-3 sequences from M5 isolate (Manfredo). P C
Figure imgf000034_0001
from Ml 2 isolate (A735).
TABLE 12: Conservation of GBS 80 and GBS 104 amino acid sequences.
TABLE 13: Conservation of GBS 322 and GBS 276 amino acid sequences.
TABLE 14: Active maternal immunization assay for a combination of fragments from GBS 322, GBS 80, GBS 104, and GBS 67.
TABLE 15: Antigen surface exposure of GBS 80, GBS 322, GBS 104, and GBS 67.
TABLE 16: Active maternal immunization assay for each of GBS 80 and GBS 322 antigens.
TABLE 17: Active maternal immunization assay for GBS 59.
TABLE 18: Summary of FACS values for surface expression of sρyM6_0159. TABLE 19: Summary of FACS values for surface expression of spyM6_0160.
TABLE 20: Summary of FACS values for surface expression of GAS 15.
TABLE 21: Summary of FACS values for surface expression of GAS 16.
TABLE 22: Summary of FACS values for surface expression of GAS 16 using a second antisera. TABLE 23 : Summary of FACS values for surface expression of GAS 18.
TABLE 24: Summary of FACS values for surface expression of GAS 18 using a second antisera.
TABLE 25: Summary of FACS values for surface expression of SpyM3_0098.
TABLE 26: Summary of FACS values for surface expression of SpyM3_0100. TABLE 27: Summary of FACS values for surface expression of SpyM3_0102 in M3 serotypes.
TABLE 28 : Summary of FACS values for surface expression of SpyM3_0102 in M6 serotypes.
TABLE 29: Summary of FACS values for surface expression of SpyM3_0104 in M3 serotypes.
TABLE 30: Summary of FACS values for surface expression of SpyM3_0104 in an M 12 serotype.
TABLE 31: Summary of FACS values for surface expression of SPs_0106 in M3 serotypes.
TABLE 32: Summary of FACS values for surface expression of SPs_0106 in an M 12 serotype.
TABLE 33: Summary of FACS values for surface expression of 19224134 in an M12 serotype.
TABLE 34: Summary of FACS values for surface expression of 19224134 in M6 serotypes.
TABLE 35: Summary of FACS values for surface expression of 19224135 in an M12 serotype.
TABLE 36: Summary of FACS values for surface expression of 19224137 in an M12 serotype. P C " ISr^BlLβliSRiiiMiibώiiy^'fjJHlΛiSSi^alues for surface expression of 19224141 in an M12 serotype.
TABLE 38: S. pneumoniae strain 670 AI sequences.
TABLE 39: Pecent identity comparison of 5". pneumoniae strains AI sequences. TABLE 40: FACS analysis of L. lactis and GBS bacteria strains expressing GBS AI-I .
TABLE 41 : Sequences of primers used to amplify AI locus.
TABLE 42: Conservation of amino acid sequences encoded by the S. pneumoniae AI locus.
TABLE 43: Protection of Mice Immunized with L. lactis expressing GBS AI-I.
TABLE 44: GAS AI-3 sequences from M49 isolate (591). TABLE 45: Comparison of Sequences Between the Four GAS AIs.
TABLE 46: Antibody Responses against GBS 80 in Serum of Mice Immunized with L. lactis Expressing GBS AI-I
TABLE 47: Anti-GBS 80 IgA Antibodies Detected in Mouse Tissues Following Immunization with L. lactis Expressing GBS AI-I TABLE 48: GBS 67 Protects Mice in an Immunization Assay
TABLE 49: Exposure Levels of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on GBS Strains
TABLE 50: High Levels of Surface Protein Expression on GBS Serotypes
TABLE 51 : Further Protection of Mice Immunized with L. lactis expressing GB S AI- 1
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, VoIs. I-IV (D.M. Weir and CC. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi, K.S. ed., CRC Press, 1997); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag); , Peters and Dalrymple, Fields Virology (2d ed), Fields et al. (eds.), B.N. Raven Press, New York, NY.
All publications, patents and patent applications cited herein, are hereby incorporated by reference in their entireties.
As used herein, an "Adhesin Island" or "AI" refers to a series of open reading frames within a bacterial genome, such as the genome for Group A or Group B Streptococcus or other gram positive bacteria, that encodes for a collection of surface proteins and sortases. An Adhesin Island may eifibSe f5br-'amind:%C!iα"gfeqtιdϊϊee^ (fomprisfng at least one surface protein. The Adhesin Island may encode at least one surface protein. Alternatively, an Adhesin Island may encode for at least two surface proteins and at least one sortase. Preferably, an Adhesin Island encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or more AI surface proteins may participate in the formation of a pilus structure on the surface of the gram positive bacteria.
Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). The transcriptional regulator may regulate the expression of the AI operon. GBS Adhesin Island 1
As discussed above, Applicants have identified a new adhesin island, "Adhesin Island 1", "AI-I", or "GBS AI-I", within the genomes of several Group B Streptococcus serotypes and isolates. AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("AI-I proteins"). Specifically, AI-I includes open reading frames encoding for two or more (i.e., 2, 3, 4 or 5) of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. One or more of the AI-I open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the AI-I open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
A schematic of AI-I is presented in Figure 1. AI-I typically resides on an approximately 16.1 kb transposon-like element frequently inserted into the open reading frame for trniA. One or more of the AI-I surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) motif or other sortase substrate motif. The AI surface proteins of the invention may affect the ability of the GBS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The AI-I sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. AI-I may encode for at least one surface protein. Alternatively, AI-I may encode for at least two surface exposed proteins and at least one sortase. Preferably, AI-I encodes for at least three surface exposed proteins and at least two sortases. The AI-I protein preferably includes GBS 80 or a fragment thereof or a sequence having sequence identity thereto.
As used herein, an LPXTG motif represents an amino acid sequence comprising at least five amino acid residues. Preferably, the motif includes a leucine (L) in the first amino acid position, a proline (P) in the second amino acid position, a threonine (T) in the fourth amino acid position and a glycine (G) in the fifth amino acid position. The third position, represented by X, may be occupied by aiyimiϊo.ώiyyeiilMiiPirefiraSifiiiitiϊI SPis occupied by lysine (K), Glutamate (E), Asparagine (N), Glutamine (Q) or Alanine (A). Preferably, the X position is occupied by lysine (K). In some embodiments, one of the assigned LPXTG amino acid positions is replaced with another amino acid. Preferably, such replacements comprise conservative amino acid replacements, meaning that the replaced amino acid residue has similar physiological properties to the removed amino acid residue. Genetically encoded amino acids may be divided into four families based on physiological properties: (1) acidic (asparatate and glutamate), (2) basic (lysine, arginine, histitidine), (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane) and (4) uncharged polar (glycine, asparagines, glutamine, cysteine, serine, threonine, and tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of a leucine with an isoleucine or valine, an asparate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity.
The first amino acid position of the LPXTG motif may be replaced with another amino acid residue. Preferably, the first amino acid residue (leucine) is replaced with an alanine (A), valine (V), isoleucine (I), proline (P), phenylalanine (F), methionine (M), glutamic acid (E), glutamine (Q), or tryptophan (Y) residue. In one preferred embodiment, the first amino acid residue is replaced with an isoleucine (I).
The second amino acid residue of the LPXTG motif may be replaced with another amino acid residue. Preferably, the second amino acid residue praline (P) is replaced with a valine (V) residue.
The fourth amino acid residue of the LPXTG motif may be replaced with another amino acid residue. Preferably, the fourth amino acid residue (threonine) is replaced with a serine (S) or an alanine (A).
In general, an LPXTG motif may be represented by the amino acid sequence XXXXG, in which X at amino acid position 1 is an L, a V, an E, an I, an F, or a Q; X at amino acid position 2 is a P if X at amino acid position 1 is an L, an I, or an F; X at amino acid position 2 is a V if X at amino acid position 1 is a E or a Q; X at amino acid position 2 is a V or a P if X at amino acid position 1 is a V; X at amino acid position 3 is any amino acid residue; X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, I, F, or Q; and X at amino acid position 4 is a T, S, or A if X at amino acid position 1 is an L.
Generally, the LPXTG motif of a GBS AI protein may be represented by the amino acid sequence XPXTG, in which X at amino acid position 1 is L, I, or F, and X at amino acid position 3 is any amino acid residue. Specific examples of LPXTG motifs in GBS AI proteins may include LPXTG (SEQ ID NO: 122) or IPXTG (SEQ ID NO: 133). As discussed further below, the threonine in the fourth amino acid position of the LPXTG motif may be involved in the formation of a bond between the LPXTG containing protein and a cell wall precursor. Accordingly, in preferred LPXTG motifs, the threonine in the fourth amino acid p'^ilioπ'iS' ιϊiofcil-Θpllee3%itH"an6tIi;er'a:mMo acid or, if the threonine is replaced, the replacement amino acid is preferably a conservative amino acid replacement, such as serine.
Instead of an LPXTG motif, the AI surface proteins of the invention may contain alternative sortase substrate motifs such as NPQTN (SEQ ID NO: 142), NPKTN (SEQ ID NO: 168), NPQTG (SEQ ID NO: 169), NPKTG (SEQ ID NO: 170), XPXTGG (SEQ ID NO: 143), LPXTAX (SEQ ID NO: 144), or LAXTGX (SEQ ID NO: 145). (Similar conservative amino acid substitutions can also be made to these membrane motifs).
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. The AI surface proteins may be polymerized into pili by sortase-catalysed transpeptidation.
(See Figure 44.) Cleavage of AI surface proteins by sortase between the threonine and glycine residues of an LPXTG motif yields a thioester-linked acyl intermediate of sortase. Many AI surface proteins include a pilin motif amino acid sequence which interacts with the sortase and LPXTG amino acid sequence. The first lysine residue in a pilin motif can serve as an amino group acceptor of the cleaved LPXTG motif and thereby provide a covalent linkage between AI subunits to form pili. For example, the pilin motif can make a nucleophilic attack on the acyl enzyme providing a covalent linkage between AI subunits to form pili and regenerate the sortase enzyme. Examples of pilin motifs may include ((YPKN(Xi0)K; SEQ ID NO: 146), (YPKN(Xg)K; SEQ ID NO: 147), (YPK(X7)K; SEQ ID NO: 148), (YPK(Xn)K; SEQ ID NO: 149), or (PKN(X9)K; SEQ ID NO: 150)). Preferably, the AI surface proteins of the invention include a pilin motif amino acid sequence.
Typically, AI surface proteins of the invention will contain an N-terminal leader or secretion signal to facilitate translocation of the surface protein across the bacterial membrane.
Group B Streptococci are known to colonize the urinary tract, the lower gastrointestinal tract and the upper respiratory tract in humans. Electron micrograph images of GBS infection of a cervical epithelial cell line (MEl 80) are presented in Figure 25. As shown in these images, the bacteria closely associate with tight junctions between the cells and appear to cross the monolayer by a paracellular route. Similar paracellular invasion of MEl 80 cells is also shown in the contrast images in Figure 26. The AI surface proteins of the invention may effect the ability of the GBS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface.
Applicants have discovered that AI-I surface protein GBS 104 can bind epithelial cells such as ME 180 human cervical cells, A549 human lung cells and Caco2 human intestinal cells (See FΪiUKs'29''aiiyiiΪOf3itr.thdiy!(ϊiϊiioiyilhe GBS 104 sequence in a GBS strain reduces the capacity of GBS to adhere to ME180 cervical epithelial cells. (See Figures 30 and 211). Deletion of GBS 104 also reduces the capacity of GBS to invade J774 macrophage-like cells. (See Figures 32 and 205). Deletion of GBS 104 also causes GBS to translocate through epithelial monolayers less efficiently. See Figure 206. GBS 104 protein therefore appears to bind to ME180 epithelial cells and to have a role in adhesion to epithelial cells and macrophage cell lines.
Similar to the GBS bacteria that are deletion mutants for GBS 104, GBS 80 knockout mutant strains also partially lose the ability to translocate through an epithelial monolayer. See Figure 207. Deletion of either GBS 80 or GBS 104 in COHl cells diminishes adherence to HUVEC endothelial cells. See Figure 208. Deletion of GBS 80 or GBS 104 in COHl does not, however, affect growth of COHl either with MEl 80 cells or in incubation medium (IM). See Figure 209. Both GBS 80 and GBS 104, therefore, appear to be involved in translocation of GBS through epithelial cells.
GBS 80 does not appear to bind to epithelial cells. Incubation of epithelial cells in the presence of GBS 80 protein followed by FACS analysis using an anti-GBS 80 polyclonal antibody did not detect GBS 80 binding to the epithelial cells. See Figure 202. Furthermore, deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade MEl 80 cervical epithelial cells. See Figure 203
Preferably, one or more of the surface proteins may bind to one or more extracellular matrix (ECM) binding proteins, such as fibrinogen, fibronectin, or collagen. As shown in Figures 5 and 204, and Example 1, GBS 80, one of the AI-I surface proteins, can bind to the extracellular matrix binding proteins fibronectin and fibrinogen. While GBS 80 protein apparently does not bind to certain epithelial cells or affect the capacity of a GBS bacteria to adhere to or invade cervical epithelial cells (See Figures 27 and 28), removal of GBS 80 from a wild type strain decreases th'e ability of that strain to translocate through an epithelial cell layer (see Figure 31). GBS 80 may also be involved in formation of biofihns. COHl bacteria overexpressing GBS
80 protein have an impaired ability to translocate through an epithelial monolayer. See Figure 212. These COHl bacteria overexpressing GBS 80 form microcolonies on epithelial cells. See Figures 213 and 214. These microcolonies may be the initiation of biofilm development.
Al Surface proteins may also demonstrate functional homology to previously identified adhesion proteins or extracellular matrix (ECM) binding proteins. For example, GBS 80, a surface protein in AI-I, exhibits some functional homology to FimA, a major fimbrial subunit of a Gram positive bacteria A. naeslundii. FimA is thought to be involved in binding salivary proteins and may be a component in a fϊmbrae on the surface of A. naeslundii. See Yeung et al. (1997) Infection & Immunity 65:2629-2639; Yeunge et al (1998) J. Bacteriol 66: 1482-1491; Yeung et al. (1988) J. Bacteriol 170:3803 - 3809; and Li et al (2001) Infection & Immunity 69:7224-7233.
A similar functional homology has also been identified between GBS 80 and proteins involved in pili formation in the Gram positive bacteria Corγnebacterium diphtheriae (SpaA, SpaD, and SpaH). See, Ton-That et al. (2003) Molecular Microbiology 50(4): 1429-1438 and Ton-That et al. (B(JI)
Figure imgf000040_0001
The C. diphtheriae proteins all included a pilin motif of WxxxVxVYPK (SEQ ID NO: 151; where x indicates a varying amino acid residue). The lysine (K) residue is particularly conserved in the C. diphtheriae pilus proteins and is thought to be involved in sortase catalized oligomerization of the subunits involved in the C. diphtheriae pilus structure. (The C. diphtheriae pilin subunit SpaA is thought to occur by sortase-catalyzed amide bond cross- linking of adjacent pilin subunits. As the thioester-linked acyl intermediate of sortase requires nucleophilic attack for release, the conserved lysine within the SpaA pilin motif might function as an amino group acceptor of cleaved sorting signals, thereby providing for covalent linkages of the C diphtheria pilin subunits. See Figure 6(d) of Ton-That et al., Molecular Microbiology (2003) 50(4): 1429-1438.)
In addition, an "E box" comprising a conserved glutamic acid residue has also been identified in the C. diphtheria pilin associated proteins as important in C. diphtheria pilin assembly. The E box motif generally comprises YxLxETxAPxGY (SEQ ID NO: 152; where x indicates a varying amino acid residue). In particular, the conserved glutamic acid residue within the E box is thought necessary for C. diphtheria pilus formation.
Preferably, the AI-I polypeptides of the immunogenic compositions comprise an E box motif. Some examples of E box motifs in the AI-I polypeptides may include the amino acid sequences YxLxExxxxxGY (SEQ ID NO: 153), YxLxExxxPxGY (SEQ ID NO: 154), or YxLxETxAPxGY (SEQ ID NO: 152). Specifically, the E box motif of the polypeptides may comprise the amino acid sequences YKLKETKAPEGY (SEQ ID NO: 155), YVLKEIETQSGY (SEQ ID NO: 156), or YKLYEISSPDGY (SEQ ID NO: 157).
As discussed in more detail below, a pilin motif containing a conserved lysine residue and an E box motif containing a conserved glutamic acid residue have both been identified in GBS 80.
While previous publications have speculated that pilus-like structures might be formed on the surface of streptococci, (see, e.g., Ton-That et al., Molecular Microbiology (2003) 50(4): 1429 - 1438), these structures have not been previously visible in negative stain (non-specific) electron micrographs, throwing such speculations into doubt. For example, Figure 34 presents electron micrographs of GBS serotype III, strain isolate COHl with a plasmid insert to facilitate the overexpression of GBS 80. This EM photo was produced with a standard negative stain - no pilus structures are distinguishable. In addition, the use of such AI surface proteins in immunogenic compositions for the treatment or prevention of infection against a Gram positive bacteria has not been previously described.
Surprisingly, Applicants have now identified the presence of GBS 80 in surface exposed pilus formations visible in electron micrographs. These structures are only visible when the electron micrographs are specifically stained against an AI surface protein such as GBS 80. Examples of these electron micrographs are srlown in Figures 11, 16 and 17, which reveal the presence of pilus structures in wild type COHl Streptococcus agalactiae. Other examples of these electron πϋϊcilEgrSphs yeShSMin'Fiiurl!' It, .siffih reveals that GBS 80 is associated with pili in a wild type clinical isolate of S. agalactiae, JM9030013. (See figure 49.)
Applicants have also constructed mutant GBS strains containing a plasmid comprising the GBS 80 sequence resulting in the overexpression of GBS 80 within this mutant. The electron micrographs of Figures 13 — 15 are also stained against GBS 80 and reveal long, oligomeric structures containing GBS 80 which appear to cover portions of the surface of the bacteria and stretch far out into the supernatant.
In some instances, the formation of pili structures on GBS appears to be correlated to surface ■ expression of GBS 80. Figure 61 provides FAC analysis of GBS 80 surface levels on bacterial strains COHl and JM9130013 using an anti-GBS 80 antisera. Immunogold electron microscopy of the
COHl and JM9130013 bacteria using anti-GBS 80 antisera demonstrates that JM9130013 bacteria, which have higher values for GBS 80 surface expression, also form longer pili structures.
The surface exposure of GBS 80 on GBS is generally not capsule-dependent. Figure 62 provides FACS analysis of capsulated and uncapsulated GBS analyzed with anti-GBS 80 and anti- GBS 322 antibodies. Surface exposure of GBS 80, unlike GBS 322, is not capsule dependent.
An Adhesin Island surface protein, such as GBS 80 appears to be required for pili formation, as well as an Adhesin Island sortase. Pili are formed in Cohl bacterial clones that overexpress GBS 80, but lack GBS 104, or one of the AI-I sortases sagO647 or sagO648. However, pili are not formed in Cohl bacterial clones that overexpress GBS 80 and lack both sagO647 and sagO648. Thus, for example, it appears that at least GBS 80 and a sortase, sagO647 or sagO648, may be necessary for pili formation. (See Figure 48.) Overexpression of GBS 80 in GBS strain 515, which lacks an AI-I, also assembles GBS 80 into pili. GBS strain 515 contains an AI-2, and thus AI-2 sortases. The AI-2 sortases in GBS strain 515 apparently polymerize GBS 80 into pili. (See Figure 42.) Overexpression of GBS 80 in GBS strain 515 cell knocked out for GBS 67 expression also apparently polymerizes GBS 80 into pili. (See Figure 72.)
While GBS 80 appears to be required for GBS AI-I pili formation, GBS 104 and sortase SAG0648 appears to be important for efficent AI-I pili assembly. For example, high-molecular structures are not assembled in isogenic COHl strains which lack expression of GBS 80 due to gene disruption and are less efficiently assembled in isogenic COHl strains which lack the expression of GBS 104 (see Figure 41). This GBS strain comprises high molecular weight pili structures composed of covalently linked GBS 80 and GBS 104 subunits. In addition, deleting SAG0648 in COHl bacteria interferes with assembly of some of the high molecular weight pili structures. Thus, indicating that SAG0648 plays a role in assembly of these pilin species. (See Figure 41).
EM photos confirm the involvement of AI surface protein GBS 104 within the hyperoligomeric structures of a GBS strain adapted for increased GBS 80 expression. (See Figures 34 - 41 and Example 6). In a wild type serotype VIII GBS strain, strain JM9030013, IEM identifies GBS 104 as forming clusters on the bacterial surface. (See Figure 50.) P' C "&BU li!Ssi!lbjp|e^a:.b"BS!cMponent of the GBS pili. Immunoblots using an anti-GBS 80 antisera on total cell extracts of Cohl and a GBS 52 null mutant Cohl reveal a shift in detected proteins in the Cohl wild type strain relative to the GBS 52 null mutant Cohl strain. The shifted proteins were also detected in the wild type Cohl bacteria with an anti-GBS 52 antisera, indicating that the GBS 52 may be present in the pilus. (See Figure 45.)
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as GBS 80. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include one or both of a pilin motif comprising a conserved lysine residue and an E box motif comprising a conserved glutamic acid residue.
More than one AI surface protein may be present in the oligomeric, pilus-like structures of the invention. For example, GBS 80 and GBS 104 may be incorporated into an oligomeric structure. Alternatively, GBS 80 and GBS 52 may be incorporated into an oligomeric structure, or GBS 80, GBS 104 and GBS 52 may be incorporated into an oligomeric structure.
In another embodiment, the invention includes compositions comprising two or more AI surface proteins. The composition may include surface proteins from the same adhesin island. For example, the composition may include two or more GBS AI-I surface proteins, such as GBS 80, GBS 104 and GBS 52. The surface proteins may be isolated from Gram positve bacteria or they may be produced recombinantly.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a GBS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more GBS Adhesin Island 1 ("AI-I") proteins and one or more GBS Adhesin Island 2 ("AI-2") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
The oligomeric, pilus-like structures of the invention may be combined with one or more additional GBS proteins. In one embodiment, the oligomeric, pilus-like structures comprise one or more AI surface proteins in combination with a second GBS protein. The second GBS protein may be a known GBS antigen, such as GBS 322 (commonly referred to as "sip") or GBS 276. Nucleotide and amino acid sequences of GBS 322 sequenced from serotype V isolated strain 2603 V/R are set
Figure imgf000043_0001
'Bifl'Md SEQ ID 8540 and in the present specification as SEQ ID NOs: 38 and 39. A particularly preferred GBS 322 polypeptide lacks the N-terminal signal peptide, amino acid residues 1-24. An example of a preferred GBS 322 polypeptide is a 407 amino acid fragment and is shown in SEQ ID NO: 40. Examples of preferred GBS 322 polypeptides are further described in PCTUS04/ , attorney docket number PP20665.002 filed September 15, 2004, hereby incorporated by reference, published as WO 2005/002619.
Additional GBS proteins which may be combined with the GBS AI surface proteins of the invention are also described in WO 2005/002619. These GBS proteins include GBS 91, GBS 184, GBS 305, GBS 330, GBS 338, GBS 361, GBS 404, GBS 690, and GBS 691. Additional GBS proteins which may be combined with the GBS AI surface proteins of the invention are described in WO 02/34771.
GBS polysaccharides which may be combined with the GBS AI surface proteins of the invention are described in WO 2004/041157. For example, the GBS AI surface proteins of the invention may be combined with a GBS polysaccharides selected from the group consisting of serotype Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII.
The oligomeric, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an AI surface protein. The invention therefore includes a method for manufacturing an oligomeric AI surface antigen comprising culturing a GBS bacterium that expresses the oligomeric AI protein and isolating the expressed oligomeric AI protein from the GBS bacteria. The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed AI protein. Preferably, the AI protein is in a hyperoligomeric form. Macromolecular structures associated with oligomeric pili are observed in the supernatant of cultured GBS strain Cohl. (See Figure 46.) These pili are found in the supernatant at all growth phases of the cultured Cohl bacteria. (See Figure 47.) The oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing an AI surface protein. The invention therefore includes a method for manufacturing an oligomeric Adhesin Island surface antigen comprising culturing a GBS bacterium adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the GBS bacteria. The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed Adhesin Island protein. Preferably, the Adhesin Island protein is in a hyperoligomeric form.
The GBS bacteria are preferably adapted to increase AI protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels. GBS bacteria may be adapted to increase AI protein expression by any means known in the art, including methods of increasing gene dosage and methods of gene upregulation. Such means include, for example, transformation of the GBS bacteria with a plasmid encoding the AI protein. The plasmid may include a strong promoter or it may include multiple copies of the sequence encoding the
Figure imgf000044_0001
the AI protein within the GBS bacterial genome may be deleted. Alternatively, or in addition, the promoter regulating the GBS Adhesin Island may be modified to increase expression.
GBS bacteria harbouring a GBS AI-I may also be adapted to increase AI protein expression by altering the number adenosine nucleotides present at two sites in the intergenic region between AraC and GBS 80. See Figure 197 A, which is a schematic showing the organization of GBS AI-I and Figure 197 B, which provides the sequence of the intergenic region between AraC and GBS 80 in the AI. The adenosine tracts which applicants have identified as influencing GBS 80 surface expression are at nucleotide positions 187 and 233 of the sequence shown in Figure 197 B (SEQ ID NO: 273). Applicants determined the influence of these adenosine tracts on GBS 80 surface expression in strains of GBS bacteria harboring four adenosines at position 187 and six adenosines at position 233, five adenosines at position 187 and six adenosines position 233, and five adenosines at position 187 and seven adenosines at position 233. FACS analysis of these strains using anti GBS 80 antiserum determined that an intergenic region with five adenosines at position 187 and six adenosines at position 233 had higher expression levels of GBS 80 on their surface than other stains. See Figure 197 C for results obtained from the FACS analysis. Therefore, manipulating the number of adenosines present at positions 187 and 233 of the AraC and GBS 80 intergenic region may further be used to adapt GBS to increase AI protein expression.
The invention further includes GBS bacteria which have been adapted to produce increased levels of AI surface protein. In particular, the invention includes GBS bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein, such as GBS 80. In one embodiment, the Gram positive bacteria of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
The invention further includes GBS bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface. The GBS bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide. Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pili proteins on the surface of Gram positive bacteria. Increased expression of a leader peptidase in GBS may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation. The GBS bacteria adapted to have increased levels of leader peptidase may additionally be adapted to express increased levels of at least one pili protein.
Alternatively, the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes" Infection and Immunity (2004) 72(6):3444-3450). As used herein, nibή-liatiog'eήic'Bi-ain'βbSϊti^e W'aderiai refer to Gram positive bacteria which are compatible with a human host subject and are not associated with human pathogenisis. Preferably, the non-pathogenic bacteria are modified to express the AI surface protein in oligomeric, or hyper-oligomeric form. Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non-pathogenic Gram positive bacterial genome or inserted into a plasmid. The non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface. Alternatively, the AI surface protein may be isolated or purified from a bacterial culture of the non-pathogenic Gram positive bacteria. For example, the AI surface protein may be isolated from cell extracts or culture supernatants. Alternatively, the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria.
The non-pathogenic Gram positive bacteria may be used to express any of the Gram positive bacterial Adhesin Island proteins described herein, including proteins from a GBS Adhesin Island, a GAS Adhesin Island, or a S pneumo Adhesin Island. The non-pathogenic Gram positive bacteria are transformed to express an Adhesin Island surface protein. Preferably, the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase. The AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with a pathogenic Gram positive bacteria, such as GBS, GAS or Streptococcus pneumoniae. The non-pathogenic Gram positive bacteria may express the Gram positive bacterial Adheshin Island proteins in oligomeric forms that further comprise adhesin island proteins encoded within the genome of the non-pathogenic Gram positive bacteria.
Applicants modified L. lactis to demonstrate that it can express GBS AI polypeptides. L. lactis was transformed with a construct encoding GBS 80 under its own promoter and terminator sequences. The transformed L. lactis appeared to express GBS 80 as shown by Western blot analysis using anti-GBS 80 antiserum. See lanes 6 and 7 of the Western Blots provided in Figures 133 A and 133B (133A and 133B are two different exposures of the same Western blot). See also Example 13. Applicants also transformed L. lactis with a construct encoding GBS AI-I polypeptides GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences. These L. lactis expressed high molecular weight structures that were immunoreactive with anti-GBS 80 in immunoblots. See Figure 134, lane 2, which shows detection of a GBS 80 monomer and higher molecular weight polymers in total transformed L. lactis extracts. Thus, it appeared that L. lactis is capable of expressing GBS 80 in oligomeric form. The high molecular weight polymers were not only detected in L. lactis extracts, but also in the culture supernatants. See Figure 135 at lane 4. See also Example 14. Thus, the GBS AI polypeptides in oligomeric form can be isolated and purified from either L. lactis cell extracts or culture supernatants. These oligomeric forms can, for instance, be isolated from cell extracts or culture supernatants by release by sonication. See Figure 136A and B. See also Figure 171, which shows purification of GBS pili from whole extracts of L. lactis expressing the GBS AI-I following sonication and gel filtration on a Sephacryl HR 400 column. F" C "f itfOMS&lUeX ilibSKIdi'fSitned with the construct encoding GBS AI-I polypeptides GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences expressed the GBS AI-I polypeptides on its surface. FACS analysis of these transformed L. lactis detected cell surface expression of both GBS 80 and GBS 104. The surface expression levels of GBS 80 and GBS 104 on the transformed L. lactis were similar to the surface expression levels of GBS 80 and GBS 104 on GBS strains COHl and JM9130013, which naturally express GBS AI-I. See Figure 169 for FACS analysis data for L. lactis transformed with GBS AI-I and wildtype JM9130013 bacteria using anti-GBS 80 and GBS 104 antisera. Table 40 provides the results of FACS analysis of transformed L. lactis, COHl, and JM9130013 bacteria using anti-GBS 80 and anti-GBS 104 antisera. The numbers provided represent the mean fluorescence value difference calculated for immune versus pre-immune sera obtained for each bacterial strain.
Table 40: FACS analysis of Z. lactis and GBS bacteria strains expressing GBS AI-I
Figure imgf000046_0001
Immunogold-electronmicroscopy performed with anti-GBS 80 primary antibodies detected the presence of pilus structures on the surface of the L. lactis bacteria expressing GBS AI-I, confirming the results of the FACS analysis. See Figure 168 B and C. Interestingly, this expression of GBS pili on the surface of the L. lactis induced L. lactis aggregation. See Figure 170. Thus, GBS AI polypeptides may also be isolated and purified from the surface of L. lactis. The ability of L. lactis to express GBS AI polypeptides on its surface also demonstrates that it may be useful as a host to deliver GBS AI antigens.
In fact, immunization of mice with L. lactis transformed with GBS AI-I was protective in a subsequent challenge with GBS. Female mice were immunized with L. lactis transformed with GBS AI-I . The immunized female mice were bred and their pups were challenged with a dose of GBS sufficient to kill 90% of non-immunized pups. Detailed protocols for intranasal and subcutaneous immunization of mice with transformed L. lactis can be found in Examples 18 and 19, respectively. Table 43 provides data showing that immunization of the female mice with L. lactis expressing GBS AI-I (LL-AI 1) greatly increased survival rate of challenged pups relative to both a negative PBS control (PBS) and a negative L. lactis control (LL 10 E9, which is wild type L. lactis not transformed to express GBS AI-I).
Table 43: Protection of Mice Immunized with L. lactis expressing GBS AI-I
Figure imgf000046_0002
Figure imgf000047_0001
Table 51 provides further evidence that immunization of mice with L. lactis transformed with GBS AI-I is protective against GBS.
Table 51: Further Protection of Mice Immunized with L. lactis expressing GBS AI-I
, Antigen ; Immunization Alive/Treated Survival % route (Aral <0.0000001)
Recombinant GBS 80 IP 48/50 92
Recombinant GBS 80 SC 21/30 70
L. lactis* AEl 1O6 cfu SC 6/66 9
L. lactis +ALl 107 cfu SC 47/70 73
L.lactis+ALl 1O8 cfu SC 116/153 76
Llaetis+ALl 1O9 cfu SC 98/118 83
LJactis+ALl 1O10 cfu SC 107/129 83
L.lactis 1O10 cfu SC 4/83 5
PBS SC 6/110 5
L.lactis+ALl 1O10 cfu IN 51/97 52
L.lactis 1O11 cfu IN 1/40 7
PBS ^ " IN - 0/37 O
Protection of immunized mice with L. lactis expressing the GBS AI-I is at least partly due to a newly raised antibody response. Table 46 provides anti-GBS 80 antibody titers detected in serum of the mice immunized with Z. lactis expressing the GBS AI-I as described above. Mice immunized with Z. lactis expressing the GBS AI-I have anti-GBS 80 antibody titres, which are not observed in mice immunized with L. lactis not transformed to express the GBS AI- 1. Further, as expected from the survival data, mice subcutaneously immunized with Z. lactis transformed to express the GBS AI-I have significantly higher serum anti-GBS 80 antibody titers than mice intranasally immunized with L. lactis transformed to express the GBS AI-I.
Table 46: Antibody Responses against GBS 80 in Serum of Mice Immunized with L. lactis Expressing GBS AI-I
Figure imgf000047_0002
Anti-GBS 80 antibodies of the IgA isotype were specifically detected in various body flμids of the mice subcutaneously or intranasally immunized with Z. lactis expressing the GBS AI-I.
Table 47: Anti-GBS 80 IgA Antibodies Detected in Mouse Tissues Following Immunization with L. lactis Expressing GBS AI-I P1 1!
Figure imgf000048_0001
Furthermore, opsonophagocytosis assays also demonstrated that at least some of the antiserum produced against the Z. lactis expressing GBS AI 1 is opsonic for GBS. See Figure 161.
To obtain protection of against GBS across a greater number of strains and serotypes, it is possible to transform L. lactis with a recombinant GBS AI encoding both GBS AI-I and AI-2, i.e., a hybrid GBS AI. By way of example, a hybrid GBS AI may be a GBS AI-I with a replacement of the GBS 104 gene with a GBS 67 gene. A schematic of such a hybrid GBS AI is depicted in Figure 231 A. A hybrid GBS AI may alternatively be a GBS AI-I with a replacement of the GBS 52 gene with a GBS 59 gene. See the schematic at Figure 231 B. Alternatively, a hybrid GBS AI may be a GBS AI- 1 with a substitution of a GBS 59 polypeptide for the GBS 52 gene and a substitution of the GBS 104 gene for genes encoding GBS 59 and the two GBS AI-2 sortases. Another example of a hybrid GBS AI is a GBS AI-I with the substitution of a GBS 59 gene for the GBS 52 gene and a GBS 67 for the GBS 104 gene. See the schematic at Figure 232. A further example of a hybrid GBS AI is a GBS AI- 1 having a GBS 59 gene and genes encoding the GBS AI-2 sortases in place of the GBS 52 gene. Yet another example of a hybrid GBS AI is a GBS AI-I with a substitution of either GBS 52 or GBS 104 with a fusion protein comprising GBS 322 and one of GBS 59, GBS 67, or GBS 150. Some of these hybrid GBS AIs may be prepared as briefly outlined in Figure 234 A-F.
Applicants have prepared a hybrid GBS AI having a GBS AI-I sequence with a substitution of a GBS 67 coding sequence for the GBS 104 gene as depicted in Figure 231 A. Transformation of L. lactis with the hybrid GBS AI-I resulted in L. lactis expression of high molecular weight polymers containing the GBS 80 and GBS 67 proteins. See Figure 233 A, which provides Western blot analysis of L. lactis transformed with the hybrid GBS AI depicted in Figure 231 A. When L. lactis transformed with the hybrid GBS AI were probed with antibodies to GBS 80 or GBS 67, high molecular weight structures were detected. See lanes labelled LL + a) in both the α-80 and α-67 immunoblots. The GBS 80 and GBS 67 proteins were confirmed to be present on the surface of L. lactis by FACS analysis. See Figure 233 B, which shows a shift in fluorescence when GBS 80 and GBS 67 antibodies are used to detect GBS 80 and GBS 67 surface expression. The same shifts in fluorescence were not observed in L. lactis control cells, cells not transformed with the hybrid GBS AI.
Alternatively, the oligomeric, pilus-like structures may be produced recombinantly. If produced in a recombinant host cell system, the AI surface protein will preferably be expressed in coordination with the expression of one or more of the AI sortases of the invention. Such AI sortases will facilitate oligomeric or hyperoligomeric formation of the AI surface protein subunits. IP C lϊ^'oiAsife'lΘ^ite-^inri'SwBϊ'^pically have a signal peptide sequence within the first 70 amino acid residues. They may also include a transmembrane sequence within 50 amino acid residues of the C terminus. The sortases may also include at least one basic amino acid residue within the last 8 amino acids. Preferably, the sortases have one or more active site residues, such as a catalytic cysteine and histidine.
As shown in Figure 1, AI-I includes the surface exposed proteins of GBS 80, GBS 52 and GBS 104 and the sortases SAG0647 and SAG0648. AI-I typically appears as an insertion into the 3' end of the trmA gene.
In addition to the open reading frames encoding the AI-I proteins, AI-I may also include a divergently transcribed transcriptional regulator such as araC (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). It is believed that araC may regulate the expression of the AI operon. (See Korbel et al., Nature Biotechnology (2004) 22(7): 911 — 917 for a discussion of divergently transcribed regulators in E. coli). AI-I may also include a sequence encoding a rho independent transcriptional terminator (see hairpin structure in Figure 1). The presence of this structure within the adhesin island is thought to interrupt transcription after the GBS 80 open reading frame, leading to increased expression of this surface protein.
A schematic identifying AI-I within several GBS serotypes is depicted in Figure 2. AI-I sequences were identified in GBS serotype V, strain isolate 2603; GBS serotype III, strain isolate NEM316; GBS serotype II, strain isolate 18RS21; GBS serotype V, strain isolate CJBl 11; GBS serotype III, strain isolate COHl and GBS serotype Ia, strain isolate A909. (Percentages shown are amino acid identity to the 2603 sequence). (An AI-I was not identified in GBS serotype Ib, strain isolate H36B or GBS serotype Ia, strain isolate 515). An alignment of AI-I polynucleotide sequences from serotype V, strain isolates 2603 and
CJBl I l; serotype II, strain isolate 18RS21; serotype III, strain isolates COHl and NEM316; and serotype Ia, strain isolate A909 is presented in Figure 18. An alignment of amino acid sequences of AI- 1 surface protein GBS 80 from serotype V, strain isolates 2603 and CJB 111; serotype 1 a, strain isolate A909; serotype III, strain isolates COHl and NEM316 is presented in Figure 22. An alignment of amino acid sequences of AI-I surface protein GBS 104 from serotype V, strain isolates 2603 and CJBl 11; serotype III, strain isolates COHl and NEM316; and serotype II, strain isolate 18RS21 is presented in Figure 23. Preferred AI-I polynucleotide and amino acid sequences are conserved among two or more GBS serotypes or strain isolates.
As shown in this figure, the full length of surface protein GBS 80 is particularly conserved among GBS serotypes V (strain isolates 2603 and CJBIII), III (strain isolates NEM316 and COHl), and Ia (strain isolate A909). The GBS 80 surface protein is missing or fragmented in serotypes II (strain isolate 18RS21), Ib (strain isolate H36B) and Ia (strain isolate 515).
Polynucleotide and amino acid sequences for AraC are set forth in FIGURE 30. GiMAcfcϊnyiMlEii / iS! 7 ≡ 3 ^
A second adhesin island, "Adhesin Island 2" or "AI-2" or "GBS AI-2" has also been identified in numerous GBS serotypes. A schematic depicting the correlation between AI-I and AI-2 within the GBS serotype V, strain isolate 2603 is shown in Figure 3. (Homology percentages in Figure 3 represent amino acid identity of the AI-2 proteins to the AI-I proteins). Alignments of AI-2 polynucleotide sequences are presented in Figures 20 and 21 (Figure 20 includes sequences from serotype V, strain isolate 2603 and serotype III, strain isolate NEM316. Figure 21 includes sequences from serotype III, strain isolate COHl and serotype Ia, strain isolate A909). An alignment of amino acid sequences of AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJBl 11; serotype Ia, strain isolate 515; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype III, strain isolate NEM316 is presented in Figure 24. Preferred AI-2 polynucleotide and amino acid sequences are conserved among two or more GBS serotypes or strain isolates.
AI-2 comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, AI-2 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5 or more) of GBS 67, GBS 59, GBS 150,
SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525. In one embodiment, AI-2 includes open reading frames encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406. Alternatively, AI-2 may include open reading frames encoding for two or more of 01520, 01521, 01522, 01523, 01523, 01524 and 01525. One or more of the surface proteins typically include an LPXTG motif (such as LPXTG (SEQ
ID NO: 122)) or other sortase substrate motif. The GBS AI-2 sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GBS AI-2 may encode for at least one surface protein. Alternatively, AI-2 may encode for at least two surface proteins and at least one sortase. Preferably, GBS AI-2 encodes for at least three surface proteins and at least two sortases. One or more of the AI-2 surface proteins may include an LPXTG or other sortase substrate motif.
One or more of the surface proteins may also typically include pilin motif. The pilin motif may be involved in pili formation. Cleavage of AI surface proteins by sortase between the threonine and glycine residue of an LPXTG motif yields a thioester-linked acyl intermediate of sortase. The first lysine residue in a pilin motif can serve as an amino group acceptor of the cleaved LPXTG motif and thereby provide a covalent linkage between AI subunits to form pili. For example, the pilin motif can make a nucleophilic attack on the acyl enzyme providing a covalent linkage between AI subunits to form pili and regenerate the sortase enzyme. Some examples of pilin motifs that may be present in the GBS AI-2 proteins include ((YPKN(X8)K; SEQ ID NO: 158), (PK(X8)K; SEQ ID NO: 159), (YPK(X9)KjSEQ ID NO: 160), (PKN(X8)K; SEQ ID NO: 161), or (PK(Xi0)K; SEQ ID NO: 162)).
One or more of the surface protein may also include an E box motif. The E box motif contains a conserved glutamic acid residue that is believed to be necessary for pilus formation. Some examples of E box motifs may include the amino acid sequences YxLxETxAPxG (SEQ ID NO: 163),
Figure imgf000051_0001
165), or YxLxETxAPxGY
(SEQ ID NO: 152).
As shown in Figure 3, GBS AI-2 may include the surface exposed proteins of GBS 67, GBS 59 and GBS 150 and the sortases of SAG1406 and SAG1405. Alternatively, GBS AI-2 may include the proteins 01521, 01524 and 01525 and sortases 01520 and 01522. GBS 067 and 01524 are preferred AI-2 surface proteins.
AI-2 may also include a divergently transcribed transcriptional regulator such as a Ro fA like protein (for example rogB). As in AI-I, rogB is thought to regulate the expression of the AI-2 operon.
A schematic depiction of AI-2 within several GBS serotypes is depicted in Figure 4. (Percentages shown are amino acid identity to the 2603 sequence). While the AI-2 surface proteins GBS 59 and GBS 67 are more variable across GBS serotypes than the corresponding AI-I surface proteins, AI-2 surface protein GBS 67 appears to be conserved in GBS serotypes where the AI-I surface proteins are disrupted or missing.
For example, as discussed above and in Figure 2, the AI-I GBS 80 surface protein is fragmented in GBS serotype II, strain isolate 18RS21. Within AI-2 for this same sequence, as shown in Figure 4, the GBS 67 surface protein has 99% amino acid sequence homology with the corresponding sequence in strain isolate 2603. Similarly, the AI-I GBS 80 surface protein appears to be missing in GBS serotype Ib, strain isolate H36B and GBS serotype Ia, strain isolate 515. Within AI-2 for these sequences, however, the GBS 67 surface protein has 97 — 99 % amino acid sequence homology with the corresponding sequence in strain isolate 2603. GBS 67 appears to have two allelic variants, which can be divided according to percent homology with strains 2603 and H36B. See figures 237-239.
Unlike for GBS 67, amino acid sequence identity of GBS 59 is variable across different GBS strains. As shown in Figures 63 and 224, GBS 59 of GBS strain isolate 2603 shares 100% amino acid residue homology with GBS strain 18RS21, 62% amino acid sequence homology with GBS strain H36B, 48% amino acid residue homology with GBS strain 515 and GBS strain CJBl 11, and 47% amino acid residue homology with GBS strain NEM316. The amino acid sequence homologies of the different GBS strains suggest that there are two isoforms of GBS 59. The first isoform appears to include the GBS 59 protein of GBS strains CJBl I l, NEM316, and 515. The second isoform appears to include the GBS 59 protein of GBS strains 18RS21, 2603, and H36B. (See Figures 63 and 224.)
As expected from the variability in GBS 59 isoforms, antibodies specific for the first GBS 59 isoform detect the first but not the second GBS 59 isoform and antibodies specific for the second GBS 59 isoform detect the second but not the first GBS 59 isoform. See Figure 226A, which shows FACS analysis of 28 GBS strains having a GBS 59 gene detected using PCR for GBS 59 surface expression. For each of the 28 GBS strains, FACS analysis was performed using either an antibody for GBS 59 isoform 1 (α-cjbl 11) or GBS 59 isoform 2 (α -2603). Only one of the two antibodies detected GBS 59 surface expression on each GBS strain. As a negative control, GBS strains in which a GBS 59 v gfieQJnόt iJtl8tΘll;by!'pS]R!1'Sii"no! nive significant GBS 59 surface expression levels. Figure 226B.
Also, GBS 59 is opsonic only against GBS strains expressing a homologous GBS 59 protein. See Figure 225. In one embodiment, the immunogenic composition of the invention comprises a first and a second isoform of the GBS 59 protein to provide protection across a wide range of GBS serotypes that express polypeptides from a GBS AI-2. The first isoform may be the GBS 59 protein of GBS strain CJBl 11, NEM316, or 515. The second isoform may be the GBS 59 protein of GBS strain 18RS21, 2603, or H36B. The gene encoding GBS 59 has been identified in a high number of GBS isolates; the GBS 59 gene was detected in 31 of 40 GBS isolates tested (77.5%). The GBS 59 protein also appears to be present as part of a pilus in whole extracts derived from GBS strains. Figure 64 shows detection of high molecular weight GBS 59 polymers in whole extracts of GBS strains CJB 111 , 7357B, COH31 , D1363C, 5408, 1999, 5364, 5518, and 515 using antiserum raised against GBS 59 of GBS strain CJB 111. Figure 65 also shows detection of these high molecular weight GBS 59 polymers in whole extracts of GBS strains D136C, 515, and CJBl 11 with anti-GBS 59 antiserum. (See also Figure 220 A for detection of GBS 59 high molecular weight polymers in strain 515.) Figure 65 confirms the presence of different isoforms of GBS 59. Antisera raised against two different GBS 59 isoforms results in different patterns of immunoreactivity depending on the GBS strain origin of the whole extract. Figure 65 further shows detection of GBS 59 monomers in purified GBS 59 preparations.
GBS 59 is also highly expressed on the surface of GBS strains. GBS 59 was detected on the surface of GBS strains CJB 111 , DKl , DK8, Davis, 515, 2986, 5551 , 1169, and 7357B by FACS analysis using mouse antiserum raised against GBS 59 of GBS CJBl 11. FACS analysis did not detect surface expression of GBS 59 in GBS strains SMU071, JM9130013, and COHl, which do not contain a GBS 59 gene. (See Figure 66.) Further confirmation that GBS 59 is expressed on the surface of GBS is detection of GBS 59 by immuno-electron microscopy on the surface of GBS strain 515 bacteria. See Figure 215.
GBS 67 and GBS 150 also appear to be included in high molecular weight structures, or pili. Figure 69 shows that anti-GBS 67 and anti-GBS 150 immunoreact with high molecular weight structures in whole GBS strain 515 extracts. (See also Figure 220 B and C.) It is also notable in Figure 69 that the anti-GBS 59 antisera, raised in a mouse following immunization with GBS 59 of GBS strain 2603, does not cross-hybridize with GBS 59 in GBS strain 515. GBS 59 of GBS stain 515 is of a different isotype than GBS 59 of GBS stain 2603. See Figure 63, which illustrates that the homology of these two GBS 59 polypeptides is 48%, and Figure 65, which confirms that GBS 59 antisera raised against GBS strain 2603 does not cross-hybridize with GBS 59 of GBS strain 515.
Formation of pili containing GBS 150 does not appear to require GBS 67 expression. Figure 70 provides Western blots showing that higher molecular weight structures in GBS strain 515 total e^iy§tslimώ^ikθlwith''a≤i"i'esl:έ'll3li anti-GBS 150 antiserum. In a GBS strain 515 lacking GBS 67 expression, anti-GBS 67 antiserum no longer immunoreacts with polypeptides in total extracts, while anti-GBS 150 antiserum is still able to cross-hybridze with high molecular weight structures. Likewise, formation of pili containing GBS 59 does not appear to require GBS 67 expression.
As expected, FACS detects GBS 67 cell surface expression on wildtype GBS strain 515, but not GBS strain 515 cells knocked out for GBS 67. FACS analysis using anti-GBS 59 antisera, however, detects GBS 59 expression on both the wildtype GBS strain 515 cells and the GBS strain 515 cells knocked out for GBS 67. Thus, GBS 59 cell surface expression is detected on GBS stain 515 cells regardless of GBS 67 expression.
GBS 67, while present in pili, appears to be localized around the surface of GBS strain 515 cells. See the immuno-electron micrographs presented in Figure 216. GBS 67 binds to fibronectin. See Figure 217.
Formation of pili encoded by GBS AI-2 does require expression of GBS 59. Deletion of GBS 59 from strain 515 bacteria eliminates detection of high molecular weight structures by antibodies that bind to GBS 59 (Figure 221 A, lane 3), GBS 67 (Figure 221 B, lane 3), and GBS 150 (Figure 221 C, lane 3). By contrast, Western blot analysis of 515 bacteria with a deletion of the GBS 67 gene detects high molecular weight structures using GBS 59 (Figure 221 A, lane 2) and GBS 150 (Figure 221 C, lane 2) antisera. Similarly, Western blot analysis of 515 bacteria with a deletion of the GBS 150 gene detects high molecular weight structures using GBS 59 (Figure 221 A, lane 4) and GBS 67 (Figure 221 B, lane 4). See also Figure 223, which provides Western blots of each of the 515 strains interrogated with antibodies for GBS 59, GBS 67, and GBS 150. FACS analysis of strain 515 bacteria deleted for either GBS 59 or GBS 67 confirms these results. See Figure 222, which shows that only deletion of GBS 59 abolishes surface expression of both GBS 59 and GBS 67. Formation of pili encoded by GBS AI-2 also requires expression of both GBS adhesin island-
2 encoded sortases. See Figure 218, which provides Western blot analysis of strain 515 bacteria lacking Srtl, Srt2, or both Srtl and Srt2. Only deletion of both Srtl and Srt2 abolishes pilus assembly as detected by antibodies that cross-hybridize with each of GBS 59, GBS 67 and GBS 150. The results of the Western blot analysis were verified by FACS, which provided similar results. See Figure 219.
As shown in Figure 4, two of the GBS strain isolates (COH 1 and A909) do not appear to contain homologues to the surface proteins GBS 59 and GBS 67. For these two strains, the percentages shown in Figure 4 are amino acid identity to the COHl protein). Notwithstanding the difference in the surface protein lengths for these two strains, AI-2 within these sequences still contains two sortase proteins and three LPXTG containing surface proteins, as well as a signal peptidase sequence leading into the first surface protein. One of the surface proteins in this variant of AI-2, spbl , has previously been identified as a potential adhesion protein. (See Adderson et al.,
Infection and Immunity (2003) 71(12):6857 - 6863). Alternatively, because of the lack of GBS 59
Figure imgf000054_0001
be a third type of AI (Adhesin Island-3, AI-3, or GBS AI-3).
More than one AI surface protein may be present in the oligomeric, pilus-like structures of the invention. For example, GBS 59 and GBS 67 may be incorporated into an oligomeric structure. Alternatively, GBS 59 and GBS 150 may be incorporated into an oligomeric structure, or GBS 59, GBS 150 and GBS 67 may be incoiporated into an oligomeric structure.
In another embodiment, the invention includes compositions comprising two or more AI surface proteins. The composition may include surface proteins from the same adhesin island. For example, the composition may include two or more GBS AI-2 surface proteins, such as GBS 59, GBS 67 and GBS 150. The surface proteins may be isolated from Gram positve bacteria or they may be produced recombinantly.
GAS Adhesin Islands
As discussed above, Applicants have identified at least four different GAS Adhesin Islands. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus. Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fascilitis. In addition, post-streptococcal autoimmune responses are still a major cause of cardiac pathology in children.
Group A Streptococcal infection of its human host can generally occur in three phases. The first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces. Generally this attachment phase begins in the throat or the skin.. The deeper the tissue level infected, the more severe the damage that can be caused. In the second stage of infection, the bacteria secretes a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. This toxin binds to susceptible host cell receptors and triggers innappropropriate immune responses by these host cells, resulting in pathology. Because the toxin can diffuse throughout the host, the necrosis directly caused by the GAS toxins may be physically located in sites distant from the bacterial infection. The final phase of GAS infection can occur long after the original bacteria have been cleared from the host system. At this stage, the host's previous immune response to the GAS bacteria due to cross reactivity between epitopes of a GAS surface protein, M, and host tissues, such as the heart. A general review of GAS infection can be found in Principles of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001). In order to prevent the pathogenic effects associated with the later stages of GAS infection, an effective vaccine against GAS will preferably facilitate host elimination of the bacteria during the initial attachment and invasion stage. IF" C "lolaAI Ii$loip.A IlrepliiθicΘ are historically classified according to the M surface protein described above. The M protein is surface exposed trypsin-sensitive protein generally comprising two polypeptide chains complexed in an alpha helical formation. The carboxyl terminus is anchored in the cytoplasmic membrane and is highly conserved among all group A streptococci. The amino terminus, which extend through the cell wall to the cell surface, is responsible for the antigenic variability observed among the 80 or more serotypes of M proteins.
A second layer of classification is based on a variable, trypsin-resistant surface antigen, commonly referred to as the T-antigen. Decades of epidemiology based on M and T serological typing have been central to studies on the biological diversity and disease causing potential of Group A Streptococci. While the M-protein component and its inherent variability have been extensively characterized, even after five decades of study, there is still very little known about the structure and variability of T-antigens. Antisera to define T types is commercially available from several sources, including Sevapharma (http://www.sevapharma.cz/en).
The gene coding for one form of T-antigen, T-type 6, from an M6 strain of GAS (D741) has been cloned and characterized and maps to an approximately 11 kb highly variable pathogenicity island. Schneewind et al., J Bacteriol. (1990) 172(6):3310 - 3317. This island is known as the Fibronectin-binding, Collagen-binding T-antigen (FCT) region because it contains, in addition to the T6 coding gene {teeδ), members of a family of genes coding for Extra Cellular Matrix (ECM) binding proteins. Bessen et al.3 Infection & Immunity (2002) 70(3): 1159-1167. Several of the protein products of this gene family have been shown to directly bind either fibronectin and/or collagen. See Hanski et al., Infection & Immunity (1992) 60(12):5119-5125; Talay et al., Infection & Immunity (1992( 60(9):3837-3844; Jaffe et al. (1996) 21(2):373-384; Rocha et al., Adv Exp Med Biol. (1997) 418:737-739; Kreikemeyer et al., J Biol Chem (2004) 279(16):15850-15859; Podbielski et al., MoI. Microbiol. (1999) 31(4):1051-64; and Kreikemeyer et al., Int. J. Med Microbiol (2004) 294(2-3):177- 88. In some cases direct evidence for a role of these proteins in adhesion and invasion has been obtained.
Applicants raised antiserum against a recombinant product of the teeδ gene and used it to explore the expression of T6 in M6 strain 2724. In immunoblot of mutanolysin extracts of this strain, the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used.
This pattern of high molecular weight products is similar to that observed in immunoblots of the protein components of the pili identified in Streptococcus agalactiae (described above) and previously in Coiγnebacterium diphtheriae. Electron microscropy of strain M6_2724 with antisera specific for the product of teeό revealed abundant surface staining and long pilus like structures extending up to 700 nanometers from the bacterial surface, revealing that the T6 protein, one of the antigens recognized in the original Lancefiled serotyping system, is located within a GAS Adhesin
Island (GAS AI-I) and forms long covalently linked pilus structures. 1" :' Il ΑpplMiϊfs
Figure imgf000056_0001
different Group A Streptococcus Adhesin Islands.
While these GAS AI sequences can be identified in numerous M types, Applicants have surprisingly discovered a correlation between the four main pilus subunits from the four different GAS AI types and specific T classifications. While other trypsin-resistant surface exposed proteins are likely also implicated in the T classification designations, the discovery of the role of the GAS adhesin islands (and the associated hyper-oligomeric pilus like structures) in T classification and GAS serotype variance has important implications for prevention and treatment of GAS infections. Applicants have identified protein components within each of the GAS adhesin islands which are associated with the pilus formation. These proteins are believed to be involved in the bacteria's initial adherence mechanisms. Immunological recognition of these proteins may allow the host immune response to slow or prevent the bacteria's transition into the more pathogenic later stages of infection.
In addition, Applicants have discovered that the GBS pili structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysaccharide matrix). Biofilms are generally associated with bacterial resistance, as antibiotic treatments and host immune response are frequently unable to erradicate all of the bacteria components of the biofilm. Direction of a host immune response against surface proteins exposed during the first steps of bacterial attachment (i.e., before complete biofilm formation) is preferable.
The invention therefore provides for improved immunogenic compositions against GAS infection which may target GAS bacteria during their initial attachment efforts to the host epithelial cells and may provide protection against a wide range of GAS serotypes. The immunogenic compositions of the invention include GAS AI surface proteins which may be formulated in an oligomeric, or hyperoligomeric (pilus) form. The invention also includes combinations of GAS AI surface proteins. Combinations of GAS AI surface proteins may be selected from the same adhesin island or they may be selected from different GAS adhesin islands. While there is surprising variability in the number and sequence of the GAS AI components across isolates, GAS AI sequences may be generally characterized as Type 1, Type 2, Type 3, and Type 4, depending on the number and type of sortase sequence within the island and the percentage identity of other proteins within the island. Schematics of the GAS adhesin islands are set forth in FIGURE 51A and FIGURE 162.- In all strains identified so far, the adhesin island region is flanked by highly conserved open reading frames Ml_123 and Ml_136. Between three and five genes in each GAS adhesin island code for ECM binding adhesin proteins containing LPXTG motifs. GAS Adhesin Island 1
As discussed above, Applicants have identified adhesin islands, "GAS Adhesin Island 1" or "GAS AI-I", within the genome Group A Streptococcus serotypes and isolates. GAS AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-I proteins"). GAS AI-I preferably comprises surface proteins, a srtB sortase, and a rofA divergently transcribed transcriptional regulator. GAS AI-
1 surface proteins may include a fibronectin binding protein, a collagen adhesion protein and a FfflylpmflWc^aiiyulSitJ'^iPelSrSilpiyafeh of these GAS AI-I surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with serine). Specifically, GAS AI-I includes open reading frames encoding for two or more (i.e., 2, 3, 4 or 5) of M6_SpyO157, M6_SpyO158, M6_SpyO159, M6_SpyO 160, M6_Spy0161.
Applicants have also identified open reading frames encoding fimbrial structural subunits in other GAS bacteria harbouring an AI-I. These open reading frames encode fimbrial structural subunits CDC SS 410j£ϊmbrial, ISS3650_fimbrial, and DSM2071_fimbrial. A GAS AI-I may comprise a polynucleotide encoding any one of CDC SS 410_fϊmbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
As discussed above, the hyper-oligomeric pilus structure of GAS AI-I appears to be responsible for the T-antigen type 6 classification, and GAS AI-I corresponds to the FCT region previously identified for teeό. As in GAS AI-I, the teeό FCT region includes open reading frames encoding for a collagen adhesion protein (cpa, capsular polysaccharide adhesion) and a fibronectin binding protein (prtFl). Immunoblots of teeό, a GAS AI-I fimbrial structural subunit corresponding to M6_Sρyl60, reveal high molecular weight structures indicative of the hyper-oligomeric pilus structures. Immunoblots with antiserum specific for Cpa also recognize a high molecular weight ladder structure, indicating Cpa involvement in the GAS AI-I pilus structure or formation. In EM photos of GAS bacteria, Cpa antiserum reveals abundant staining on the surface of the bacteria and occasional gold particles extended from the surface of the bacteria. In contrast, immunoblots with antiserum specific for PrtFl recognize only a single molecular species with electrophoretic mobility corresponding to its predicted molecular mass, indicating that PrtFl may not be associated with the oligomeric pilus structure. A preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which may be formulated or purified in an oligomeric (pilis) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Another preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which has been isolated in an oligomeric (pilis) form. The oligomer or hyperoligomeric pilus structures comprising the GAS AI-I surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
One or more of the GAS AI-I open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-I open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the GAS AI-I surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. The LPXTG sortase substrate motif of a GAS AI surface protein may be generally represented by the formula XXXXG, wherein X at amino acid position 1 is an L, a V, an E, or a Q, wherein X at amino acid position 2 is a P if X at amino acid position 1 is an L, wherein X at amino acid position 2 is a V if X at amino acid position 1 is a E or a Q, wherein X at amino acid position 2 is
Figure imgf000058_0001
V, wherein X at amino acid position 3 is any amino acid residue, wherein X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, or Q, and wherein X at amino acid position 4 is a T, S, or A if X at amino acid position 1 is an L. Some examples of LPXTG motifs present in GAS AI surface proteins include LPSXG (SEQ ID NO: 134), VVXTG (SEQ ID NO: 135), EVXTG (SEQ ID NO: 136), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138), LPXAG (SEQ ID NO: 139), QVPTG (SEQ ID NO: 140), and FPXTG (SEQ ID NO: 141).
The GAS AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer. Preferably, one or more GAS AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. GAS AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The GAS AI-I sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-I may encode for at least one surface protein. Alternatively, GAS AI-I may encode for at least two surface exposed proteins and at least one sortase. Preferably, GAS AI-I encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. GAS AI-I preferably includes a srtB sortase. GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a GAS AI-I surface protein such as M6_Spy0157, M6_SpyO159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, or DSM2071_fimbrial. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The ■ oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively. AI surface proteins or fragments thereof to be incorporated into the oligomeπc, pilus-hke structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 1 ("GAS AI-I") proteins and one or more GAS Adhesin Island 2 ("GAS AI-2"), GAS Adhesin Island 3 ("GAS AI-3"), or GAS Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the GAS Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form. In addition to the open reading frames encoding the GAS AI-I proteins, GAS AI-I may also include a divergently transcribed transcriptional regulator such as RofA {i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). GAS Adhesin Island 2 A second adhesin island, "GAS Adhesin Island 2" or "GAS AI-2" has also been identified in
Group A Streptococcus serotypes and isolates. GAS AI-2 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-2 proteins"). Specifically, GAS AI-2 includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, 7, or 8) of GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SρyO133, and GAS20.
A preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which may be formulated or purified in an oligomeric (pills) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Another preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which has been isolated in an oligomeric (pilis) form. The oligomer or hyperoligomeric pilus structures comprising the GAS AI-2 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
One or more of the GAS AI-2 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-2 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the GAS AI-2 surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. The AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. AI surface proteins may also be able to bind to or associate with fibrinogen, fϊbronectin, or collagen. H"" !'-"
Figure imgf000060_0001
predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-2 may encode for at least one surface protein. Alternatively, GAS AI-2 may encode for at least two surface exposed proteins and at least one sortase. Preferably, GAS AI-2 encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as GAS 15, GAS 16, or GAS 18. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif. The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 2 ("GAS AI-2") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 3 ("GAS AI-3"), or GAS Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the GAS AI-2 proteins, GAS AI-2 may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). GAS Adhesin Island 3
A third adhesin island, "GAS Adhesin Island 3" or "GAS AI-3" has also been identified in several Group A Streptococcus serotypes and isolates. GAS AI-3 comprises a series of approximately sdvefirόperf'reMuϊg mine's eh'cό'αirig for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-3 proteins"). Specifically, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, SPsOlOO, orf78, orf79, orfSO, orf81, orf82, orf83, orf84, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, sρyM18_0131, spyM18_0132, SpyoM01000156, SpyoMO 1000155, SpyoMO 1000154, SpyoMO 1000153, SpyoMO 1000152, SpyoM01000151, SpyoM01000150, and SpyoMO 1000149. In one embodiment, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyM3_0098, SpyM3_0099, SρyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104. In another embodiment, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPsO104, SPs0105, and SPsOlOO. In a further embodiment, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of orf78, orf79, orfSO, orfδl, orf82, orf83, and orf84. In yet another embodiment, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132. In yet another embodiment, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyoMO 1000156, SpyoMO 1000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151, SpyoMO 1000150, and SpyoMO 1000149. Applicants have also identified open reading frames encoding fimbrial structural subunits in other GAS bacteria harbouring an AI-3. These open reading frames encode fimbrial structural subunits ISS3040Jϊmbrial, ISS3776_fimbrial, and ISS4959_fimbrial. A GAS AI-3 may comprise a polynucleotide encoding any one of ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959j5mbrial.
One or more of the GAS AI-3 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-3 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
A preferred immunogenic composition of the invention comprises a GAS AI-3 surface protein which may be formulated or purified in an oligomeric (pilis) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Another preferred immunogenic composition of the invention comprises a GAS AI-3 surface protein which has been isolated in an oligomeric (pilis) form. The oligomer or hyperoligomeric pilus structures comprising the GAS AI-3 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
One or more of the GAS AI-3 surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. The AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer.
Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an IP, Ii Ij'" / Il H c; in Kj; ,.■■' p> "7 ψ$ ""-I! CI)I epitH'elϊaϊ tfell 'Surface. '"Al surface protein's may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The GAS AI-3 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-3 may encode for at least one surface protein. Alternatively, GAS AI-3 may encode for at least two surface exposed proteins and at least one sortase. Preferably, GAS AI-3 encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine or alanine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., ' Infection & Immunity (2004) 72(5): 2710 - 2722. The invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPs0106, orf78, orfSO, orf82, orf84, spyM18_0126, spyM18_0128, spyM18_0130, spyM18_0132, SpyoM01000155, Sp'yoMO 1000153, SpyoMO 1000151, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyM3_0098, SpyM3_0100, SρyM3_0102, and SpyM3_0104. In another embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SPsOlOO, SPs0102, SPs0104, and SPsOlOo. In another embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as orf78, orf80, orf82, and orf84. In yet another embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as spyM18_0126, spyM18_0128, sρyM18_0130, and sρyM18_0132. In a further embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyoMO 1000155, SpyoM01000153, SρyoM01000151, and SpyoM01000149. In yet a further embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric siibiitrits may%e''covaleritly as'sό'cia'ϊed'via an LPXTG motif, preferably, via the threonine amino acid residue.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif. The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 3 ("GAS AI-3") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 2 ("GAS AI-2"), or GAS Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the GAS AI-3 proteins, GAS AI-3 may also include a transcriptional regulator such as Nra. GAS Adhesin Island 4 A fourth adhesin island, "GAS Adhesin Island 4" or "GAS AI-4" has also been identified in
Group A Streptococcus serotypes and isolates. GAS AI-4 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-4 proteins"). Specifically, GAS AI-4 includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, 7, or 8) of 19224134, 19224135, 19223136, 19223137, 19224138, 19224139, 19224140, and 19224141.
Applicants have also identified open reading frames encoding fimbrial structural subunits in other GAS bacteria harbouring an AI-4. These open reading frames encode fimbrial structural subunits 20010296_fimbrial, 20020069_fimbrial, CDC SS 635Jimbrial, ISS4883_fimbrial, and ISS4538_fimbrial. A GAS AI-4 may comprise a polynucleotide encoding any one of 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
One or more of the GAS AI-4 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the GAS AI-4 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
A preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which may be formulated or purified in an oligomeric (pilis) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Another preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which has been isolated in an oligomeric (pilis) form. The oligomer or hyperoligomeric pilus structures comprising the GAS AI-4 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
One or more of the GAS AI-4 surface protein sequences typically include an LPXTG motif
(such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. The AI surface proteins of the irϋelttioϊ iήaγ'4SJtlti:e! ability '1Of r'tie:;GA;έ bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer. Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The GAS AI-4 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-4 may encode for at least one surface protein. Alternatively, GAS AI-4 may encode for at least two surface exposed proteins and at least one sortase. Preferably, GAS AI-4 encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid IL The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fϊmbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 4 ("GAS AI-4") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I "), GAS Adhesin Island 2 ("GAS AI-2"), or GAS Adhesin Island 3 ("GAS AI-3") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form. ,n, it i!"1 .•■ Ii Ij li;;:;1 in li:;;;; , ■ :;:,:!> "';;ιr rn ":"!' iq
H" " IrradMiOn W thVbp'en fealmg1 frames encoding the GAS AI-4 proteins, GAS AI-4 may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction). The oligomeric, pilus-like structures of the invention may be combined with one or more additional GAS proteins. In one embodiment, the oligomeric, pilus-like structures comprise one or more AI surface proteins in combination with a second GAS protein.
The oligomeric, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an AI surface protein. The invention therefore includes a method for manufacturing an oligomeric AI surface antigen comprising culturing a GAS bacterium that expresses the oligomeric AI protein and isolating the expressed oligomeric AI protein from the GAS bacteria.
The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed AI protein.
Preferably, the AI protein is in a hyperoligomeric form. The oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing an AI surface protein. The invention therefore includes a method for manufacturing an oligomeric Adhesin Island surface antigen comprising culturing a GAS bacterium adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the GAS bacteria. The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed
Adhesin Island protein. Preferably, the Adhesin Island protein is in a hyperoligomeric form.
The GAS bacteria are preferably adapted to increase AI protein expression by at least two
(e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels. GAS bacteria may be adapted to increase AI protein expression by any means known in the art, including methods of increasing gene dosage and methods of gene upregulation. Such means include, for example, transformation of the GAS bacteria with a plasmid encoding the AI protein.
The plasmid may include a strong promoter or it may include multiple copies of the sequence encoding the AI protein. Optionally, the sequence encoding the AI protein within the GAS bacterial genome may be deleted. Alternatively, or in addition, the promoter regulating the GAS Adhesin
Island may be modified to increase expression.
The invention further includes GAS bacteria which have been adapted to produce increased levels of AI surface protein. In particular, the invention includes GAS bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein. In one embodiment, the Gram positive bacteria of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
The invention further includes GAS bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface. The GAS bacteria may be adapted
Figure imgf000066_0001
AI proteins on its surface by increasing expression levels of LepA polypeptide, or an equivalent signal peptidase, in the GAS bacteria. Applicants have shown that deletion of LepA in strain SF370 bacteria, which harbour a GAS AI-2, abolishes surface exposure of M and pili proteins on the GAS, Increased levels of LepA expression in GAS are expected to result in increased exposure of M and pili proteins on the surface of GAS. Increased expression of LepA in GAS may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation. The GAS bacteria adapted to have increased levels of LepA expression may additionally be adapted to express increased levels of at least one pili protein. Alternatively, the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes" Infection and Immunity (2004) 72(6):3444-3450). As used herein, non-pathogenic Gram positive bacteria refer to Gram positive bacteria which are compatible with a human host subject and are not associated with human pathogenisis. Preferably, the non-pathogenic bacteria are modified to express the AI surface protein in oligomeric, or hyper-oligomeric form. Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non-pathogenic Gram positive bacterial genome or inserted into a plasmid. The non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface. Alternatively, the AI surface protein may be isolated or purified from a bacterial culture of the non-pathogenic Gram positive bacteria. For example, the AI surface protein may be isolated from cell extracts or culture supernatants. Alternatively, the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria.
The non-pathogenic Gram positive bacteria may be used to express any of the GAS Adhesin Island proteins described herein. The non-pathogenic Gram positive bacteria are transformed to express an Adhesin Island surface protein. Preferably, the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase. The AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with pathogenic GAS.
Applicants modified L. lactis to demonstrate that, like GBS polypeptides, it can express GAS AI polypeptides. L. lactis was transformed with pAM401 constructs encoding entire pili gene clusters of AI-I, AI-2, and AI-4 adhesin islands. Briefly, the ρAM401 is a promoterless high-copy plasmid. The entire pili gene clusters of anM6 (AI-I), Ml (AI-2), and M12 (AI-4) bacteria were inserted into the pAM401 construct. The gene clusters were transcribed under the control their own (M6, Ml, or M12) promoter or the GBS promoter that successfully initiated expression of the GBS AI-I adhesin islands in Z. lactis, described above. Figure 172 provides a schematic depiction of GAS M6 (AI-I), ]yll1'VM-I);'ail!d'MΪϊtAΪ-4) idhέsSfisϊiϊmϊi and indicates the portions of the adhesin island sequences inserted in the pAM401 construct.
Each of the L. lactis transformed with one of the M6, Ml5 or M12 adhesin island gene clusters expressed high molecular weight structures that were immunoreactive with antibodies that bind to polypeptides present in their respective pili. Figures 173 A-C provide results of Western blot analysis of surface protein-enriched extracts of L. lactis transformed with M6 (Figure 173 A), Ml (Figure 173 B), or Ml 2 (Figure 173 C) adhesin island gene clusters using antibodies that bind to the fimbrial structural subunit encoded by each cluster. Figure 173 A at lanes 3 and 4 shows detection of high molecular structures in L. lactis transformed with an adhesin island pilus gene cluster from an Ml AI-2 using an antibody that binds to fimbrial structural subunit SpyO128. Figure 173B at lanes 3 and 4 shows detection of high molecular weight structures in L. lactis transformed with an adhesin island pilus gene cluster from an M 12 AI-4 using an antibody that binds to fimbrial structural subunit EftLSL.A. Figure 173C at lane 3 shows detection of high molecular weight structures in L. lactis transformed with an adhesin island pilus gene cluster from an M6 AI- 1 using an antibody that binds to fimbrial structural subunit M6_Spy0160. In figures 173 A-C, "pi" immediately following the notation of AI subtype indicates that the promoter present in the Adhesin Island is used to drive transcription of the adhesin island gene cluster and "p2" indicates that the promoter was the GBS promoter described above. Thus, it appears that I. lactis is capable of expressing the fimbrial structural subunits encoded by GAS adhesin islands in an oligomeric form. Alternatively, the oligomeric, pilus-like structures may be produced recombinantly. If produced in a recombinant host cell system, the AI surface protein will preferably be expressed in coordination with the expression of one or more of the AI sortases of the invention. Such AI sortases will facilitate oligomeric or hyperoligomeric formation of the AI surface protein subunits. S. pneumoniae from TIGR4 Adhesin Island As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae from TIGR4. The S. pneumoniae from TIGR4 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae from TIGR4 AI proteins includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, or 7) of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, and SP0468.
A preferred immunogenic composition of the invention comprises a S. pneumoniae from TIGR4 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Another preferred immunogenic composition of the invention comprises a S. pneumoniae from TIGR4 AI surface protein which has been isolated in an oligomeric (pilis) form. The oligomer or hyperoligomer pilus structures comprising S. pneumoniae surface proteins may be purified or otherwise formulated for use in immunogenic compositions. I'"1" **
Figure imgf000068_0001
TIGR4 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae from TIGR4 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF. One or more of the S. pneumoniae from TIGR4 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae from TIGR4 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae from TIGR4 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae from TIGR4 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The S. pneumoniae from TIGR4 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae from TIGR4 AI may encode for at least one surface protein. Alternatively, S. pneumoniae from TIGR4 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae from TIGR4 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae from TIGR4 AI surface protein such as SP0462, SP0463, SP0464, or SP0465. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif B" ' Trie oTigomeric'j'pilus'lfke sMctures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae from TIGR4 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae from TIGR4 AI proteins and one or more S. pneumoniae strain 670 AI proteins, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae from TIGR4 AI proteins, S. pneumoniae from TIGR4 AI may also include a transcriptional regulator. S. pneumoniae strain 670 Adhesin Island As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 670. The S. pneumoniae strain 670 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 670 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of orfl_670, or£3_670, orf4_670, orf5_670, orf6_670, orf7_670, orf8_670.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 670 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 670 AI surface protein which has been isolated in an oligomeric (pilis) form. One or more of the S. pneumoniae strain 670 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 670 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 670 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 670 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 670 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 670 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The S. pneumoniae strain 670 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 670 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 670 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae strain 670 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, prefeMbly M'twee'n lie threδήineW glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 670 AI surface protein such as orf3_670, orf4_670, or orf5_670. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 670 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 670 AI proteins and one or more S. pneumoniae from TIGR4 AI proteins, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 670 AI proteins, S. pneumoniae strain 670 AI may also include a transcriptional regulator. S. pneumoniae strain 14 CSR 10 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 14 CSR 10. The S. pneumoniae strain 14 CSR 10 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 14 CSR 10 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2J4CSR, ORF3_14CSR, ORF4J4CSR, ORF5_14CSR, ORF6_14CSR, ORF7_14CSR, ORF8J4CSR.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 14 CSR 10 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 14 CSR 10 AI surface protein which has been isolated in an oligomeric (pilis) form. ft"" " One oi" ώSMjf'tøe Erpiτe&όmάi strain 14 CSR 10 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 14 CSR 10 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF. One or more of the S. pneumoniae strain 14 CSR 10 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 14 CSR 10 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 14 CSR 10 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 14 CSR 10 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The S. pneumoniae strain 14 CSR 10 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 14 CSR 10 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 14 CSR 10 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae strain 14 CSR 10 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 14 CSR 10 AI surface protein such as orf3_CSR, orf4_CSR, or orf5_CSR. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif. in. ii T' / 11 1 iq; n iir ,■" p> 71 ;p Ti; qι
""" "■"" The ol'igδϊiiSfie; jiϊluSiike sTructufes may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 14 CSR 10 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 14 CSR 10 AI proteins, and one or more AI proteins of any of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 14 CSR 10AI proteins, S. pneumoniae strain 14 CSR 10 AI may also include a transcriptional regulator. S. pneumoniae strain 19A Hungary 6 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 19A Hungary 6. The S. pneumoniae strain 19A Hungary 6 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 19A Hungary 6 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF6_19AH, ORF7_19AH, ORF8_19AH.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 19A Hungary 6 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 19A Hungary 6 AI surface protein which has been isolated in an oligomeric (pilis) form.
One or more of the S. pneumoniae strain 19A Hungary 6 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 19A Hungary 6 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 19A Hungary 6 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 19A Hungary 6 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 19A Hungary 6 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 19A Hungary 6 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The S. pneumoniae strain 19A Hungary 6 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 19A Hungary 6 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 19A
Hungary 6 AI may encode for at least two surface exposed proteins and at least one sortase. Pi)'έiybabJy/S. |?keϊ?]wrøSe''stfSiiϊ'"ΪPΑ"ϊlύii!gary 6 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 19A Hungary 6 AI surface protein such as orβ_19AH, orf4_19AH, or orf5_19AH. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 19A Hungary 6 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 19A Hungary 6 AI proteins and one or more AI proteins from one of any one of S. pneumoniae from TIGR4, 670, 14 CSR 10, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI GR4 AI proteins, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 19A Hungary 6 AI proteins, S. pneumoniae strain 19A Hungary 6 AI may also include a transcriptional regulator. S. pneumoniae strain 19F Taiwan 14 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 19F Taiwan 14. The S. pneumoniae strain 19F Taiwan 14 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 19F
Taiwan 14 AI proteins includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, or 7) o|TOrøj#Jwii QSbI iM^oIPP 19FTW, ORF5_19FTW, ORF6_19FTW, ORF7J9FTW, ORF8_19FTW.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 19F Taiwan 14 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a 5. pneumoniae strain 19F Taiwan 14 AI surface protein which has been isolated in an oligomeric (pilis) form.
One or more of the S. pneumoniae strain 19F Taiwan 14 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORP. Alternatively, one or more of the S. pneumoniae strain 19F Taiwan 14 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 19F Taiwan 14 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 19F Taiwan 14 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 19F Taiwan 14 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 19F Taiwan 14 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The JS. pneumoniae strain 19F Taiwan 14 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 19F Taiwan 14 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 19F Taiwan 14 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae strain 19F Taiwan 14 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an Al sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 19F Taiwan 14 AI surface protein such as orf3_19FTW, orf4_19FTW, or orf5_19FTW. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 3|;14S/Ϊ5/5d,.Kj'l|:ii)^(i:'"!li6j92i| B-O, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 19F Taiwan 14 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 19F Taiwan 14 AI proteins and one or more AI proteins of any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 14 CSR 10, 23F Taiwan 15, or 23F Poland 16, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 19F Taiwan 14 AI proteins, S. pneumoniae strain 19F Taiwan 14 AI may also include a transcriptional regulator. S. pneumoniae strain 23F Poland 16 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 23F Poland 16. The S. pneumoniae strain 23F Poland 16 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 23F Poland 16 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_23FP, ORF3J23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, and ORF8_23FP.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Poland 16 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Poland 16 AI surface protein which has been isolated in an oligomeric (pilis) form. One or more of the S. pneumoniae strain 23F Poland 16 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 23F Poland 16 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 23F Poland 16 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 23F Poland 16 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may ^[afffcf.'fHbJkϊilit^ Εϊ S.'' pOevifiv&Mie li translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 23F Poland 16 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 23 F Poland 16 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The S. pneumoniae strain 23 F Poland 16 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 23F ' Poland 16 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 23F Poland 16 Al may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae strain 23F Poland 16 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface ' protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 23F Poland 16 AI surface protein such as orβ_23FP, orf4_23FP, or orf5_23FP. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, ' respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 23F Poland 16 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 23F Poland 16 AI proteins and one or more AI proteins from any one or more S. pneumoniae strains of TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 14 CSR 10, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form. p1 C 'In,a(MyoyBEe.''o^i!n'%SiniMmes encoding the S. pneumoniae strain 23F Poland 16 AI proteins, S. pneumoniae strain 23F Poland 16 AI may also include a transcriptional regulator. S. pneumoniae strain 23F Taiwan 15 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 23F Taiwan 15. The S. pneumoniae strain 23F Taiwan 15 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 23F Taiwan 15 AI proteins includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, or 7) of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, ORF8_23FTW.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Taiwan 15 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 23F Taiwan 15 AI surface protein which has been isolated in an oligomeric (pilis) form. One or more of the S. pneumoniae strain 23F Taiwan 15 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 23F Taiwan 15 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 23F Taiwan 15 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 23F Taiwan 15 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 23F Taiwan 15 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 23 F Taiwan 15 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The S. pneumoniae strain 23F Taiwan 15 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 23F Taiwan 15 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 23 F Taiwan 15 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae strain 23F Taiwan 15 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the asl^yffiuli^Iibaiiipepiliiialtiin^ilactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment,. the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 23F Taiwan 15 AI surface protein such as orG_23FTW, orf4_23FTW, or orf5_23FTW. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 23F Taiwan 15 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 23F Taiwan 15 AI proteins and one or more AI proteins from any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 14 CSR 10, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 23F Taiwan 15 AI proteins, S. pneumoniae strain 23F Taiwan 15 AI may also include a transcriptional regulator. ■!>■ pneumoniae strain 6B Finland 12 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 6B Finland 12. The S. pneumoniae strain 6B Finland 12 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 6B
Finland 12 AI proteins includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, or 7) of ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, ORF8_6BF.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Finland 12 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Finland 12 AI surface protein which has been isolated in an oligomeric (pilis) form.
One or more of the S. pneumoniae strain 6B Finland 12 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. ^ψe!^!^ypψlJι^S^SΛvø'<^BS^&^3ϋ^^ιoniae strain 6B Finland 12 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 6B Finland 12 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The >S. pneumoniae strain 6B Finland 12 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 6B Finland 12 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 6B Finland 12 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The S. pneumoniae strain 6B Finland 12 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 6B Finland 12 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 6B Finland 12 AI may encode for at least two surface exposed proteins and at least one sortase.
Preferably, S. pneumoniae strain 6B Finland 12 AI encodes for at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 6B Finland 12 AI surface protein such as orf3_6BF, orf4_6BF, or orf5_6BF. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif. ii; '" C l^?''°lil©iIli':iP,^i:lip' IMiMlS may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 6B Finland 12 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 6B Finland 12 AI proteins and one or more AI proteins of any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 6B Finland 12 AI proteins, S. pneumoniae strain 6B Finland 12 AI may also include a transcriptional regulator. S. pneumoniae strain 6B Spain 2 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 6B Spain 2. The S. pneumoniae strain 6B Spain 2 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 6B Spain 2 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_6BSP, ORF3_6BSP, ORF4 6BSP, ORF5 6BSP, ORF6_6BSP, ORF7_6BSP, and ORF8_6BSP.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Spain 2 AI surface protein which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 6B Spain 2 AI surface protein which has been isolated in an oligomeric (pilis) form.
One or more of the S. pneumoniae strain 6B Spain 2 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 6B Spain 2 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 6B Spain 2 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 6B Spain 2 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more S. pneumoniae strain 6B Spain 2 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 6B Spain 2 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen. The S. pneumoniae strain 6B Spain 2 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 6B Spain 2 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 6B Spain 2 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S.
Figure imgf000081_0001
at least three surface exposed proteins and at least two sortases.
The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 6B Spain 2 AI surface protein such as orβ_6BSP, orf4_6BSP, or orf5_6BSP. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively.
AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 6B Spain 2 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 6B Spain 2 AI proteins and one or more AI proteins of any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 14 CSR 10, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form.
In addition to the open reading frames encoding the S. pneumoniae strain 6B Spain 2 AI proteins, S. pneumoniae strain 6B Spain 2 AI may also include a transcriptional regulator. S. pneumoniae strain 9V Spain 3 Adhesin Island
As discussed above, Applicants have identified adhesin islands within the genome of S. pneumoniae strain 9V Spain 3. The S. pneumoniae strain 9V Spain 3 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases. Specifically, the S. pneumoniae strain 9V Spain 3 AI proteins includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP, and ORF8_9VSP.
A preferred immunogenic composition of the invention comprises a S. pneumoniae strain 9V Spain 3 AI surface protein which may be formulated or purified in an oligomeric (pilis) form. Another preferred immunogenic composition of the invention comprises a S. pneumoniae strain 9V Spain 3 Al surface protein which has been isolated in an oligomeric (pilis) form.
One or more of the S. pneumoniae strain 9 V Spain 3 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae strain 9V Spain 3 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae strain 9V Spain 3 AI surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
The S. pneumoniae strain 9V Spain 3 AI surface proteins of the invention may affect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. Preferably, one or more & pneumoniae strain 9V Spain 3 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. S. pneumoniae strain 9V Spain 3 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
The S. pneumoniae strain 9V Spain 3 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. S. pneumoniae strain 9 V Spain 3 AI may encode for at least one surface protein. Alternatively, S. pneumoniae strain 9V Spain 3 AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, S. pneumoniae strain 9V Spain 3 AI encodes for at least three surface exposed proteins and at least two sortases. The AI surface proteins may be covalently attached to the bacterial cell wall by membrane- associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a S. pneumoniae strain 9V Spain 3 AI surface protein such as orf3_9VSP, orf4_9VSP, or orf5_9VSP. The oligomeric, pilus-like structure may comprise numerous units of AI surface protein. Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively. AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a S. pneumoniae strain 9V Spain 3 AI protein in oligomeric form, preferably in a hyperoligomeric form. In one embodiment, the invention comprises a composition comprising one or more S. pneumoniae strain 9 V Spain 3 AI proteins and one or more AI proteins from any one or more of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S. pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeric form. In addition to the open reading frames encoding the S. pneumoniae strain 9V Spain 3 AI proteins, S. pneumoniae strain 9V Spain 3 AI may also include a transcriptional regulator.
The S. pneumoniae oligomeric, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an S. pneumoniae AI surface protein. The invention therefore includes a method for manufacturing an oligomeric AI surface antigen comprising culturing a S. pneumoniae bacterium that expresses the oligomeric AI protein and isolating the expressed oligomeric AI protein from the S. pneumoniae bacteria. The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed AI protein. Preferably, the AI protein is in a hyperoligomeric form. The oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing an AI surface protein. The invention therefore includes a method for manufacturing an S. pneumoniae oligomeric Adhesin Island surface antigen comprising culturing a S. pneumoniae bacterium adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the S. pneumoniae bacteria. The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed Adhesin Island protein. Preferably, the Adhesin Island protein is in a hyperoligomeric form.
The S. pneumoniae bacteria are preferably adapted to increase AI protein expression by at least two {e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels.
S. pneumoniae bacteria may be adapted to increase AI protein expression by any means known in the art, including methods of increasing gene dosage and methods of gene upregulation.
Such means include, for example, transformation of the S. pneumoniae bacteria with a plasmid enco pdin igz t'jhe , A/I u prosteiπn. s Thye p ≡lasm'zidβ m3ayB inc,lud ,e a strong promoter or i .t may i .nc,lud,e mu ,lti.p,le copies of the sequence encoding the AI protein. Optionally, the sequence encoding the AI protein within the S. pneumoniae bacterial genome may be deleted. Alternatively, or in addition, the promoter regulating the S. pneumoniae Adhesin Island may be modified to increase expression. The invention further includes S. pneumoniae bacteria which have been adapted to produce increased levels of AI surface protein. In particular, the invention includes S. pneumoniae bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein. In one embodiment, the S. pneumoniae of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface. The invention further includes S. pneumoniae bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface. The S. pneumoniae bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide. Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pili proteins on the surface of Gram positive bacteria. Increased expression of a leader peptidase in S. pneumoniae may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation. The S. pneumoniae bacteria adapted to have increased levels of leader peptidase may additionally be adapted to express increased levels of at least one pili protein. Alternatively, the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes" Infection and Immunity (2004) 72(6):3444-3450). As used herein, non-pathogenic Gram positive bacteria refer to Gram positive bacteria which are compatible with a human host subject and are not associated with human pathogenisis. Preferably, the non-pathogenic bacteria are modified to express the AI surface protein in oligomeric, or hyper-oligomeric form. Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non-pathogenic Gram positive bacterial genome or inserted into a plasmid. The non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface. Alternatively, the AI surface protein may be isolated or purified from a bacterial culture of the non-pathogenic Gram positive bacteria. For example, the AI surface protein may be isolated from cell extracts or culture supernatants. Alternatively, the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria.
The non-pathogenic Gram positive bacteria may be used to express any of the S. pneumoniae
Adhesin Island proteins described herein. The non-pathogenic Gram positive bacteria are transformed to e PxpCresTs anz A- UdheSsinO IsElaiIn1Zd sEurfyaceE prBoteQin. Preferably, the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase. The AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with pathogenic S. pneumoniae.
Figures 190 A and B, and 193-195 provide examples of three methods successfully practiced by applicants to purify pili from S. pneumoniae TIGR4.
Immunogenic Compositions
The Gram positive bacteria AI proteins described herein are useful in immunogenic compositions for the prevention or treatment of Gram positive bacterial infection. For example, the GBS AI surface proteins described herein are useful in immunogenic compositions for the prevention or treatment of GBS infection. As another example, the GAS AI surface proteins described herein may be useful in immunogenic compositions for the prevention or treatment of GAS infection. As another example, the S. pneumoniae AI surface proteins may be useful in immunogenic cojmpositions for the prevention or treatment of S. pneumoniae infection. Gram positive bacteria AI surface proteins that can provide protection across more than one serotype or strain isolate may be used to increase immunogenic effectiveness. For example, a particular GBS AI surface protein having an amino acid sequence that is at least 50% {i.e., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous to the particular GBS AI surface protein of at least 2 (i.e., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) other GBS serotypes or strain isolates may be used to increase the effectiveness of such compositions.
As another example, fragments of Gram positive bacteria AI surface proteins that can provide protection across more than one serotype or strain isolate may be used to increase immunogenic effectiveness. Such a fragment may be identified within a consensus sequence of a full length amino acid sequence of a Gram positive bacteria AI surface protein. Such a fragment can be identified in the consensus sequence by its high degree of homology or identity across multiple (Le, at least 3, 4, 5, 6, 7, 8, 9, 10, or more) Gram positive bacteria serotypes or strain isolates. Preferably, a high degree of ' homology is a degree of homology of at least 90% (Le., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) across Gram positive bacteria serotypes or strain isolates. Preferably, a high degree of identity is a degree of identity of at least 90% (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) across Gram positive bacteria serotypes or strain isolates. In one embodiment of the invention, such a fragment of a Gram positive bacteria AI surface protein may be used in the immunogenic compositions.
In addition, the AI surface protein oligomeric pilus structures may be formulated or purified for use in immunization. Isolated AI surface protein oligomeric pilus structures may also be used for immunization.
The invention includes an immunogenic composition comprising a first Gram positive bacteria AI protein and a second Gram positive bacterial AI protein. One or more of the AI proteins p |j I"' ,•■" ji Ii >ι»::: if 1Ii ii,::;; ,, ■■ :::::n "7 ;:;:'n r,\' iqi may be a surface protein Sudh" surface p rdteins may contain an LPXTG motif or other sortase substrate motif.
The first and second AI proteins may be from the same or different genus or species of Gram positive bacteria. If within the same species, the first and second AI proteins may be from the same or different AI subtypes. If two AIs are of the same subtype, the AIs have the same numerical designation. For example, all AIs designated as AI-I are of the same AI subtype. If two AIs are of a different subtype, the AIs have different numerical designations. For example, AI-I is of a different AI subtype from AI-2, AI-3, AI-4, etc. Likewise, AI-2 is of a different AI subtype from AI-I, AI-3, and AI-4, etc. For example, the invention includes an immunogenic composition comprising one or more
GBS AI-I proteins and one or more GBS AI-2 proteins. One or more of the AI proteins may be a surface protein. Such surface proteins may contain an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) and may bind fibrinogen, fibronectin, or collagen. One or more of the AI proteins may be a sortase. The GBS AI-I proteins may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. Preferably, the GBS AI-I proteins include GBS 80 or GBS 104. The GBS AI-2 proteins may be selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525. In one embodiment, the GBS AI-2 proteins are selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406. In another embodiment, the GBS AI-2 proteins may be selected from the group consisting of 01520, 01521, 01522, 01523, 01523, 01524 and 01525. Preferably, the GBS AI-2 protein includes GBS 59 or GBS 67.
As another example, the invention includes an immunogenic composition comprising one or more of any combination of GAS AI-I, GAS AI-2, GAS AI-3, or GAS AI-4 proteins. One or more of the GAS AI proteins may be a sortase. The GAS AI-I proteins may be selected from the group consisting of M6_SpyO156, M6_SpyO157, M6_Spy0158, M6_SpyO159, M6_Spy0160, M6_SpyO161, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial. Preferably, the GAS AI-I proteins are selected from the group consisting of M6_SpyO157, M6_SpyO159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
The GAS AI-2 proteins may be selected from the group consisting of SpyO124, GAS15, SpyO127, GAS16, GAS17, GAS18, SpyOBl, SpyO133, and GAS20. Preferably, the GAS AI-2 proteins are selected from the group consisting of GAS 15, GAS 16, and GAS 18.
The GAS AI-3 proteins may be selected from the group consisting of SρyM3_0097, SpyM3_OO98, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SPs0099, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf77, orf78, orf79, orfSO, orfδl, orf82, orf83, orf84, spyM18_0125, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, sρyM18_0131, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151,
SpyoMO 1000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. In on IPe 1 e Cmb: '"o!!d'"i,m■•'"e 1n,1t S me Q GAS'S1 '/ A jI-i:;3;ι!' p 7r'o Bti:e:!!in 3s a »»rle! selected from the group consisting of SpyM3_0097,
SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104. In another embodiment, the GAS AI-3 proteins are selected from the group consisting of SPs0099, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, and SPs0106. In yet another embodiment, the GAS AI-3 proteins are selected from the group consisting of orf77, orf78, orf79, orfδO, orfδl, orf82, orf83, and orf84. In a further embodiment, the GAS AI-3 proteins are selected from the group consisting of spyM18_0125, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132. In yet another embodiment the GAS AI-3 proteins are selected from the group consisting of SpyoMO 1000156, SpyoMO 1000155, SpyoMO 1000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoMO 1000150, and SpyoM01000149.
The GAS AI-4 proteins may be selected from the group consisting of 19224133, 19224134, 19224135, 19224136, 19224137, 19224138, 19224139, 19224140, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial. Preferably, the GAS-AI4 proteins are selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
As yet another example, the invention includes an immunogenic composition comprising one or more of any combination of S. pneumonaie from TIGR4, S. pneumonaie strain 670, S. pneumonaie from 19A Hungary 6, S. pneumonaie from 6B Finland 12, S. pneumonaie from 6B Spain 2, S. pneumonaie from 9V Spain 3, S. pneumonaie from 14 CSR 10, S. pneumonaie from 19F Taiwan 14, S. pneumonaie from 23F Taiwan 15, or S. pneumonaie from 23F Poland 16 AI proteins. One or more of the AI proteins may be a surface protein. Such surface proteins may contain an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) and may bind fibrinogen, fibronectin, or collagen. One or more of the AI proteins may be a sortase.
The S. pneumonaie from TIGR4 AI proteins may be selected from the group consisting of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, SP0468. Preferably, the S. pneumonaie from TIGR4 AI proteins include SP0462, SP0463, or SP0464.
The S. pneumonaie strain 670 AI proteins may be selected from the group consisting of Orfl_670, Orf3_670, Orf4_670, Orf5_670, Orf6_670, Orf7_670, and Orf8_670. Preferably, the S. pneumonaie strain 670 AI proteins include Orf3_670, Orf4_670, or Orf5_670.
The S. pneumonaie from 19A Hungary 6 AI proteins may be selected from the group consisting of ORF2_19AH, ORF3_19AH, ORF4J9AH, ORF5_19AH, ORF6_19AH, ORF7J9AH, or ORF8_19AH. The S. pneumonaie from 6B Finland 12 AI proteins may be selected from the group consisting of ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF , or ORF8_6BF. P C T Th Ze S U. pn SeuImJoBnai/e f ≡rom 765B, S3pa9in 2 AI proteins may be selected from the group consisting of ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP , or ORF8_6BSP.
The S. pneumonaie from 9V Spain 3 AI proteins may be selected from the group consisting of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP , or ORF8_9VSP.
The S. pneumonaie from 14 CSR 10 AI proteins may be selected from the group consisting of ORF2J4CSR, ORF3_14CSR, ORF4J4CSR, ORF5J4CSR, ORF6_14CSR, ORF7_14CSR , or ORF8_14CSR. The S. pneumonaie from 19F Taiwan 14 AI proteins may be selected from the group consisting of ORF2_19FTW, ORF3J9FTW, ORF4_19FTW, ORF5J9FTW, ORF6_19FTW, ORF7_19FTW , or ORF8_19FTW.
The S. pneumonaie from 23F Taiwan 15 AI proteins may be selected from the group consisting of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, or ORF8_23FTW.
The S. pneumonaie from 23F Poland 16 AI proteins may be selected from the group consisting of ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP , or ORF8_23FP.
Preferably, the Gram positive bacteria AI proteins included in the immunogenic compositions of the invention can provide protection across more than one serotype or strain isolate. For example, the immunogenic composition may comprise a first AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 91, 98, 99 or 100%) homologous to the amino acid sequence of a second AI protein, and wherein said first AI protein and said second AI protein are derived from the genomes of different serotypes of a Gram positive bacteria. The first AI protein may also be homologous to the amino acid sequence of a third AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different serotypes of a Gram positive bacteria. The first AI protein may also be homologous to the amino acid sequence of a fourth AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different serotypes of a Gram positive bacteria.
For example, preferably, the GBS AI proteins included in the immunogenic compositions of the invention can provide protection across more than one GBS serotype or strain isolate. For example, the immunogenic composition may comprise a first GBS AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e.,, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) homologous to the amino acid sequence of a second GBS AI protein, and wherein said first AI protein and said second AI protein are derived from the genomes of different GBS serotypes. The first GBS AI protein may also be homologous to the amino acid sequence of a third GBS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of P C T/ Ii EIi O 5 / Ξ 723 «3 different GBS serotypes. The first AI protein may also be homologous to the amino acid sequence of a fourth GBS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different GBS serotypes.
The first AI protein may be selected from an AI-I protein or an AI-2 protein. For example, the first AI protein may be a GBS AI-I surface protein such as GBS 80. The amino acid sequence of GBS 80 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 80 amino acid sequence from GBS serotype III, strain isolates NEM316 and COHl and the GBS 80 amino acid sequence from GBS serotype Ia, strain isolate A909.
As another example, the first AI protein may be GBS 104. The amino acid sequence of GBS 104 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 104 amino acid sequence from GBS serotype III, strain isolates NEM316 and COHl, the GBS 104 amino acid sequence from GBS serotype Ia, strain isolate A909, and the GBS 104 amino acid sequence serotype II, strain isolate 18RS21.
Table 12 provides the amino acid sequence identity of GBS 80 and GBS 104 across GBS serotypes Ia, Ib, II, III, V, and VIII. The GBS strains in which genes encoding GBS 80 and GBS 104 were identified share, on average, 99.88 and 99.96 amino acid sequence identity, respectively. This high degree of amino acid identity indicates that an immunogenic composition comprising a first protein of GBS 80 or GBS 104 may provide protection across more than one GBS serotype or strain isolate. Table 12. Conservation of GBS 80 and GBS 104 amino acid sequences
Figure imgf000089_0001
p i| 11 / ! (I > [ II:::J; ,.■" pi ";;;I' j ::ιι ■■:•: qi
Serotype Strains GBS 80 GBS 104 cGH %AA identity cGH %AA identity total 14/22 99.88+/-0.19 15/22 99.96 +/-0.056
As another example, the first AI protein may be an AI-2 protein such as GBS 67. The amino acid sequence of GBS 61 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 67 amino acid sequence from GBS serotype III, strain isolate NEM316, the GBS 67 amino acid sequence from GBS serotype Ib, strain isolate H36B, and the GBS 67 amino acid sequence from GBS serotype II, strain isolate 17RS21.
As another example, the first AI protein may be an AI-2 protein such as spbl . The amino acid sequence of spbl from GBS serotype III, strain isolate COHl is greater than 90% homologous to the spbl amino acid sequence from GBS serotype Ia, strain isolate A909. As yet another example, the first AI protein may be an AI-2 protein such as GBS 59. The amino acid sequence of GBS 59 from GBS serotype II, strain isolate 18RS21 is 100% homologous to the GBS 59 amino acid sequence from GBS serotype V, strain isolate 2603. The amino acid sequence of GBS 59 from GBS serotype V, strain isolate CJBl 11 is 98% homologous to the GBS 59 amino acid sequence from GBS serotype III, strain isolate NEM316. The compositions of the invention may also be designed to include Gram positive AI proteins from divergent serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of serotypes or strain isolates of a Gram positive bacteria and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.
For example, the invention may include an immunogenic composition comprising a first and second Gram positive bacteria AI protein, wherein a polynucleotide sequence encoding for the full length sequence of the first AI protein is not present in a similar Gram positive bacterial genome comprising a polynucleotide sequence encoding for the second AI protein.
The compositions of the invention may also be designed to include AI proteins from divergent GBS serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of GBS serotypes or strain isolates and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.
For example, the invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein a polynucleotide sequence encoding for the full length sequence of the first GBS Al protein is not present in a genome comprising a polynucleotide sequence encoding for the second GBS AI protein. For example, the first AI protein could be GBS 80 (such as the GBS 80 sequence from GBS serotype V, strain isolate 2603). As previously discussed (and depicted in Figure T), the sequence for GBS 80 in GBS sertoype II, strain isolate 18RS21 is disrupted. In this instance, the second AI protein could be GBS 104 or GBS 67 (sequences selected from the GBS serotype II, strain isolate 18RS21). PC IV ϋ J S O 5 / E 7 E .31O
Further, the the invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein the first GBS AI protein has detectable surface exposure on a first GBS strain or serotype but not a second GBS strain or serotype and the second GBS AI protein has detectable surface exposure on a second GBS strain or serotype but not a first GBS strain or serotype. For example, the first AI protein could be GBS 80 and the second AI protein could be GBS 67. As seen in Table 15, there are some GBS serotypes and strains that have surface exposed GBS 80 but that do not have surface exposed GBS 67 and vice versa. An immunogenic composition comprising a GBS 80 and a GBS 67 protein may provide protection across a wider group of GBS strains and serotypes.
and GBS 67
Figure imgf000092_0001
Figure imgf000092_0002
Alternatively, the invention may include an immunogenic composition comprising a first and second Gram positive bacteria AI protein, wherein the polynucleotide sequence encoding the sequence of the first AI protein is less than 90 % (i.e., less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second AI protein. PC T/ U S O 5 ,■■ " E: 7 EE 3 W
The invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein the polynucleotide sequence encoding the sequence of the first GBS AI protein is less than 90 % (i.e., less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second GBS AI protein. For example, the first GBS AI protein could be GBS 67 (such as the GBS 67 sequence from GBS serotype Ib, strain isolate H36B). As shown in Figures 2 and 4, the GBS 67 sequence for this strain is less than 90% homologous (87%) to the corresponding GBS 67 sequence in GBS serotype V, strain isolate 2603. In this instance, the second GBS AI protein could then be the GBS 80 sequence from GBS serotype V, strain isolate 2603.
An example immunogenic composition of the invention may comprise adhesin island proteins GBS 80, GBS 104, GBS 67, and GBS 59, and non-AI protein GBS 322. FACS analysis of different GBS strains demonstrates that at least one of these five proteins is always found to be expressed on the surface of GBS bacteria. An initial FACS analysis of 70 strains of GBS bacteria, obtained from the CDC in the United States (33 strains), ISS in Italy (17 strains), and Houston/Harvard (20 strains), detected surface exposure of at least one of GBS 80, GBS 104, GBS 322, GBS 67, or GBS 59 on the surface of the GBS bacteria. Figure 227 provides the FACS data obtained for surface exposure of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on each of 37 GBS strains. Figure 228 provides the FACS data obtained for surface exposure of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on each of 41 GBS strains obtained from the CDC. As can be seen from Figures 227 and 228, each GBS strain had surface expression of at least one of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59. The surface exposure of at least one of these proteins on each bacterial strain indicates that an immunogenic composition coinprising these proteins will provide wide protection across GBS strains and serotypes.
The surface exposed GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 proteins are also present at high levels as determined by FACS. Table 49 summarizes the FACS results for the initial 70 GBS strains examined for GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 surface expression. A protein was designated as having high levels of surface expression of a protein if a five-fold shift in fluorescence was observed when using antibodies for the protein relative to preimmune control serum. Table 49: Exposure Levels of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on GBS Strains
Figure imgf000093_0001
Table 50 details which of the surface proteins is highly expressed on the different GBS serotype. Table 50: High Levels of Surface Protein Expression on GBS Serotypes
Figure imgf000093_0002
PCT / US O 5/ E! 7 iE 31Qi
Alternatively, the immunogenic composition of the invention may include GBS 80, GBS 104,
GBS 67, and GBS 322. Assuming that protein antigens that are highly accessible to antibodies confer 100% protection with suitable adjuvants, an immunogenic composition containing GBS 80, GBS 104, GBS 67, GBS 59 and GBS 322 will provide protection for 89% of GBS strains and serotypes, the same percentage as an immunogenic composition containing GBS 80, GBS 104, GBS 67, and GBS 322 proteins. See Figure 229. However, it may be preferable to include GBS 59 in the composition to increase its immunogenic strength. As seen from Table 50, GBS 59 is highly expressed on the surface two-thirds of GBS bacteria examined by FACS analysis, unlike GBS 80, GBS 104, and GBS 322, which are highly expressed in less than half of GBS bacteria examined. GBS 59 opsonophagocytic activity is also comparable to that of a mix of GBS 322, GBS 104, GBS 67, and GBS 80 proteins. See Figure 230.
By way of another example, preferably, the GAS AI proteins included in the immunogenic compositions of the invention can provide protection across more than one GAS serotype or strain isolate. For example, the immunogenic composition may comprise a first GAS AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) homologous to the amino acid sequence of a second GAS AI protein, and wherein said first AI protein and said second AI protein are derived from the genomes of different GAS serotypes. The first GAS AI protein may also be homologous to the amino acid sequence of a third GAS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different GAS serotypes. The first AI protein may also be homologous to the amino acid sequence of a fourth GAS AI protein, such that the first AI protein, the second AI protein and the third AI protein are derived from the genomes of different GAS serotypes.
The compositions of the invention may also be designed to include GAS AI proteins from divergent serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of serotypes or strain isolates of a GAS bacteria and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.
For example, the first AI protein could be a ρrtF2 protein (such as the 19224141 protein from GAS serotype M12, strain isolate A735). As previously discussed (and depicted in Figure 164), the sequence for a prtF2 protein is not present in GAS AI types 1 or 2. In this instance, the second AI protein could be collagen binding protein M6_SpyO159 (from M6 isolate (MGAS10394), which comprises an AI-I) or GAS 15 (from Ml isolate (SF370), which comprises an AI-2).
Further, the invention may include an immunogenic composition comprising a first and second GAS AI protein, wherein the first GAS AI protein has detectable surface exposure on a first GAS strain or serotype but not a second GAS strain or serotype and the second GAS AI protein has detectable surface exposure on a second GAS strain or serotype but not a first GAS strain or serotype. The invention may include an immunogenic composition comprising a first and second GAS AI protein, wherein the polynucleotide sequence encoding the sequence of the first GAS AI protein is less than 90 % (i.e., less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second GAS AI protein. Preferably the first and second GAS AI proteins are subunits of the pilus. More preferably the first and second GAS AI proteins are selected from the major pilus forming proteins (i.e., M6_Sρy0160 from M6 strain 10394, SPyO128 from Ml strain SF370, SpyM3_0100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM18_0128 from M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from M12 strain A735, fimbrial structural subunit from M77 strain ISS4959, fimbrial structural subunit from M44 strain ISS3776, fimbrial structural subunit from M50 strain ISS3776 ISS 4538, fimbrial structural subunit from M12strain CDC SS635, fimbrial structural subunit from M23 strain DSM2071, fimbrial structural subunit from M6 strain CDC SS410). Table 45 provides the percent identity between the amino acidic sequences of each of the main pilus forming subunits from GAS AI-I, AI-2, AI-3, and AI-4 representative strains (i.e., M6_Spy0160 from M6 strain 10394, SPyO128 from Ml strain SF370, SρyM3_0100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM18_0128 from M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from M12 strain A735, Fimbrial structural subunit from M77 strain ISS4959, fimbrial structural subunit from M44 strain ISS3776, fimbrial structural subunit from M50 strain ISS3776 ISS 4538, fimbrial structural subunit from M12strain CDC SS635, fimbrial structural subunit from M23 strain DSM2071, fimbrial structural subunit from M6 strain CDC SS410). Table 45: Comparison of Amino Acid Sequences of Major Pilus Proteins in the Four GAS
AIs
Figure imgf000095_0001
Figure imgf000096_0001
For example, the first main pilus subunit may be selected from bacteria of GAS serotype M6 strain 10394 and the second main pilus subunit may be selected from bacteria of GAS serotype Ml strain 370. As can be seen from Table 45, the main pilus subunits encoded by these strains of bacteria share only 23% nucleotide identity. An immunogenic composition comprising pilus main subunits from each of these strains of bacteria is expected to provide protection across a wider group of GAS strains and serotypes. Other examples of main pilus subunits that can be used in combination to provide increased protection across a wider range of GAS strains and serotypes include proteins encoded by GAS serotype M5 Manfredo isolate and serotype M6 strain 10394, which share 23% sequence identity, GAS serotype M18 strain 8232 and serotype Ml strain 370, which share 38% sequence identity, GAS serotype M3 strain 315 and serotype M12 strain A735, which share 61% sequence identity, and GAS serotype M3 strain 315 and serotype M6 strain 10394 which share 25% sequence identity.
As also can be seen from Table 45, the amino acid sequences of the four types of main pilus subunits present in GAS are relatively divergent. Figures 198-201 provide further tables comparing the percent identity of adhesin island-encoded surface exposed proteins for different GAS serotypes relative to other GAS serotypes harbouring an adhesin island of the same or a different subtype (GAS AI-I, GAS AI-2, GAS AI-3, and GAS AI-4). See also further discussion below.
Immunizations with the Adhesin Island proteins of the invention are discussed further in the Examples. v Co-expression of GBS Adhesin Island proteins and role of GBS AI proteins in surface presentation In addition to the use of the GBS adhesin island proteins for cross strain and cross serotype protection, Applicants have identified interactions between adhesin island proteins which appear to affect the delivery or presentation of the surface proteins on the surface of the bacteria.
In particular, Applicants have discovered that surface exposure of GBS 104 is dependent on the concurrent expression of GBS 80. As discussed further in Example 2, reverse transcriptase PCR analysis of AI-I shows that all of the AI genes are co-transcribed as an operon. Applicants constructed a series of mutant GBS containing in frame deletions of various AI-I genes. (A schematic of the GBS mutants is presented in Figure 7). FACS analysis of the various mutants comparing mean shift values using anti-GBS 80 versus anti-GBS 104 antibodies is presented in Figure 8. Removal of the GBS 80 operon prevented surface exposure of GBS 104; removal of the
Figure imgf000097_0001
80. While not being limited to a specific theory, it is thought that GBS 80 is involved in the transport or localization of GBS 104 to the surface of the bacteria. The two proteins may be oligomerized or otherwise associated. It is possible that this association involves a conformational change in GBS 104 that facilitates its transition to the surface of the GBS bacteria.
PiIi structures that comprise GBS 104 appear to be of a lower molecular weight than pili structures lacking GBS 104. Figure 68 shows that polyclonal anti-GBS 104 antibodies (see lane marked α-104 POLIC.) cross-hybridize with smaller structures than do polyclonal anti-GBS 80 antibodies (see lane marked α-GBS 80 POLIC). In addition, Applicants have shown that removal of GBS 80 can cause attenuation, further suggesting the protein contributes to virulence. As described in more detail in Example 3, the LD50's for the Δ80 mutant and the Δ80, Δ104 double mutant were reduced by an order of magnitude compared to wildtype and Δ104 mutant.
The sortases within the adhesin island also appear to play a role in localization and presentation of the surface proteins. As discussed further in Example 4, FACS analysis of various sortase deletion mutants showed that removal of sortase SAG0648 prevented GBS 104 from reaching the surface and slightly reduced the surface exposure of GBS 80. When sortase SAG0647 and sortase SAG0648 were both knocked out, neither GBS 80 nor GBS 104 were surface exposed. Expression of either sortase alone was sufficient for GBS 80 to arrive at the bacterial surface. Expression of SAG0648, however, was required for GBS 104 surface localization.
Accordingly, the compositions of the invention may include two or more AI proteins, wherein the AI proteins are physically or chemically associated. For example, the two AI proteins may form an oligomer. In one embodiment, the associated proteins are two AI surface proteins, such as GBS 80 and GBS 104. The associated proteins may be AI surface proteins from different adhesin islands, including host cell adhesin island proteins if the AI surface proteins are expressed in a recombinant system. For example, the associated proteins may be GBS 80 and GBS 67. Adhesin Island proteins from other Gram positive bacteria
Applicants' identification and analysis of the GBS adhesin islands and the immunological and biological functions of these AI proteins and their pilus structures provides insight into similar structures in other Gram positive bacteria.
As discussed above, "Adhesin Island" or "AI" refers to a series of open reading frames within a bacterial genome that encode for a collection of surface proteins and sortases. An Adhesin Island may encode for amino acid sequences comprising at least one surface protein. The Adhesin Island may encode at least one surface protein. Alternatively, an Adhesin Island may encode for at least two surface proteins and at least one sortase. Preferably, an Adhesin Island encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or more AI ip J]" '■ "'ι|'" _,.>■■ (I j| c',j; !|'"[j ιi"'j; _,,■■■ jj:::iι ■■;;;" i'p ":::[; ιi"| surface protein's may participate in me formation of a pilus structure on the surface of the Gram positive bacteria.
Gram positive adhesin islands of the invention preferably include a divergently transcribed transcriptional regulator. The transcriptional regulator may regulate the expression of the AI operon. The invention includes a composition comprising one or more Gram positive bacteria AI surface proteins. Such AI surface proteins may be associated in an oligomeric or hyperoligomeric structure.
Preferred Gram positive adhesin island proteins for use in the invention may be derived from Staphylococcus (such as S. aureus), Streptococcus (such as S. agalactiae (GBS), S. pyogenes (GAS), S. pneumonaie, S. mutans), Enterococcus (such as E.faecalis and E.faecium), Clostridium (such as C. difficile), Listeria (such as L. monocytogenes) and Corynebacterium (such as C. diphtheria).
One or more of the Gram positive AI surface protein sequences typically include an LPXTG motif or other sortase substrate motif. Gram positive AI surface proteins of the invention may affect the ability of the Gram positive bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of Gram positive bacteria to translocate through an epithelial cell layer.
Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. Gram positive AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
Gram positive AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. A Gram positive bacteria AI may encode for at least one surface exposed protein. The Adhesin Island may encode at least one surface protein. Alternatively, a Gram positive bacteria AI may encode for at least two surface exposed proteins and at least one sortase. Preferably, a Gram positive AI encodes for at least three surface exposed proteins and at least two sortases. Gram positive AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710 - 2722. Typically, Gram positive bacteria AI surface proteins of the invention will contain an N-terminal leader or secretion signal to facilitate translocation of the surface protein across the bacterial membrane.
Gram positive bacteria AI surface proteins of the invention may affect the ability of the Gram positive bacteria to adhere to and invade target host cells, such as epithelial cells. Gram positive bacteria AI surface proteins may also affect the ability of the gram positive bacteria to translocate through an epithelial cell layer. Preferably, one or more of the Gram positive AI surface proteins are P C T/ U.Ξ O 5 /1E! 7 E! B 1Qi capable of binding to or other associating with an epithelial cell surface. Further, one or more Gram positive AI surface proteins may bind to fibrinogen, fibronectin, or collagen protein.
In one embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising a Gram positive bacteria AI surface protein. The oligomeric, pilus-like structure may comprise numerous units of the AI surface protein. Preferably, the oligomeric, pilus- like structures comprise two or more AI surface proteins. Still more preferably, the oligomeric, pilus- like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acid residue.
Gram positive bacteria AI surface proteins or fragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include one or both of a pilin motif comprising a conserved lysine residue and an E box motif comprising a conserved glutamic acid residue.
The oligomeric, pilus like structures may be used alone or in the combinations of the invention. In one embodiment, the invention comprises a Gram positive bacteria Adhesin Island in oligomeric form, preferably in a hyperoligomeric form. The oligomeric, pilus-like structures of the invention may be combined with one or more additional Gram positive AI proteins (from the same or a different Gram positive species or genus). In one embodiment, the oligomeric, pilus-like structures comprise one or more Gram positive bacteria AI surface proteins in combination with a second Gram positive bacteria protein. The second Gram positive bacteria protein may be a known antigen, and need not normally be associated with an AI protein.
The oligomeric, pilus-like structures may be isolated or purified from bacterial cultures overexpressing a Gram positive bacteria AI surface protein. The invention therefore includes a method for manufacturing an oligomeric Adhesin Island surface antigen comprising culturing a Gram positive bacteria adapted for increased AI protein expression and isolation of the expressed oligomeric Adhesin Island protein from the Gram positive bacteria. The AI protein may be collected from secretions into the supernatant or it may be purified from the bacterial surface. The method may further comprise purification of the expressed Adhesin Island protein. Preferably, the Adhesin Island protein is in a hyperoligomeric form.
Gram positive bacteria are preferably adapted to increase AI protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200) times wild type expression levels.
Gram positive bacteria may be adapted to increase AI protein expression by means known in the art, including methods of increasing gene dosage and methods of gene upregulation. Such means . PC T/ Il J S O Eii ./ ≤ 7' iΞ! 39 include, for example, transformation of the Gram positive bacteria with a plasmid encoding the AI protein. The plasmid may include a strong promoter or it may include multiple copies of the sequence encoding the AI protein. Optionally, the sequence encoding the AI protein within the Gram positive bacterial genome may be deleted. Alternatively, or in addition, the promoter regulating the Gram positive Adhesin Island may be modified to increase expression.
The invention further includes Gram positive bacteria which have been adapted to produce increased levels of AI surface protein. In particular, the invention includes Gram positive bacteria which have been adapted to produce oligomeric or hyperoligomeric AI surface protein. In one embodiment, the Gram positive bacteria of the invention are inactivated or attenuated to permit in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface.
The invention further includes Gram positive bacteria which have been adapted to have increased levels of expressed AI protein incorporated in pili on their surface. The Gram positive bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide. Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pili proteins on the surface of Gram positive bacteria. Increased expression of a leader peptidase in Gram positive may be achieved by any means known in the art, such as increasing gene dosage and methods of gene upregulation. The Gram positive bacteria adapted to have increased levels of leader peptidase may additionally be adapted to express increased levels of at least one pili protein.
Alternatively, the AI proteins of the invention may be expressed on the surface of a nonpathogenic Gram positive bacteria, such as Streptococus gordonii (See, e.g., Byrd et al., "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002) 20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al., "Mucosal VaccineMade from Live, Recombinant Lactococcus lactis Protects Mice against Pharangeal Infection with Streptococcus pyogenes" Infection and Immunity (2004) 72(6):3444-3450). It has already been demonstrated, above, that L. lactis expresses GBS and GAS AI polypeptides in oligomeric form and on its surface.
Alternatively, the oligomeric, pilus-like structures may be produced recombinantly. If produced in a recombinant host cell system, the Gram positive bacteria AI surface protein will preferably be expressed in coordination with the expression of one or more of the AI sortases of the invention. Such AI sortases will facilitate oligomeric or hyperoligomeric formation of the AI surface protein subunits. ,
Gram positive AI Sortases of the invention will typically have a signal peptide sequence within the first 70 amino acid residues. They may also include a transmembrane sequence within 50 amino acid residues of the C terminus. The sortases may also include at least one basic amino acid residue within the last 8 amino acids. Preferably, the sortases have one or more active site residues, such as a catalytic cysteine and histidine. Ihesinlslaiάd'siirfa'e'e' protein's from two or more Gram positive bacterial genus or species may be combined to provide an immunogenic composition for prophylactic or therapeutic treatment of disease or infection of two more Gram positive bacterial genus or species. Optionally, the adhesin island surface proteins may be associated together in an oligomeric or hyperoligomeric structure. In one embodiment, the invention comprises an adhesin island surface proteins from two or more Streptococcus species. For example, the invention includes a composition comprising a GBS AI surface protein and a GAS adhesin island surface protein. As another example, the invention includes a composition comprising a GAS adhesin island surface protein and a S. pneumoniae adhesin island surface protein. In one embodiment, the invention comprises an adhesin island surface protein from two or more Gram positive bacterial genus. For example, the invention includes a composition comprising a Streptococcus adhesin island protein and a Corymb acterium adhesin island protein.
Examples of AI sequences in several Gram positive bacteria are discussed further below. Streptococcus pyogenes TGAS) As discussed above, Applicants have identified at least four different GAS Adhesin Islands.
These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus. Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fasciitis. In addition, post-streptococcal autoimmune responses are still a major cause of cardiac pathology in children.
Group A Streptococcal infection of its human host can generally occur in three phases. The first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces. Generally this attachment phase begins in the throat or the skin. The deeper the tissue level infected, the more severe the damage that can be caused. In the second stage of infection, the bacteria secrete a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. This toxin binds to susceptible host cell receptors and triggers innappropropriate immune responses by these host cells, resulting in pathology. Because the toxin can diffuse throughout the host, the necrosis directly caused by the GAS toxins may be physically located in sites distant from the bacterial infection. The final phase of GAS infection can occur long after the original bacteria have been cleared from the host system. At this stage, the host's previous immune response to the GAS bacteria due to cross reactivity between epitopes of a GAS surface protein, M, and host tissues, such as the heart. A general review of GAS infection can be found in Principles of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
In order to prevent the pathogenic effects associated with the later stages of GAS infection, an effective vaccine against GAS will preferably facilitate host elimination of the bacteria during the initial attachment and invasion stage. p. |j" f / 1 1| ;q; Ji 5; /' P ';;/ 5; 3 cji
'"" Isolates of Group' A Streptococcus are historically classified according to the M surface protein described above. The M protein is surface exposed trypsin-sensitive protein generally comprising two polypeptide chains complexed in an alpha helical formation. The carboxyl terminus is anchored in the cytoplasmic membrane and is highly conserved among all group A streptococci. The amino terminus, which extends through the cell wall to the cell surface, is responsible for the antigenic variability observed among the 80 or more serotypes of M proteins.
A second layer of classification is based on a variable, trypsin-resistant surface antigen, commonly referred to as the T-antigen. Decades of epidemiology based on M and T serological typing have been central to studies on the biological diversity and disease causing potential of Group A Streptococci. While the M-protein component and its inherent variability have been extensively characterized, even after five decades of study, there is still very little known about the structure and variability of T-antigens. Antisera to define T types are commercially available from several sources, including Sevapharma (http://www.sevapharma.cz/en).
The gene coding for one form of T-antigen, T-type 6, from an M6 strain of GAS (D741) has been cloned and characterized and maps to an approximately 11 kb highly variable pathogenicity island. Schneewind et al., J Bacteriol. (1990) 172(6):3310 - 3317. This island is known as the Fibronectin-binding, Collagen-binding T-antigen (FCT) region because it contains, in addition to the T6 coding gene (teeό), members of a family of genes coding for Extra Cellular Matrix (ECM) binding proteins. Bessen et al., Infection & Immunity (2002) 70(3):1159-1167. Several of the protein products of this gene family have been shown to directly bind either fϊbronectin and/or collagen. See Hanski et al., Infection & Immunity (1992) 60(12):5119-5125; Talay et al., Infection & Immunity (1992( 60(9):3837-3844; Jaffe et al. (1996) 21(2):373-384; Rocha et al., Adv Exp Med Biol. (1997) 418:737-739; Kreikemeyer et al., J Biol Chem (2004) 279(16): 15850-15859; Podbielski et al., MoI. Microbiol. (1999) 31(4): 1051-64; and Kreikemeyer et al., Int. J. Med Microbiol (2004) 294(2-3):177- 88. In some cases direct evidence for a role of these proteins in adhesion and invasion has been obtained.
Applicants raised antiserum against a recombinant product of the teeό gene and used it to explore the expression of T6 in M6 strain ISS3650. In immunoblot of mutanolysin extracts of this strain, the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the teeό gene product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used. See Figure 163 A, last lane labeled "M6_Tee6."
This pattern of high molecular weight products is similar to that observed in immunoblots of the protein components of the pili identified in Streptococcus agalactiae (described above) and previously in Corynebacterium diphtheriae. Electron microscropy of strain M6 ISS3650 with antisera specific for the product of teeό revealed abundant surface staining and long pilus like structures extending up to 700 nanometers from the bacterial surface, revealing that the T6 protein, one of the an PtigCensT recZogynizSed1O in1S theZ orBiginJa1lE LaBnce1Giiie1ld, sero ^typm. g sys ftem, i .s , located . wi .Athi.n a ^ GAΛ SQ A . d„hesi .n Island (GAS AI-I) and forms long covalently linked pilus structures. See Figure 1631.
In addition to the teeό gene, the FCT region in M6JSS3650 (GAS AI-I) contains two other genes (prtFl and cpa) predicted to code for surface exposed proteins; these proteins are characterized as containing the cell wall attachment motif LPXTG. Western blot analysis using antiserum specific for PrtFl detected a single molecular species with electrophoretic mobility corresponding to the predicted molecular mass of the protein and one smaller band of unknown origin. Western blot analysis using antisera specific for Cpa recognized a high molecular weight covalently linked ladder (Fig 163 A, second lane). Immunogold labelling of Cpa with specific antiserum followed by transmission electron microscopy detected an abundance of Cpa at the cell surface and only occasional structures extending from the cell surface (Fig. 163J).
Four classes of FCT region can be discerned by the types and order of the genes contained within the region. The FCT region of strains of types M3, M5, M 18 and M49 have a similar organization whereas those of M6, Ml and M12 differ. See Figure 164. As discussed below, these four FCT regions correlate to four GAS Adhesin Island types (AI-I, AI-2, AI-3 and AI-4).
Applicants discovery of genes coding for pili in the FCT region of strain M6_ISS3650 prompted them to examine the predicted surface exposed proteins in the variant FCT regions of three other GAS strains of having different M-type (M1J3F370, M5JSS4883 and Ml 2_20010296) representing the other three FCT variants. Each gene present in the FCT region of each bacteria was cloned and expressed. Antisera specific for each recombinant protein was then used to probe mutanolysin extracts of the respective strains (6). In Ml strain SF370, there are three predicted surface proteins (Cpa (also referred to as Ml_126 and GAS 15), Ml_128 (a fϊmbrial protein also referred to as SpyO128 and GAS 16), and Ml_130 (also referred to as Spy0130 and GAS 18)) (GAS AI-2). Antisera specific for each surface protein reacted with a ladder of high molecular weight material (Fig. 163B). Immunogold staining of Ml strain SF370 with antiserum specific for Ml_128 revealed pili structures similar to those seen when M6 strain ISS3650 was immunogold stained with antiserum specific for tee6 (See Fig 1163K). Antisera specific for surface proteins Cpa and Ml_130 revealed abundant surface staining and occasional structures extending from the surface of Ml strain SF370 bacteria (Fig. 163S).
The Ml_128 protein appears to be necessary for polymerization of Cpa and Ml_130 proteins. If the Ml_128 gene in Ml_SF370 was deleted, Western blot analysis using antibodies that hybridize to Cpa and Ml_130 no longer detected high molecular weight ladders comprising the Cpa and Ml_130 proteins (Fig. 163 E). See also Figures 177 A-C which provide the results of Western blot analysis of the Ml_128 (Δ128) deleted bacteria using anti-Ml_130 antiserum (Figure 177 A), anti-Ml_128 antiserum (Figure 177 B), and anti-Ml_126 antiserum (Figure 177 C^" nigh 'molecular weighH'adders;iήdϊcative of pilus formation on the surface of Ml strain SF370, could not be detected by any of the three antisera in Δ 128 bacteria. If the Δl28 bacteria were transformed with a plasmid containing the gene for Ml_128, Western blot analysis using antisera specific for Cpa and Ml_130 again detected high molecular weight ladders (Figure 163 H).
In agreement with the Western blot analysis, immunoelectron microscopy failed to detect pilus assembly on the Δ128 strain SF370 bacteria using Ml_128 antisera (Figure 178 B). Although Δ128 SF370 bacteria were unable to form pili, Ml_126 (cpa) and Ml_130, which contain sortase substrate motifs, were present on the bacteria's surface. FACS analysis of the Ml_128 deleted (Δ128) strain SF370 bacteria also detected both Ml_126 and Ml_130 on the surface of the Δ128 strain SF370 bacteria. See Figure 179 D and F, which show a shift in fluorescence when antibodies immunoreactive to Ml_126 and Ml_130 are used on Δ128 bacteria. As expected, virtually no shift in fluorescence is observed when antibodies immunoreactive to Ml_128 are used with the Δ128 bacteria (Figure 179 E). By contrast, deletion of the Ml_130 gene did not effect polymerization of Ml_128 (Fig.
163 F). See also Figures 177 A-C, which provide Western blot analysis results of the Ml_130 deleted (Δ130) strain SF370 bacteria using anti-Ml_130 (Figure 177 A), anti-Ml_128 (Figure 177 B), and anti-Ml_126 antiserum (Figure 177 C). The anti-Ml_128 and anti-Ml_126 antiserum both detected the presence of high molecular weight ladders in the Δ130 strain SF370 bacteria, indicating that the Δ130 bacteria form pili that comprise Ml_126 and Ml_128 polypeptides in the absence of Ml_130. As expected, the Western blot probed with antiserum immunoreactive with Ml_130 did not detect any proteins for the Δ130 bacteria(Figure 177A).
Hence, the composition of the pili in GAS resembles that previously described for both C. diphtheria (7, 8) and S. agalactiae (described above) (9) in that each pilus is formed by a backbone component which abundantly stains the pili in EM and is essential for the incorporation of the other components.
Also similar to C. diphtheria, elimination of the srtCl gene from the FCT region of Ml_SF370 abolished polymerization of all three proteins and assembly of pili (Fig. 163 G). See also Figures 177 A-C, which provide Western blot analysis of the SrtCl deleted (ΔSrtCl) strain SF370 bacteria using anti-Ml_130 (Figure 177 A), anti-Ml_128 (Figure 177 B), and anti- Ml_126 antiserum (Figure 177 C). None of the three antisera immunoreacted with high molecular weight structures (pili) in the ΔSrtCl bacteria. Confirming that deletion of the SrtCl gene abrogates pilus assembly in strain SF370, immunoelectron microscopy using antisera against Ml_128 failed to detect pilus formation on the bacteria surface. See Figure 178 C. Although no assembled pili were detected on ΔSrtCl SF370, Ml_128 proteins could be detected on the surface of SF370. Thus, it appeared that SrtCl deletion prevented pilus assembly on the siϊfϊicfe 6f"thέ"'SF§7ϋ bidterfa', WFnδt1' anchoring of the proteins that comprise pili to the bacterial cell wall. FACS analysis of the ΔSrtCl strain SF370 confirmed that deletion of SrtCl does not eliminate cell surface expression of Ml_126, Ml_128 or Ml_130. See Figure 179 G-I, which show a shift in fluorescence when antibodies immunoreactive to Ml_126 (Figure 179 G), Ml_128 (Figure 179 H), and Ml_130 (Figure 179 I) are used to detect cell surface protein expression on ΔSrtCl bacteria. Thus, SrtCl deletion prevents pilus formation, but not surface anchoring of proteins involved in pilus formation on the surface of bacteria. Another sortase is possibly involved in anchoring of the proteins to the bacteria surface. Pilus polymerization in C. diphtheriae is also dependent on particular sortase enzyme whose gene resides at the same genetic locus as the pilus components (7, 8).
The LepA signal peptidase, SpyO127, also appears to be essential for pilus assembly in strain SF370. LepA deletion mutants (ΔLepA) of strain SF370 fail to assemble pili on the cell surface. Not only are the ΔLepA mutants unable to assemble pili, they are also deficient at cell surface Ml expression. See Figure 180, which provides a FACS analysis of the wildtype (A) and ΔLepA mutant (B) SF370 bacteria using Ml antisera. No shift in fluorescence is observed for the ΔLepA mutant bacteria in the presence of Ml immune serum. It is possible that these deletion mutants of LepA will be useful for detecting non-M, non-pili, surface exposed antigens on the surface of GAS, or any Gram positive bacteria. These antigens may also be useful in immunogenic compositions. Pili were also observed in M5 strain ISS4882 and M12 strain 20010296. The M5 strain
ISS4882 contains genes for four predicted surface exposed proteins (GAS ΛI-3). Antisera against three of the four products of the FCT region (GAS AI-3) of M5JSS4883 (Cpa, M5_orf80, M5_orf82) stained high molecular weight ladders in Western blot analysis (Figure 163 C). Long pili were visible when antisera against M5_orf80 was used in immunogold staining followed by electron microscopy (Figure 163L).
The M12 strain 20010296 contains genes for five predicted surface exposed proteins. (GAS AI-4) Antisera against three of the five products of the FCT region (GAS AI-4) of Ml 2_20010296 (Cpa, EftLSL.A, Orf2) stained high molecular weight ladders in Westen blot analysis (Figure 163 D). Long pili were visible when antisera against EftLSL.A were used (Fig. 163 M).
The major pilus forming proteins identified in the four strains studied by applicants (T6, Ml_128, M5_orf80 and EftLSL.A) share between 23% and 65% amino acid identity in any pairwise comparison, indicating that each pilus may represent a different Lancefϊeld T-antigen. Each pilus is part of a trypsin resistant structure on the GAS bacteria surface, as is the case for the Lancefield T antigens. See Figure 165, which provides a FACS analysis of bacteria harboring each of the FCT types that had or had not been treated with trypsin (<5). Following treatment, surface expression of the
Figure imgf000106_0001
assayed ti'yϊώffecf Immunofluorescence and flow cytometry using antibodies specific for the pilus proteins, the bacteria's respective M proteins, or surface proteins not associated with the pili (Figure 165). Staining the cells with sera specific for proteins associated with the pili was not effected by trypsin treatment, whereas trypsin treatment substantially reduced detection of M- proteins or surface proteins not associated with pili.
The pili structures identified on the surface of the GAS bacteria were confirmed to be Lancefϊeld T antigens when commercially available T-serotyping sera detected the pili on the surface of bacteria. Western blot analysis was initially performed to determine if polyvalent serum pools (designated T, U, W, X, andY) could detect recombinant proteins for each of the major pilis components (T6, Ml_128, M5_orf80 and EftLSL.A) identified in the strains of bacteria discussed above. Pool U, which contains the T6 serum, recognized the T6 protein specifically (a surface exposed pilus protein from GAS AI-l)(Fig. 166 B). Pool T specifically recognized Ml_128 (a surface exposed pilus protein from GAS AI-2) (Fig. 166 A). Pool W recognized both M5_orf80 and EftLSL.A (Fig. 166 C). Using monovalent sera representative of each of the components of each polyvalent pool, applicants confirmed the specificity of the T6 antigen (corresponding to a surface exposed pilus protein from GAS AI-l)(Fig. 166 E) and identified Ml_128 as antigen Tl (corresponding to a surface exposed pilus protein from GAS AI-2) (Fig. 166 D), EftLSL.A as antigen T12 (corresponding to a surface exposed pilus protein from GAS AI-4) (Fig. 166 G) and M5_orf80 as a common antigen recognized by the related sera T5, T27 and T44 (corresponding to a surface exposed pilus protein from GAS AI-3).
Confirming applicants observations, discussed above, that deleting the Ml_128 gene from Ml_SF370 abolishes pilus formation, the pool T sera stained whole Ml_SF370 bacteria (Fig. 166 H) but failed to stain Ml_SF370 bacteria lacking the Ml_128 gene (Fig. 166 1).
As discussed above, Applicants have identified at least four different Group A Streptococcus Adhesin Islands. While these GAS AI sequences can be identified in numerous M types, Applicants have surprisingly discovered a correlation between the four main pilus subunits from the four different GAS AI types and specific T classifications. While other trypsin-resistant surface exposed proteins are likely also implicated in the T classification designations, the discovery of the role of the GAS adhesin islands (and the associated hyper-oligomeric pilus like structures) in T classification and GAS serotype variance has important implications for prevention and treatment of GAS infections. Applicants have identified protein components within each of the GAS adhesin islands which are associated with the pilus formation. These proteins are believed to be involved in the bacteria's initial adherence mechanisms. Immunological recognition of these proteins may allow the host immune response to slow or prevent the bacteria's transition into the more pathogenic later stages of infection. In addition, the GAS pili may be involved in formation of biofilms. Applicants have discovered that the GBS pili structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysaccharide matrix). Biofilms are generally associated with bacterial resistance, as antibiotic treatments and host immune response are frequently un'ail'e to 6iτaαic;'aie"air"δf the'bactefia components of the biofilm. Direction of a host immune response against surface proteins exposed during the first steps of bacterial attachment (i.e., before complete biofilm formation) is preferable.
The invention therefore provides for improved immunogenic compositions against GAS infection which may target GAS bacteria during their initial attachment efforts to the host epithelial cells and may provide protection against a wide range of GAS serotypes. The immunogenic compositions of the invention include GAS AI surface proteins which may be formulated in an oligomeric, or hyperoligomeric (pilus) form. The invention also includes combinations of GAS AI surface proteins. Combinations of GAS AI surface proteins may be selected from the same adhesin island or they may be selected from different GAS adhesin islands.
The invention comprises compositions comprising a first GAS AI protein and a second GAS AI protein wherein the first and second GAS Al proteins are derived from different GAS adhesin islands. For example, the invention includes a composition comprising at least two GAS AI proteins wherein the GAS AI proteins are encoded by the adhesin islands selected from the group consisting of GAS AI-I and AI-2; GAS AI-I and GAS AI-3; GAS AI-I and GAS AI-4; GAS AI-2 and GAS AI-3; GAS AI-2 and GAS AI-4; and GAS AI-3 and GAS AI-4. Preferably the two GAS AI proteins are derived from different T-types.
A schematic arrangement of GAS Adhesin Island sequences is set forth in FIGURE 162. In all strains, the AI region is flanked by the highly conserved open reading frames Ml_123 and Ml- 136. Between three and five genes in each locus code for surface proteins containing LPXTG motifs. These surface proteins also all belong to the family of genes coding for ECM binding adhesins.
Adhesin island sequences can be identified in numerous M types of Group A Streptococcus. Examples of AI sequences within Ml, M6, M3, M5, M12, M18, and M49 serotypes are discussed below. GAS Adhesin Islands generally include a series of open reading frames within a GAS genome that encode for a collection of surface proteins and sortases. A GAS Adhesin Island may encode for amino acid sequences comprising at least one surface protein. Alternatively, a GAS Adhesin Island may encode for at least two surface proteins and at least one sortase. Preferably, a GAS Adhesin Island encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or more GAS AI surface proteins may participate in the formation of a pilus structure on the surface of the Gram positive bacteria.
GAS Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator. The transcriptional regulator may regulate the expression of the GAS AI operon. Examples of transcriptional regulators found in GAS AI sequences include RofA and Nra.
The GAS AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen. One or more of the GAS AI surface proteins may comprise a fimbrial structural subunit. "" Orie of1 more of me GAS* Al'surface proteins may include an LPXTG motif or other sortase substrate motif. The LPXTG motif may be followed by a hydrophobic region and a charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. See Barnett, et al., J. Bacteriology (2004) 186 (17): 5865-5875.
GAS AI sequences may be generally categorized as Type 1, Type 2, Type 3, or Type 4, depending on the number and type of sortase sequences within the island and the percentage identity of other proteins (with the exception of RofA and cpa) within the island. Figure 167 provides a chart indicating the number and type of sortase sequences identified within the adhesin islands of various strains and serotypes of GAS. As can be seen in this figure, all GAS strains and serotypes thus far characterized as an AI-I have a SrtB type sortase, all GAS strains and serotypes thus far characterized as an AI-2 have SrtB and SrtCl type sortases, all GAS strains and serotypes thus far characterized as an AI-3 have a SrtC2 type sortase, and all GAS strains and serotypes thus far characterized as an AI-4 have SrtB and SrtC2 type sortases. A comparison of the percentage identity of sequences within the adhesin islands was presented in Table 45, see above.
(1) Adhesin Island sequence within M6: GAS Adhesin Island 1 ("GAS AI-I")
A GAS Adhesin Island within M6 serotype (MGAS 10394) is outlined in Table 4 below. This GAS adhesin island 1 ("GAS AI-I") comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator.
GAS AI-I surface proteins include SρyO157 (a fibronectin binding protein), SpyO159 (a collagen adhesion protein) and Spy0160 (a fimbrial structural subunit). Preferably, each of these GAS AM surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with serine).
GAS AI-I includes a srtB type sortase. GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO: 166), particularly where the motif is followed by a serine.
Table 4: GAS AI-I sequences from M6 isolate (MGAS10394)
Figure imgf000108_0001
M6_Sρy0160 appears to be present on the surface of GAS as part of oligomeric (pilus) structures. Figures 127-132 present electron micrographs of GAS serotype M6 strain 3650 immunogold stained for M6_SpyO16O using anti-M6_Sρy0160 antiserum. Oligomeric or hyperoligmeπc structures labelled witn gold particles can be seen extending from the surtace of the GAS in each of these figures, indicating the presence of multiple M6_Spy0160 polypeptides in the oligomeric or hyperoligomeric structures. Figure 176 A-F present electron micrographs of GAS M6 strain 2724 immunogold stained for M6_Spy0160 using anti-M6_Spy0160 antiserum (Figures 176 A- E) or immunogold stained for M6_Spy0159 using anti-M6_SpyO159 antiserum (Figure 176 F). Oligomeric or hyperoligomeric structures labelled with gold particles can again be seen extending from the surface of the M6 strain 2724 GAS bacteria immunogold stained for M6_Spy0160. M6_SpyO159 is also detected on the surface of the M6 strain 2724 GAS.
FACS analysis has confirmed that the GAS AI-I surface proteins spyM6_0159 and spyM6_0160 are indeed expressed on the surface of GAS. Figure 73 provides the results of FACS analysis for surface expression of spyM6_0159 on each of GAS serotypes M6 2724, M6 3650, and M62894. A shift in fluorescence is observed for each GAS serotype when anti-spyM6_0159 antiserum is present, demonstrating cell surface expression. Table 18, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-spyM6_0159 antiserum, and the difference in fluorescence value between the pre-immune and anti-spyM6_0159 antiserum.
Table 18; Summary of FACS values for surface expression of sρyM6_0159
Figure imgf000109_0001
Figure 74 provides the results of FACS analysis for surface expression of spyM6_0160 on each of GAS serotypes M6 2724, M6 3650, and M6 2894. In the presence of of anti-spyM6_0160 antiserum, a shift in fluorescence is observed for each GAS serotype, which demonstrates its cell surface expression. Table 19, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-spyM6_0160 antiserum, and the change in fluorescence value between the pre-immune and anti-sρyM6_0160 antiserum.
Table 19: Summary of FACS values for surface expression of spyM6_0160
Figure imgf000109_0002
Surface expression of M6_Sρy0159 and M6_Spy0160 on M6 serotype GAS has also been confirmed by Western blot analysis. Figure 98 shows that while pre-immune sera (P α-0159) does not detect expression of M6_SρyO159 in GAS serotype M6, anti-M6_SpyO159 immune sera (I α- 0159) is able to detect M6_SρyO159 protein in both total GAS M6 extracts (M6 tot) and GAS M6 fractions enriched for cell surface proteins (M6 surf prot). The M6_SpyO159 proteins detected in the tόtal"GAS"M6'extracts"bf the"GΑέ'Mo extracts enriched for surface proteins are also present as high molecular weight structures, indicating that M6_SpyO159 may be in an oligomeric (pilus) form.
Figure 112 shows that while preimmune sera (Preimmune Anti 106) does not detect expression of M6_Spy0160 in GAS serotype M6 strain 2724, anti-M6_Spy0160 immune sera (Anti 160) does in both total GAS M6 strain 2724 extracts (M6 2724 tot) and GAS M6 strain 2724 fractions enriched for surface proteins. The M6_Spy0160 proteins detected in the total GAS M6 strain 2724 extracts or the GAS M6 strain 2724 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that M6_SpyO16O may be in an oligomeric (pilus) form.
Figures 110 and 111 both further verify the presence of M6_SρyO 159 and M6_Spy0160 in higher molecular weight structures on the surface of GAS. Figure 110 provides a Western blot performed to detect M6_SpyO159 and M6_Spy0160 in GAS M6 strain 2724 extracts enriched for surface proteins. Antiserum raised against either M6_Spy0159 (Anti- 159) or M6_SρyO16O (Anti- 160) cross-hybridizes with high molecular weight structures (pili) in these extracts. Figure 111 provides a similar Western blot that verifies the presence of M6_ SρyO159 and M6_Spy0160 in high molecular weight structures in GAS M6 strain 3650 extracts enriched for surface proteins.
SpyM6_0157 (a fibronectin-binding protein) may also be expressed on the surface of GAS serotype M6 bacteria. Figure 174 shows the results of FACS analysis for surface expression of spyM6_0157 on M6 strain 3650. A slight shift in fluorescence is observed, which demonstrates that some spyM6_0157 may be expressed on the GAS cell surface. Adhesin Island sequence within M6: GAS Adhesin Island 2 ("GAS AI-2")
A GAS Adhesin Island within Ml serotype (SF370) is outlined in Table 5 below. This GAS adhesin island 2 ("GAS AI-2") comprises surface proteins, a SrtB sortase, a SrtCl sortase and a RofA divergently transcribed transcriptional regulator.
GAS AI-2 surface proteins include GAS 15 (Cpa), SpyO128 (thought to be a fimbrial protein) and Spy0130 (a hypothetical protein). Preferably, each of these GAS AI-2 surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), WXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
GAS AI-2 includes a srtB type sortase and a srtCl sortase. As discussed above, GAS SrtB sortases may preferably anchor surface proteins with an LPSTG (SEQ ID NO: 166) motif, particularly where the motif is followed by a serine. GAS SrtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO: 167) motif. GAS SrtCl may be differentially regulated by RofA.
GAS AI-2 may also include a LepA putative signal peptidase I protein. Table 5 : GAS AI-2 sequence from Ml isolate (SF370)
Figure imgf000110_0001
Figure imgf000111_0001
GAS 15, GAS 16, and GAS 18 appear to be present on the surface of GAS as part of oligomeric (pilus) structures. Figures 113-115 present electron micrographs of GAS serotype Ml strain SF370 immunogold stained for GAS 15 using anti-GAS 15 antiserum. Figures 116-121 provide electron micrographs of GAS serotype Ml strain SF370 immunogold stained for GAS 16 using anti- GAS 16 antiserum. Figures 122-125 present electron micrograph of GAS serotype Ml strain SF370 immunogold stained for GAS 18 using anti-GAS 18 antiserum. Oligomers of these proteins can be seen on the surface of SF370 bacteria in the immuno-gold stained micrographs.
Figure 126 reveals a hyperoligomer on the surface of a GAS serotype Ml strain SF370 bacterium immunogold stained for GAS 18. This long hyperoliogmeric structure comprising GAS 18 stretches far out into the supernatant from the surface of the bacteria.
FACS analysis has confirmed that the GAS AI-2 surface proteins GAS 15, GAS 16, and GAS 18 are expressed on the surface of GAS. Figure 75 provides the results of FACS analysis for surface expression of GAS 15 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348. A shift in fluorescence is observed for each GAS serotype when anti-GAS 15 antiserum is present, demonstrating cell surface expression. Table 20, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-GAS 15 antiserum, and the difference in fluorescence value between the pre- immune and anti-GAS 15 antiserum.
Table 20: Summary of FACS values for surface expression of GAS 15
Figure imgf000111_0002
Figures 76 and 79 provide the results of FACS analysis for surface expression of GAS 16 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348. The FACS data in Figure 76 was obtained using antisera was raised against full length GAS 16. In the presence of this anti-GAS 16 antiserum, a shift in fluorescence is observed for each GAS serotype, dfemtonsfrating-'its-'beα slrfade'expr'essϊon:1 Table 21, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti- GAS 16 antiserum, and the change in fluorescence value between the pre-immune and anti-GAS 16 antiserum.
Table 21: Summary of FACS values for surface expression of GAS 16
Figure imgf000112_0002
The FACS data in Figure 79 was obtained using antisera was raised against a truncated GAS 16, which is encoded by SEQ ID NO: 179, shown below.
SEQ ID NO: 179:
Figure imgf000112_0001
ACTGATAAAGATATGACCATTACTTTTACAAATAAAΆAAGATTT
In the presence of this anti-GAS 16 antiserum, a shift in fluorescence is observed for each GAS serotype, demonstrating its cell surface expression. Table 22, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-GAS 16 antiserum, and the change in fluorescence value between the pre-immune and anti-GAS 16 antiserum.
Table 22: Summary of FACS values for surface expression of GAS 16 using a second antisera
Figure imgf000112_0003
i;:;ii C" JϊguM I? Olis-pivS'eUiyiesnlts of FACS analysis for surface expression of GAS 18 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348. The antiserum us"ed to obtain the FACS data in each of Figures 77 and 78 was different, although each was raised against full length GAS 18. In the presence of each of the anti-GAS 18 antisera, a shift in fluorescence is observed for each GAS serotype, demonstrating its cell surface expression. Tables 23 and 24, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, first or second anti-GAS 18 antiserum, and the change in fluorescence value between the pre-immune and first or second anti-GAS 18 antiserum. Table 23: Summary of FACS values for surface expression of GAS 18
Figure imgf000113_0001
Table 24: Summary of FACS values for surface expression of GAS 18 using a second antisera
Figure imgf000113_0002
Surface expression of GAS 15, GAS 16, and GAS 18 on Ml serotype GAS has also been confirmed by Western blot analysis. Figure 89 shows that while pre-immune sera does not detect GAS Ml expression of GAS 15, anti-GAS 15 immune sera is able to detect GAS 15 protein in both total GAS Ml extracts and GAS Ml proteins enriched for cell surface proteins. The GAS 15 proteins detected in the Ml extracts enriched for surface proteins are also present as high molecular weight structures, indicating that GAS 15 may be in an oligomeric (pilus) form. Figure 90 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 15 antisera. Again, the lanes that contain GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 15. Figure 91 provides an additional Western blot identical to that of Figure 90, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
Figure 92 provides a Western blot that was probed for GAS 16 protein. While pre-immune sera does not detect GAS Ml expression of GAS 16, anti-GAS 16 immune sera is able to detect GAS
Figure imgf000114_0001
eVϊcΗecl for cell surface proteins. The GAS 16 proteins detected in the Ml extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 16 may be in an oligomeric (pilus) form. Figure 93 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 16 antisera. The lanes that contain total GAS Ml protein (Ml tot new and Ml tot old) and the lane that contains GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 16. Figure 94 provides an additional Western blot identical to that of Figure 93, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane. Figure 95 provides a Western blot that was probed for GAS 18 protein. While pre-immune sera does not detect GAS Ml expression of GAS 18, anti-GAS 18 immune sera is able to detect GAS 18 protein in GAS Ml extracts enriched for cell surface proteins. The GAS 18 proteins detected in the Ml extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 18 may be in an oligomeric (pilus) form. Figure 96 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 18 antisera. The lane that contains GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 18. Figure 97 provides an additional Western blot identical to that of Figure 96, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
Figures 102-106 provide additional Western blots to verify the presence of GAS 15, GAS 16, and GAS 18 in high molecular weight structures in GAS. Each Western blot was performed using proteins from a different GAS Ml strain, 2580, 2913, 3280, 3348, and 2719. Each Western blot was probed with antisera raised against each of GAS 15, GAS 16, and GAS 18. As can be seen in Figures 102-106, none of the Western blots shows detection of proteins using pre-immune serum (Pα-158, Pα-15, Pα-16, or Pα-18), while each Western blot shows cross-hybridization of the GAS 15 (Iα-15), GAS 16 (Ia- 16), and GAS 18 (Ia- 18) antisera to high molecular weight structures. Thus, these Western blots confirm that GAS 15, GAS 16, and GAS 18 can be present in pili in GAS Ml.
Figure 107 provides a similar Western blot performed to detect GAS 15, GAS 16, and GAS 18 proteins in a GAS serotype Ml strain SF370 protein fraction enriched for surface proteins. This Western blot also shows detection of GAS 15 (Anti-15), GAS 16 (Anti-16), and GAS 18 (Anti-18) as high molecular weight structures.
(3) Adhesin Island sequence within M3. M5. and M18: GAS Adhesin Island 3 ("GAS AI-3") GAS Adhesin Island sequences within M3, M5, and M18 serotypes are outlined in Tables 6 — 8 and 10 below. This GAS adhesin island 3 ("GAS AI-3") comprises surface proteins, a SrtC2 sortase, and a Negative transcriptional regulator (Nra) divergently transcribed transcriptional regulator.
GAS AI-3 surface proteins within include a collagen binding protein, a fimbrial protein, a F2 like fibronectin-binding protein. GAS AI-3 surface proteins may also include a hypothetical surface l
Figure imgf000115_0001
surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
GAS AI-3 includes a SrtC2 type sortase. GAS SrtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail. GAS SrtC2 may be differentially regulated by Nra.
GAS AI-3 may also include a LepA putative signal peptidase I protein.
GAS AI-3 may also include a putative multiple sugar metabolism regulator.
Table 6: GAS AI-3 sequences from M3 isolate (MGAS315)
Figure imgf000115_0002
Table 7: GAS AI-3 sequence from M3 isolate (SSI-I)
Figure imgf000115_0003
Figure imgf000115_0004
Figure imgf000116_0001
Table 8: GAS AI-3 se uences from M18 isolate (MGAS8232)
Figure imgf000116_0002
Table 44: GAS AI-3 sequences from M49 isolate (591)
Figure imgf000116_0003
A schematic of AI-3 serotypes M3, M5, M18, and M49 is shown in Figure 51A. Each contains an open reading frame encoding a SrtC2-type sortase of nearly identical amino acid sequence. See Figure 52B for an amino acid sequence alignment for each of the SrtC2 amino acid sequences.
The protein F2-like fibronectin-binding protein of each these type 3 adhesin islands contains a pilin motif and an E-box. Figure 60 indicates the amino acid sequence of the pilin motif and E-box of each of GAS AI-3 serotype M3 MGAS315 (SpyM3_0104/21909640), GAS AI-3 serotype M3 SSI (SpsO 106/28895018), GAS AI-3 serotype M18 (SpyM18_0132/19745307), and GASAI-3 serotype
FACS analysis has confirmed that the GAS AI-3 surface proteins SρyM3_0098, SpyM3_0100, SρyM3_0102, and SpyM3_0104 are expressed on the surface of GAS. Figure 80 provides the results of FACS analysis for surface expression of SρyM3_0098 on each of GAS
Figure imgf000117_0001
ϊtϊi. S lϊiSϊn fluorescence is observed for each GAS serotype when anti-SpyM3_0098 antiserum is present, demonstrating cell surface expression. Table 25, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0098 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0098 antiserum.
Table 25: Summary of FACS values for surface expression of SpyM3_0098
Figure imgf000117_0002
Figure 81 provides the results of FACS analysis for surface expression of SpyM3_0100 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0100 antiserum is present, demonstrating cell surface expression. Table 26, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0100 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0100 antiserum.
Table 26: Summary of FACS values for surface expression of SpyM3_0100
Figure imgf000117_0003
Figure 82 provides the results of FACS analysis for surface expression of SpyM3_0102 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SρyM3_0102 antiserum is present, demonstrating cell surface expression. Table 27, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0102 antiserum.
Table 27: Summary of FACS values for surface expression of SpyM3_0102 in M3 serotypes
Figure imgf000117_0004
Figure 82 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM3_0102 identified in a different GAS serotype, M6. FACS analysis conducted with the SpyM3_0102 antisera was able to detect surface expression of the homologous SpyM3_0102 antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894. Table 28, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SρyM3_0102 antiserum.
Table 28: Summary of FACS values for surface expression of SpyM3_0102 in M6 serotypes
2724 3650 2894
Figure imgf000118_0001
SpyM3_0102 is also homologous to pilin antigen 19224139 of GAS serotype M12. Antisera raised against SpyM3_0102 is able to detect high molecular weight structures in GAS serotype M12 strain 2728 protein fractions enriched for surface proteins, which would contain the 19224139 antigen. See Figure 109 at the lane labelled M12 2728 surf prot.
Figure 83 provides the results of FACS analysis for surface expression of SpyM3_0104 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0104 antiserum is present, demonstrating cell surface expression. Table 29, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-SpyM3_0104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0104 antiserum.
Table 29: Summary of FACS values for surface expression of SpyM3_0104 in M3 serotypes
Figure imgf000118_0002
Figure 83 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM3_0104 identified in a different GAS serotype, Ml 2. FACS analysis conducted with the SpyM3_0104 antisera was able to detect surface expression of the homologous SpyM3_0104 antigen on GAS serotype M12 2728. Table 30, below, quantitatively summarizes the FACS fluorescence values obtained for this GAS serotype in the presence of pre-immune antiserum, anti-SρyM3_0104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0104 antiserum.
Table 30: Summary of FACS values for surface expression of SpyM3_0104 in an M12 serotype
Figure imgf000118_0003
Figure 84 provides the results of FACS analysis for surface expression of SPs_0106 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SPs_0106 antiserum is present, demonstrating cell surface expression. Table 31, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SPs_0106 antiserum, and the difference in fluorescence value between the pre-immune and anti-SPs_0106 antiserum.
Table 31 : Summary of FACS values for surface expression of SPs_0106 in M3 serotypes
Figure imgf000118_0004
Figure 84 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SPs_0106 identified in a different GAS serotype, M12. FACS analysis
Figure imgf000119_0001
able to detect surface expression of the homologous SPs_0106 antigen on GAS serotype M12 2728. Table 32, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SPs_0106 antiserum, and the difference in fluorescence value between the pre-immune and anti- SPs_0106 antiserum.
Table 32: Summary of FACS values for surface expression of SPs_0106 in an M12 serotype
Figure imgf000119_0002
(4) Adhesin Island sequence within Ml 2: GAS Adhesin Island 4 ("GAS AI-4")
GAS Adhesin Island sequences within M12 serotype are outlined in Table 11 below. This GAS adhesin island 4 ("GAS AI-4") comprises surface proteins, a SrtC2 sortase, and a RofA regulatory protein.
GAS AI-4 surface proteins within may include a fimbrial protein, an F or F2 like fibronectin- binding protein, and a capsular polysaccharide adhesion protein (Cpa). GAS AI-4 surface proteins may also include a hypothetical surface protein in an open reading frame (orf). Preferably, each of these GAS AI-4 surface proteins include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO: 139).
GAS AI-4 includes a SrtC2 type sortase. GAS SrtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO: 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail.
GAS AI-4 may also include a LepA putative signal peptidase I protein and a MsmRL protein. Table 11; GAS AI-4 se uences from M12 isolate A735
Figure imgf000119_0003
A schematic of AI-4 serotype M12 is shown in Figure 51A.
One of the open reading frames encodes a SrtC2-type sortase having an amino acid sequence nearly identical to the amino acid sequence of the SrtC2-type sortase of the AI-3 serotypes described atfdV&r sWFlflufe1 sifffof afrϊ'aminό a'δicF'sequence alignment for each of the SrtC2 amino acid sequences.
Other proteins encoded by the open reading frames of the AI-4 serotype Ml 2 are homologous to proteins encoded by other known adhesin islands in S. pyogenes, as well as the GAS AI-3 serotype M5 (Manfredo). Figure 52 is an amino acid alignment of the capsular polysaccharide adhesion protein (cpa) of AI-4 serotype M12 (19224135), GAS AI-3 serotype M5 (ORF78), S. pyogenes strain MGAS315 serotype M3 (21909634), S. pyogenes SSI-I serotype M3 (28810257), S. pyogenes MGAS8232 serotype M3 (19745301), and GAS AI-2 serotype Ml (GAS15). The amino acid sequence of the AI-4 serotype M12 cpa shares a high degree of homology with other cpa proteins. Figure 53 shows that the F-like fibronectin-binding protein encoded by the AI-4 serotype
M12 open reading frame (19224134) shares homology with a F-like fibronectin-binding protein found in S. pyogenes strain MGAS 10394 serotype M6 (50913503).
Figure 54 is an amino acid sequence alignment that illustrates that the F2-like fibronectin- binding protein of AI-4 serotype Ml 2 (19224141) shares homology with the F2-like fibronectin- binding protein of S. pyogenes strain MGAS8232 serotype M3 (19745307), GAS AI-3 serotype M5 (ORF84), S. pyogenes strain SSI serotype M3 (28810263), and S. pyogenes strain MGAS315 serotype M3 (21909640).
Figure 55 is an amino acid sequence alignment that illustrates that the fimbrial protein of AI-4 serotype'M12 (19224137) shares homology with the fimbrial protein of GAS AI-3 serotype M5 (ORF80), and the hypothetical protein of S. pyogenes strain MGAS315 serotype M3 (21909636), S. pyogenes strain SSI serotype M3 (28810259), S. pyogenes strain MGAS8732 serotype M3 (19745303), and S. pyogenes strain Ml GAS serotype Ml (13621428).
Figure 56 is an amino acid sequence alignment that illustrates that the hypothetical protein of GAS AI-4 serotype M12 (19224139) shares homology with the hypothetical protein of S. pyogenes strain MGAS315 serotype M3 (21909638), S. pyogenes strain SSI-I serotype M3 (28810261), GAS AI-3 serotype M5 (ORF82), and S. pyogenes strain MGAS8232 serotype M3 (19745305).
The protein F2-like fibronectin-binding protein of the type 4 adhesin island also contains a highly conserved pilin motif and an E-box. Figure 60 indicates the amino acid sequence of the pilin motif and E-box in AI-4 serotype Ml 2. FACS analysis has confirmed that the GAS AI-4 surface proteins 19224134, 19224135,
19224137, and 19224141 are expressed on the surface of GAS. Figure 85 provides the results of FACS analysis for surface expression of 19224134 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224134 antiserum is present, demonstrating cell surface expression. Table 33, below, quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M12 2728 in the presence of pre-immune antiserum, anti-19224134 antiserum, and the difference in fluorescence value between the pre-immune and anti-19224134 antiserum.
Table 33: Summary of FACS values for surface expression of 19224134 in an M12 serotype
|~ 2728 [ !D> i 'Li' ::;:;i! i
Figure imgf000121_0001
Figxire 85 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to 19224134 identified in a different GAS serotype, M6. FACS analysis conducted with the 19224134 antisera was able to detect surface expression of the homologous 19224134 antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894. Table 34, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-19224134 antiserum, and the difference in fluorescence value between the pre-immune and anti-19224134 antiserum.
Table 34: Summary of FACS values for surface expression of 19224134 in M6 serotypes
Figure imgf000121_0002
Figure 86 provides the results of FACS analysis for surface expression of 19224135 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224135 antiserum is present, demonstrating cell surface expression. Table 35, below, quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M12 2728 in the presence of pre-immune antiserum, anti-19224135 antiserum, and the difference in fluorescence value between the pre-immune and anti- 19224135 antiserum.
Table 35: Summary of FACS values for surface expression of 19224135 in an M12 serotype
Figure imgf000121_0003
Figure 87 provides the results of FACS analysis for surface expression of 19224137 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224137 antiserum is present, demonstrating cell surface expression. Table 36, below, quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M12 2728 in the presence of pre-immune antiserum, anti-19224137 antiserum, and the difference in fluorescence value between the pre-immune and anti- 19224137 antiserum.
Table 36: Summary of FACS values for surface expression of 19224137 in an M12 serotype
Figure imgf000121_0004
Figure 88 provides the results of FACS analysis for surface expression of 19224141 on GAS serotype M12 2728. A shift in fluorescence is observed when anti-19224141 antiserum is present, demonstrating cell surface expression. Table 37, below, quantitatively summarizes the FACS fluorescence values obtained for GAS serotype M 12 2728 in the presence of pre-immune antiserum, anti-19224141 antiserum, and the difference in fluorescence value between the pre-immune and anti- 19224141 antiserum. P
Figure imgf000122_0001
surface expression of 19224141 in an M12 serotype
Figure imgf000122_0002
19224139 (designated as orf2) may also be expressed on the surface of GAS serotype M12 bacteria. Figure 175 shows the results of FACS analysis for surface expression of 19224139 on M12 strain 2728. A slight shift in fluorescence is observed, which demonstrates that some 19224139 may be expressed on the GAS cell surface.
Surface expression of 19224135 on M12 serotype GAS has also been confirmed by Western blot analysis. Figure 99 shows that while pre-immune sera (P α-4135) does not detect GAS M12 expression of 19224135, anti-19224135 immune sera (I α-4135) is able to detect 19224135 protein in both total GAS M12 extracts (M12 tot) and GAS M12 fractions enriched for cell surface proteins (M12 surf prot). The 19224135 proteins detected in the total GAS M12 extracts or the GAS M12 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that 19224135 may be in an oligomeric (pilus) form. See also Figure 108, which provides a further Western blot showing that anti-19224135 antiserum (Anti-19224135) imrnunoreacts with high molecular weight structures in GAS M12 strain 2728 protein extracts enriched for surface proteins. Surface expression of 19224137 on M12 serotype GAS has also been confirmed by Western blot analysis. Figure 100 shows that while pre-immune sera (P α-4137) does not detect GAS M 12 expression of 19224137, anti-19224137 immune sera (I α-4137) is able to detect 19224137 protein in both total GAS M 12 extracts (M 12 tot) and GAS M12 fractions enriched for cell surface proteins (M12 surf prot). The 19224137 proteins detected in the total GAS M12 extracts or the GAS M12 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that 19224137 may be in an oligomeric (pilus) form. See also Figure 108, which provides a further Western blot showing that anti-19224137 antiserum (Anti-19224137) immunoreacts with high molecular weight structures in GAS M12 strain 2728 protein extracts enriched for surface proteins. Streptococcus pneumoniae Adhesin island sequences can be identified in Streptococcus pneumoniae genomes. Several of these genomes include the publicly available Streptococcus pneumoniae TIGR4 genome or Streptococcus pneumoniae strain 670 genome. Examples of these S. pneumoniae AI sequence are discussed below.
S. pneumoniae Adhesin Islands generally include a series of open reading frames within a S. pneumoniae genome that encode for a collection of surface proteins and sortases. A S. pneumoniae Adhesin Island may encode for amino acid sequences comprising at least one surface protein. Alternatively, an S. pneumoniae Adhesin Island may encode for at least two surface proteins and at least one sortase. Preferably, a S. pneumoniae Adhesin Island encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or more S. pneumoniae AI i C: surface pro'te: iϊMbiiaώ;iiϊ;i ation of a pilus structure on the surface of the S. pneumoniae bacteria.
S. pneumoniae Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator. The transcriptional regulator may regulate the expression of the S. pneumoniae AI operon.
The S. pneumoniae AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen.
A schematic of the organization of a S. pneumoniae AI locus is provided in Figure 137. The locus comprises open reading frames encoding a transcriptional regulator (rlrA), cell wall surface proteins (rrgA, rrgB, rrgC), and sortases (srtB, srtC, srtD). Figure 137 also indicates the S. pneumoniae strain TIGR4 gene name corresponding to each of these open reading reading frames.
Tables 9 and 38 identify the genomic location of each of these open reading frames in S. pneumoniae strains TIGR.4 and 670, respectively. Table 9: S. pneumoniae AI sequences from TIGR4
Genomic Location Strand Length PID Synonym (AI Sequence Functional description Identifier)
436302..437831 509 15900377 SP0461 transcriptional regulator
438326..441007 + 893 15900378 SP0462 cell wall surface anchor family protein
441231..443228! 665 15900379 SP0463 cell wall surface anchor family protein
443275..444456 393 15900380 SP0464 cell wall surface anchor family protein
444675..444806! 43 15900381 SP0465 hypothetical protein
444857..445096 279 15900382SP0466 sortase
445791..446576 261 15900383 SP0467 sortase
446563..447414I 283 15900384SP0468 sortase
Table 38: S. pneumoniae strain 670 AI sequences
Figure imgf000123_0001
The full-length nucleotide sequence of the S. pneumoniae strain 670 AI is also shown in Figure 101, as is its translated amino acid sequence.
At least eight other S. pneumoniae strains contain an adhesin island locus described by the locus depicted in Figure 137. These strains were identified by an amplification analysis. The genomes of different S. pneumoniae strains were amplified with eleven separate sets of primers. The sequence of each of these primers is provided below in Table 41.
Table 41: Sequences of primers used to amplify AI locus
Primer Forward Primer Sequence Reverse Primer Sequence
Figure imgf000124_0001
These primers hybridized along the entire length of the AI locus to generate amplification products representative of sequences throughout the locus. See Figure 138, which is a schematic of the location where each of these primers hybridizes to the S. pneumoniae AI locus. Figure 139A provides the set of amplicons obtained from amplification of the AI locus in S. pneumoniae strain TIGR4. Figure 139B provides the length, in base pairs, of each amplicon in S. pneumoniae strain TIGR4. Amplification of the genome of S. pneumoniae strains 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9 V Spain 3, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, and 23F Poland 16 produced a set of eleven amplicons for the eleven primer pairs, indicating that each of these strains also contained the S. pneumoniae AI locus.
The S. pneumoniae strains were also identified as containing the AI locus by comparative genome hybridization (CGH) analysis. The genomes of sixteen S. pneumoniae strains were interrogated for the presence of the AI locus by comparison to unique open reading frames of strain TIGR4. The AI locus was detected by this method in strains 19A Hungary 6 (19AHUN), 6B Finland 12 (6BFIN12), 6B Spain 2 (6BSP2), 14CSR10 (14 CSRlO), 9V Spain 3 (9VSP3), 19F Taiwan 14 (19FTW14), 23F Taiwan 15 (19FTW15), and 23F Poland 16 (23FP16). See Figure 140.
The AI locus has been sequenced for each of these strains and the nucleotide and encoded amino acid seqeunce for each orf has been determined. An alignment of the complete nucleotide sequence of the adhesin island present in each of the ten strains is provided in Figure 196. Aligning the amino acid sequences encoded by the orfs reveals conservation of many of the AI polypeptide amino acid sequences. For example, Table 39 provides a comparison of the percent identities of the polypeptides encoded within the S. pneumoniae strain 670 and TIGR4 adhesin islands.
Table 39: Pecent identit com arison of S. neumoniae strains AI se uences
Figure imgf000124_0002
P C 1F/ U S O 5 ,,■■' 2 7 Ei; 3 SI
Figures 141-147 each provide a multiple sequence alignment for the polypeptides encoded by one of the open reading frames in all ten AI-positive S. pneumoniae strains. In each of the sequence alignments, light shading indicates an LPXTG motif and dark shading indicates the presence of an E- box motif with the conserved glutamic acid residue of the E-box motif in bold.
The sequence alignments also revealed that the polypeptides encoded by most of the open reading frames may be divided into two groups of homology, S. pneumoniae AI-a and AI-b. S. pneumoniae strains that comprise AI-a include 14 CSR 10, 19A Hungary 6, 23F Poland 15, 670, 6B Finland 12, and 6B Spain 2. S. pneumoniae strains that comprise AI-b include 19F Taiwan 14, 9V Spain 3, 23F Taiwan 15, and TIGR4. An immunogenic composition of the invention may comprise one or more polypeptides from within each of S. pneumoniae AI-a and AI-b. For example, polypeptide RrgB, encoded by open reading frame 4, may be divided within two such groups of homology. One group contains the RrgB sequences of six S. pneumoniae strains and a second group contains the RrgB sequences of four S. pneumoniae strains. While the amino acid sequence of the strains within each individual group is 99-100 percent identical, the amino acid sequence identity of the strains in the first relative to the second group is only 48%. Table 41 provides the identity comparisons of the amino acid sequences encoded by each open reading frame for the ten S. pneumoniae strains.
Table 42: Conservation of amino acid sequences encoded by the S. pneumoniae AI locus
Figure imgf000125_0001
The division of homology between the RrgB polypeptide in the S. pneumoniae strains is due a lack of amino acid sequence identity in the central amino acid residues. Amino acid residues 1-30 and 617-665 are identical for each of the ten S. pneumoniae strains. However, amino acid residues 31-616 share between 42 and 100 percent identity between strains. See Figure 149. The shared N- and C-terminal regions of identity in the RrgB polypeptides may be preferred portions of the RrgB polypeptide for use in an immunogenic composition. Similarly, shared regions of identity in any of the polypeptides encoded by the S. pneumoniae AI locus may be preferable for use in immunogenic compositions. One of skill in the art, using the amino acid alignments provided in Figures 141-147, would readily be able to determine these regions of identity.
The S. pneumoniae comprising these AI loci do, in fact, express high molecular weight polymers on their surface, indicating the presence of pili. See Figure 182, which shows detection of high molecular weight structures expressed by S. pneumoniae strains that comprise the adhesin island lqcli£SepGtelll:FipiFiia7iplhSyitiilnsi!are indicated as rlrA+. Confirming these findings, electron microscopy and negative staining detects the presence of pili extending from the surface of S. pneumoniae. See Figure 185. To demonstrate that the adhesin island locus was responsible for the pili, the rrgA-srtD region of TIGR 4 were deleted. Deletion of this region of the adhesin island resulted in a loss of pili expression. See Figure 186. See also Figure 235, which provides an electron micrograph of 5. pneumoniae lacking the rrgA-srtD region immunogold stained using anti-RrgB and anti-RrgC antibodies. No pili can be seen. Similarly to that described above, a S. pneumoniae bacteria that lacks a transcriptional repressor, mgrA, of genes in the adhesin island expresses pili. See Figure 187. However, and as expected, a S. pneumoniae bacteria that lacks both the mgrA and adhesin island genes in the rrgA-srtD region does not express pili. See Figure 188.
These high molecular weight pili structures appear to play a role in adherence of S. pneumoniae to cells. S. pneumoniae TIGR4 that lack the pilus operon have significantly diminished ability to adhere to A549 alveolar cells in vitro. See Figure 184.
The SpO463 (S. pneumoniae TIGR4 rrgB) adhesion island polypeptide is expressed in oligomeric form. Whole cell extracts were analyzed by Western blot using a SpO463 antiserum. The antiserum cross-hybridized with high molecular weight SpO463 polymers. See Figure 156. The antiserum did not cross-hybridize with polypeptides from D39 or R6 strains of S. pneumoniae, which do not contain the AI locus depicted in Figure 137. Immunogold labelling of S. pneumoniae TIGR 4 using RrgB antiserum confirms the presence of RrgB in pili. Figure 189 shows double-labeling of S. pneumoniae TIGR 4 bacteria with immunolabeling for RrgB (5 nm gold particles) and RrgC (10 nm gold particles) protein. The RrgB protein is detected as present at intervals along the pilus structure. The RrgC protein is detected at the tips of the pili. See Figure 234 at arrows; Figure 234 is a close up of a pilus in Figure 189 at the location indicated by *.
The RrgA protein appears to be present in and necessary for formation of high molecular weight structures on the surface of 5. pneumoniae TIGR4. See Figure 181 which provides the results of Western blot analysis of TIGR4 S. pneumoniae lacking the gene encoding RrgA. No high molecular weight structures are detected in S. pneumoniae that do not express RrgA using antiserum raised against RrgB. See also Figure 183.
A detailed diagram of the amino acid sequence comparions of the RrgA protein in the ten 5.. pneumoniae strains is shown in Figure 148. The diagram reveals the division of the individual S. pneumoniae strains into the two different homology groups.
The cell surface polypeptides encoded by the S. pneumoniae TIGR4 AI, SpO462 (rrgA), SρO463 (rrgB), and SpO464 (rrgC), have been cloned and expressed. See examples 15-17. A polyacrylamide gel showing successful recombinant expression of RrgA is provided in Figure 190A. Detection of the RrgA protein, which is expressed in pET21b with a histidine tag, is also shown by Western blot analysis in Figure 190B, using an anti-histidine tag antibody.
Antibodies that detect RrgB and RrgC antibodies have been produced in mice. See Figures
191 and 192, which show detection of RrgB and RrgC, respectively, using the raised antibodies. Ii"''' !i to'ad'dtooirto tnteidertiridatid'rϊ oϊ these S. pneumoniae adhesion islands, coding sequences for
SrtB type sortases have been identified in several S. pneumoniae clinical isolates, demonstrating conservation of a SrtB type sortase across these isolates. Recombinantlv Produced AI polypeptides It is also an aspect of the invention to alter a non-AI polypeptide to be expressed as an AI polypeptide. The non-AI polypeptide may be genetically manipulated to additionally contain AI , polypeptide sequences, e.g., a sortase substrate, pilin, or E-box motif, which may cause expression of the non-AI polypeptide as an AI polypeptide. Alternatively the non-AI polypeptide may be genetically manipulated to replace an amino acid sequence within the non-AI polypeptide for AI polypeptide sequences, e.g., a sortase substrate, pilin, or E-box motif, which may cause expression of the non-AI polypeptide as an AI polypeptide. Any number of amino acid residues may be added to the non-AI polypeptide or may be replaced within the non-AI polypeptide to cause its expression as an AI polypeptide. At least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 75, 100, 150, 200, or 250 amino acid residues may be replaced or added to the non-AI polypeptide amino acid sequence. GBS 322 may be one such non-AI polypeptide that may be expressed as an AI polypeptide. GBS Adhesin Island Sequences
The GBS AI polypeptides of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from GBS, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form.
The GBS AI proteins of the invention may include polypeptide sequences having sequence identity to the identified GBS proteins. The degree of sequence identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more). Polypeptides having sequence identity include homologs, orthologs, allelic variants and functional mutants of the identified GBS proteins. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith- Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affinity gap search with parameters gap open penalty=12 and gap extension penalty =1.
The GBS adhesin island polynucleotide sequences may include polynucleotide sequences having sequence identity to the identified GBS adhesin island polynucleotide sequences. The degree of sequence identity may vary depending on the polynucleotide sequpnce in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9.7%, 98%, 99%, 99.5% or more).
The GBS adhesin island polynucleotide sequences of the invention may include polynucleotide fragments of the identified adhesin island sequences. The length of the fragment may vify'tøepending'øiii! fee-pblynϋelώfia&lfcqϊlence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
The GBS adhesin island amino acid sequences of the invention may include polypeptide fragments of the identified GBS proteins. The length of the fragment may vary depending on the amino acid sequence of the specific GBS antigen, but the fragment is preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferably the fragment comprises one or more epitopes from the sequence. Other preferred fragments include (1) the N-terminal signal peptides of each identified GBS protein, (2) the identified GBS protein without their N-terminal signal peptides, and (3) each identified GBS protein wherein up to 10 amino acid residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the N- tenninus and/or the C-terminus e.g. the N-terminal amino acid residue may be deleted. Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain). GBS 80
Examples of preferred GBS 80 fragments are discussed below. Polynucleotide and polypeptide sequences of GBS 80 from a variety of GBS serotypes and strain isolates are set forth in Figures 18 and 22. The polynucleotide and polypeptide sequences for GBS 80 from GBS serotype V, strain isolate 2603 are also included below as SEQ ID NOS 1 and 2: SEQ ID NO. 1
AATATCTATAAATTACAAGCTGATAGTTATAAATCGGAAATTACTTCTAATGGTGGTATCGAGAATAAAGACGGC
Figure imgf000128_0001
AGTAATGTGAGATACTTGTATGTAGAAGATTTAAAGAATTCACCTTCAAACATTACCAAAGCTTATGCTGTACCG
Figure imgf000128_0002
CCAGAAGTTCATACTGGTGGGAAACGATTTGTAAAGAAAGACTCAACAGAAACACAAACACTAGGTGGTGCTGAG
Figure imgf000128_0003
GGTATTGGTACGGCTATCTTTGTCGCTATCGGTGCTGCGGTGATGGCTTTTGCTGTTAAGGGGATGAAGCGTCGT ACAAAAGΆTAAC SEQ ID NO: 2
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
Figure imgf000129_0001
PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK GLAYAVDANAEGTAVT YKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG GI GTAI FVAI GAAVMAFAVKGMKRRTKDN
As described above, the compositions of the invention may include fragments of AI proteins.
In some instances, removal of one or more domains, such as a leader or signal sequence region, a transmembrane region, a cytoplasmic region or a cell wall anchoring motif, may facilitate cloning of the gene encoding the protein and/or recombinant expression of the GBS AI protein. In addition, fragments comprising immunogenic epitopes of the cited GBS AI proteins may be used in the compositions of the invention.
For .example, GBS 80 contains an N-terminal leader or signal sequence region which is indicated by the underlined sequence at the beginning of SEQ ID NO: 2 above. In one embodiment, one or more amino acids from the leader or signal sequence region of GBS 80 are removed. An example of such a GBS 80 fragment is set forth below as SEQ ID NO: 3:
SEQ ID NO: 3
AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKK LTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTG TGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVG KIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVAS TINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVK WTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEF TVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN GBS 80 contains a C-terminal transmembrane region which is indicated by the underlined sequence near the end of SEQ ID NO: 2 above. In one embodiment, one or more amino acids from the transmembrane region and/or a cytoplasmic region are removed. An example of such a GBS 80 fragment is set forth below as SEQ ID NO: 4:
SEQ ID NO: 4 MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK SNVRYLYVEDLKNSPSNITKAYΆVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK EIAELLKGMTLVKNQDΆLDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPWTG
GBS 80 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 5 IPNTG (shown in italics in SEQ ID NO: 2 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 80 protein from the host cell. Accordingly, in one preferred fragment of GBS 80 for use in the invention, the transmembrane and/or cytoplasmic regions and the cell wall anchor motif are removed from GBS 80. An example of such a GBS 80 fragment is set forth below as SEQ ID NO: 6.
SEQ IDNO: 6 MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG
EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
" SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG
EEFKWFLKSTIPANLGDYEKFEITDKFΆDGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK KL L~'L ^ ,ESKSA1NTBMKM1EI PVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK PEVHTGGKRFVKKDSTETQTLGGAEFDLLAS DGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
In one embodiment, the leader or signal sequence region, the transmembrane and cytoplasmic regions and the cell wall anchor motif are removed from the GBS 80 sequence. An example of such a GBS 80 fragment is set forth below as SEQ ID NO: 7.
SEQ ID NO: 7
AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKK LTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTG TGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVG KIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVAS TINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTΆVK WTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEF TVSQTSYNTKPTDITVDSADATPDTIKNNKRPS
Applicants have identified a particularly immunogenic fragment of the GBS 80 protein. This immunogenic fragment is located towards the N-terminus of the protein and is underlined in the GBS 80 SEQ ID NO: 2 sequence below. The underlined fragment is set forth below as SEQ ID NO: 8.
SEQ ID NO: 2 MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKΆIENTFELQYDHTPDKADNPKPSNPPRK PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNΓG GIGTAIFVAIGAAVMAFAVKGMKRRTKDN
SEQ ID NO: 8 AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDI SVDELKK LTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTG TGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVG KIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKG The immunogenicity of the protein encoded by SEQ ID NO: 7 was compared against PBS,
GBS whole cell, GBS 80 (full length) and another fragment of GBS 80, located closer to the C- terminus of the peptide (SEQ ID NO: 9, below).
SEQ ID NO: 9
MTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGK RFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDA NAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS
Both an Active Maternal Immunization Assay and a Passive Maternal Immunization Assay were conducted on this collection of proteins. iP C llA.δ''ulJ[l';ie!rein;;|taii A-etivd'Malernal Immunization assay refers to an in vivo protection assay where female mice are immunized with the test antigen composition. The female mice are then bred and their pups are challenged with a lethal dose of GBS. Serum titers of the female mice during the immunization schedule are measured as well as the survival time of the pups after challenge. Specifically, the Active Maternal Immunization assays referred to herein used groups of four
CD-I female mice (Charles River Laboratories, Calco Italy). These mice were immunized intraperitoneally with the selected proteins in Freund's adjuvant at days 1, 21 and 35, prior to breeding. 6-8 weeks old mice received 20 μg protein/dose when immunized with a single antigen, 30- 45 μg protein/dose (15 μg each antigen) when immunized with combination of antigens. The immune response of the dams was monitored by using serum samples taken on day 0 and 49. The female mice were bred 2-7 days after the last immunization (at approximately 1?= 36 - 37), and typically had a gestation period of 21 days. Within 48 hours of birth, the pups were challenged via LP. with GBS in a dose approximately equal to a amount which would be sufficient to kill 70 - 90 % of unimmunized pups (as determined by empirical data gathered from PBS control groups). The GBS challenge dose is preferably administered in 50μl of THB medium. Preferably, the pup challenge takes place at 56 to 61 days after the first immunization. The challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB prior to use. Survival of pups was monitored for 5 days after challenge.
As used herein, the Passive Maternal Immunization Assay refers to an in vivo protection assay where pregnant mice are passively immunized by injecting rabbit immune sera (or control sera) approximately 2 days before delivery. The pups are then challenged with a lethal dose of GBS.
Specifically, the Passive Maternal Immunization Assay referred to herein used groups of pregnant CDl mice which were passively immunized by injecting 1 ml of rabbit immune sera or control sera via I.P., 2 days before delivery. Newborn mice (24-48 hrs after birth) are challenged via LP. with a 70 - 90% lethal dose of GBS serotype III COHl . The challenge dose, obtained by diluting a frozen mid log phase culture, was administered in 50 μl of THB medium.
For both assays, the number of pups surviving GBS infection was assessed every 12 hrs for 4 days. Statistical significance was estimated by Fisher's exact test.
The results of each assay for immunization with SEQ ID NO: 7, SEQ ID NO: 8, PBS and GBS whole cell are set forth in Tables 1 and 2 below.
Figure imgf000131_0001
P C T SW S U ΈΪ S d TLjiibtelie "TiMi " P*assi.ve Maternal Immunization
Antigen Alive/total %Survival Fisher's exact test
PBS (neg control) 12/42 28%
GBS (whole cell) 48/52 92% PO.00000001
GBS80 (intact) 48/55 87% P<0.00000001
GBS80 (fragment) SEQ ID 7 45/57 79% P=0.0000006
GBS80 (fragment) SEQ ID 8 13/54 24% P=I
As shown in Tables 1 and 2, immunization with the SEQ ID NO: 7 GBS 80 fragment provided a substantially improved survival rate for the challenged pups than the comparison SEQ ID NO: 8 GBS 80 fragment. These results indicate that the SEQ ID NO: 7 GBS 80 fragment may comprise an important immunogenic epitope of GBS 80.
As discussed above, pilin motifs, containing conserved lysine (K) residues have been identified in GBS 80. The pilin motif sequences are underlined in SEQ ID NO: 2, below. Conserved lysine (K) residues are marked in bold, at amino acid residues 199 and 207 and at amino acid residues 368 and 375. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of GBS 80. Preferred fragments of
GBS 80 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO: 2
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG
EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
PEVHTGGKRFVKKDΞTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG
GIGTAIFVAIGAAVMAFAVKGMKRRTKDN
E boxes containing conserved glutamic residues have also been identified in GBS 80. The E box motifs are underlined in SEQ ID NO: 2 below. The conserved glutamic acid (E) residues, at amino acid residues 392 and 471 , are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of GBS 80. Preferred fragments of GBS 80 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 2 MKLSKKLLFΞAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG GIGTAIFVAIGAAVMAFAVKGMKRRTKDN
GBS 104 IP C- '"§'iώϊaliyi;
Figure imgf000133_0001
of preferred GBS 104 fragments. Nucleotide and amino acid sequences of GBS 104 sequenced from serotype V isolated strain 2603 are set forth below as SEQ ID NOS 10 and 11:
Figure imgf000133_0002
GGTTCTGGAGAAGCAACCTTTGAAAACATAAAACCTGGAGACTACACATTAAGAGAAGAAACAGCACCAATTGGT
Figure imgf000133_0003
GATGGTCGAAGAGΆGATTGCTGAAGGTTGGTTATCAAAAAAAATTACAGGGGTCAΆTGATCTCGATAAGAATAAA
TATAAAA1FTGAATTAACTGTTGAGGGTAAAACCACTGTTGAAACGAAAGAACTTAATCAACCACTAGATGTCGTT
Figure imgf000133_0004
ACTTTTATGATACTTACCATTTGTTCTTTCCGTCGTAAACAATTG
SEQ ID NO.11
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVE GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIR PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN LKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKRGGIGTIVYILVGSTFMILTICSFRRKQL
GBS 104 contains an N-terminal leader or signal sequence region which is indicated by the underlined sequence at the beginning of SEQ ID NO 11 above. In one embodiment, one or more Jmin'o
Figure imgf000134_0001
sequence region of GBS 104 are removed. An example of such a GBS 104 fragment is set forth below as SEQ ID NO 12.
SEQ ED NO 12
GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKK TDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGR REIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVtLDNSNSMNNERANNSQRALKAGEAVE KLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEV NILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSY QNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAI NSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFM QSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQL KNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEK EPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTT KNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPK RPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL
GBS 104 contains a C-terminal transmembrane and/or cytoplasmic region which is indicated by the underlined region near the end of SEQ ID NO 11 above. In one embodiment, one or more amino acids from the transmembrane or cytomplasmic regions are removed. An example of such a GBS 104 fragment is set forth below as SEQ ID NO 13. SEQ ID NO: 13
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVE GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIR PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTΞQTIKINHLNLGSGQKVVLTY DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN LKADPNANKNQIGYLEGNGKHLITNT In one embodiment, one or more amino acids from the leader or signal sequence region and one or more amino acids from the transmembrane or cytoplasmic regions are removed. An example of such a GBS 104 fragment is set forth below as SEQ ID NO 14.
SEQ ID NO: 14
GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKK TDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGR REIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVE KLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEV NILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSY QNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAI NSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFM QSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQL KNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPKSEK EPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTT KNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNT GBS 104, like GBS 80, contains an amino acid motif indicative of a cell wall anchor: SEQ
ID NO: 123 FPKTG (shown in italics in SEQ ID NO: 11 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 104 protein from the host cell. Accordingly, in one preferred fragment of GBS 104 for use in the invefitioli/only 'the tfaSriieiiSbf'anFMα'oϊ'' cytoplasmic regions and the cell wall anchor motif are removed from GBS 104. Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. Two pilin motifs, containing conserved lysine (K) residues, have been identified in GBS 104. The pilin motif sequences are underlined in SEQ ID NO: 11 , below. Conserved lysine (K) residues are marked in bold, at amino acid residues 141 and 149 and at amino acid residues 499 and 507. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of GBS 104. Preferred fragments of GBS 104 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO. 11
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVE GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIR PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN LKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL Two E boxes containing a conserved glutamic residues have also been identified in GBS 104.
The E box motifs are underlined in SEQ ID NO: 11 below. The conserved glutamic acid (E) residues, at amino acid residues 94 and 798, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of GBS 104. Preferred fragments of GBS 104 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. \
SEQ ED NO. 11
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVE GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIR PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL LGΆKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN LKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL GBS 067
The following offers examples of preferred GBS 067 fragments. Nucleotide and amino acid sequence of GBS 067 sequences from serotype V isolated strain 2603 are set forth below as SEQ ID NOS: 15 and 16.
Figure imgf000136_0001
CTAAATAAAAGTAATTTTCTACTTACTGATAAGCCCGAGGATATAAAAGGΆAATGGGGAGAGTTACTTTTTGTTT
Figure imgf000136_0002
GAT SEQ ID NO: 16
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVT AELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
QKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEA DDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEE YKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVT
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGS iKNiiAVNKQisEYHEEGDKHLiTNTHipPKGiJPMΓGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK
D GBS 067 contains a C-terminus transmembrane region which is indicated by the underlined region closest to the C-terminus of SEQ ID NO: 16 above. In one embodiment, one or more amino acids from the transmembrane region is removed and or the amino acid is truncated before the tϊ2πsiyfnb'rlαl';ri|iiln?Xnixafo^ie'σ!f sώ!Λ a GBS 067 fragment is set forth below as SEQ ID NO:
17.
SEQ ED NO: 17
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVT AELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
QKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEA DDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEE YKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIΆTGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVT
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGS IKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILS
GBS 067 contains an amino acid motif indicative of a cell wall anchor (an LPXTG (SEQ ID NO: 122) motif): SEQ ID NO: 18 IPMTG. (shown in italics in SEQ ID NO: 16 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 067 protein from the host cell. Accordingly, in one preferred fragment of GBS 067 for use in the invention, the transmembrane and the cell wall anchor motif are removed from GBS 67. An example of such a GBS 067 fragment is set forth below as SEQ ID NO: 19. SEQ ID NO: 19
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVT AELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
QKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEΆPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEA DDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEE YKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVT
LLKGATFELQEFNEDYKL YLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKI TNKPILTFEVVKGS IKNIIAVNKQISEYHEEGDKHLITNTHIPPKGI
Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Three pilin motifs, containing conserved lysine (K) residues have been identified in GBS 67. The pilin motif sequences are underlined in SEQ ID NO: 16, below. Conserved lysine (K) residues are marked in bold, at amino acid residues 478 and 488, at amino acid residues 340 and 342, and at amino acid residues 703 and 717. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of GBS 67. Preferred fragments of GBS 67 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence. SEQ ED NO: 16 MlR.y£Qϊ3lFSiκldyBS&lϊ, S'Q JHLNTK^CSEiS1TVPENGAKGKL VVKKTDDQNKPLSKATFVLKTTAHPESKIEKVT
AELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
QKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEA DDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYHEE YKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIΆTGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVT
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGS IKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK
D
Two E boxes containing conserved glutamic residues have also been identified in GBS 67. The E box motifs are underlined in SEQ ID NO: 16 below. The conserved glutamic acid (E) residues, at amino acid residues 96 and 801, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of GBS 67. Preferred fragments of GBS 67 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ID NO: 16
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVT AELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
QKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGΆNSDNRVALVTYGSDIFDGRSVDVVKGFKE DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEA DDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEE YKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVT
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGS IKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK
D
Predicted secondary structure for the GBS 067 amino acid sequence is set forth in FIGURE 33. As shown in this figure, GBS 067 contains several regions predicted to form alpha helical structures. Such alpha helical regions are likely to form coiled-coil structures and may be involved in oligomerization of GBS 067.
The amino acid sequence for GBS 067 also contains a region which is homologous to the Cna_B domain of the Staphylococcus aureus collagen-binding surface protein (ρfam05738). Although the Cna_B region is not thought to mediate collagen binding, it is predicted to form a beta sandwich structure. In the Staph aureus protein, this beta sandwich structure is through to form a stalk that presents the ligand binding domain away from the bacterial cell surface. This same amino acid sequence region is also predicted to be an outer membrane protein involved in cell envelope biogenesis. The amino acid sequence for GBS 067 contains a region which is homologous to a von
Willebrand factor (vWF) type A domain. The vWF type A domain is present at amino acid residues 229-402 of GBS 067 as shown in SEQ ID NO: 16. This type of sequence is typically found in ^faKffleiiular'jprofifliSslcri a^ϊnϊepins'arfd it thoughtto mediate adhesion, including adhesion to collagen, fϊbronectin, and fibrinogen, discussed above.
Because applicants have identified GBS 61 as a surface exposedprotein on GBS and because GBS 67 maybe involved in GBS adhesion, the immunogenicity ofthe GBS 67 proteinwas examined inmice. The results ofan immunization assaywith GBS 67 are set forth in Table 48, below.
Table 48: GBS 67 Protects Mice in an Immunization Assay
Figure imgf000139_0001
As shown in Table 48, immunization with GBS 67 provides a substantially improved survival 10 rate for challengedmice relative to negative control, PBS, immunized mice. These results indicate that GBS 67 may comprise an immunogenic composition ofthe invention. GBS 59
The following offers examples ofGBS 59 fragments. Nucleotide and amino acid sequences ofGBS 59 sequenced from serotype V isolated strain2603 are set forthbelow as SEQ ID NOS: 125 , 15 and 126. The GBS 59 polypeptide ofSEQ IDNO: 126 is referred to as SAG1407. SEQ ID NO: 125 ttaagcttcctttgattggcgtcttttcatgataactactgctccaagcataatgcttaaaccaataattgtgaa aagaattgtaccaataccacctgtttgtgggattgttacctttttattttctacacgtgtcgcatctttttggtt gctgttagcaacgtagtcaatgttaccacctgttatgtatgacccttgattaactacaaacttaatattacctgc
20 caacttagcaaatcctgctggagcaagtgtttcttcaaggttgtaagtaccgtctgcaagacctgtaacttcaaa ttgaccttgatcgtttgaagtgtaggtaatggctctagccttatctgttatccactcataagctgtacgagcctc aatgaaggctgcatcgtaatctgcttgtttagttttgataagttcttttgcagtaattcctttttcacctttttg gtctgttgcagacaacttgttataagcagcgatagcttcatctaaagctattttcttagcagctaaagttttttg accttctgattgatctgctttaagagcaaggtatttacctgctgagtttttcacaacgaattgtgcaccagccaa
25 acggtcaccttgttcattagttttgacaaatttcttaccatgagtttcaacttttggttcagttgggttcaatgg tgttgggttatcagaatctttggtattggtaatggttactttaccattttctagatttattgcacttccgtaacc agaaacacgttctgagatcatgtatgatttgttttctagaccagtgaatttacccgagaagttaccagatacttc aaatttgataccatttccaaggtcgattgtacctttagatgtttttgtcaatgatactgaagcaacagttttatc tttatctttcaatgtgtaaacaacgtttacaccatcaggtgcaattccgtcagaccaagttttagcaactgttac
30 ttcaccctttgaaggtgtaacaggaagttcagtcaagtctttacctggtttgttaccatacgacaatttgatatc attggattctggattatcaataattgcttgaccattaacagtagcactataagtcaatgtaaattcaatatcagc tgttttagctgctttttccaatttgcccaatccatcagctgtgaattttaatgtgaaaccacgggcatcaatgct aagttcatagtctgtatccttagcaaaagtttctgtagttcctgaagctttaaggctaacagttgaacccattgt caaaccatttgacattatatctgtccaaaccaagttttcgtatttagaacctttgtgaatttttgttttaacttc , 35 ataaggaacaactttaccgatttcagcagtagcagttgctttgtcacgtgcataattaccataatttgcgccagc tgtcaaaagtctattaacatctgtcaatgctgtcaaatcgtttgttttagcaaagtttttatcaatttctggttt ttcttcagtgttctttggataaacatgggcatcagcaacaacaccatcttcatttaccaatggaagagtgatgtt aactggaaccgcttttgaagcagccaggagggaaccattattgttgtaagtagattttgatttaacttcaacaat tttaaactcgcctttcaatcctttggtgttgaaaacaagtccagtatctccctctggtgtcaatccagacacggc
40 ctcatcaatatttactgttatttcaggagtaccatctttattaattaaggctggtgttaatttgttaccttcttt tgccttaacatattgcactttaccacttttatcttctttcaaagctaaagcaaagaacgcaccttcgatttcttt agatccctcgccaaagtaaccagcaaggtcagaaatagctccacctttgtagtcttttccgttaagacctgtagt tcctgggaagttacttttgttaagatttgattcggtttgcaaaatcttgtgcaaagtcactgtattagttgttgc i|St|i|t'(|cgέ4J4sgEit:I;gf'giI4SW&ά&SSaatgacgttaaagtcagtaacaatgccgagaacattgcaaaata tttgttgattcttttcat
SEQ ID NO: 126 MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKGGAISDLAG YFGEGSKEIEGAFFALALKEDKSGKVQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAIIDNPESNDIKLSYGNKPGKDLTELPV
ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKTNEQGDRLAGAQFVVKNSAGKYLALK ADQSEGQKTLAAKKIALDEAIAAYNKLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNIDYVANSNQKDATRVENKKVTIPQTG GIGTILFTIIGLSIMLGAVVIMKRRQSKEA
Nucleotide and amino acid sequences of GBS 59 sequenced from serotype V isolated strain
CJBl 11 are set forth below as SEQ ID NOS: 127 and 128. The GBS 59 polypeptide of SEQ ID NO: 128 is referred to as BO1575, SEQ ID NO: 127 ATGAAAAAAATCAACAAATGTCTTACAATGTTCTCGACACTGCTATTGATCTTAACGTCACTATTCTCAGTTGCA
Figure imgf000140_0001
GAATACATAGTTGGAACAAAAATTCTTAAAGGCTCAGACTATAAGAAACTGGTTTGGACTGATAGCATGACTAAA
Figure imgf000140_0002
SEQ ED NO: 128
MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFDNFTEGTKGKNDΞDYVGKQINDLKSY FGSTDAKEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNFADKDLDYTDNRKDKGVVSATVGDKK
EYIVGTKILKGSDYKKLVWTDSMTKGLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEIKVIKDWAVDGTITDANVAVKAIFTL
QEKQTDGTWVNVASHEATKPSRFEHTFTGLDNAKTYRVVERVSGYTPEYVSFKNGWTIKNNKNSNDPTPINPSE PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAVKTAKLALDEAVKAYNDLTKEKQEGQ
EGKTALATVDQKQKAYNDAFVKANYSYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKKRQSEEA S"'" C ffW^feiteiljpiiyp'epidSslBoriiay an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 129 IPQTG (shown in italics in SEQ ID NOs: 126 and 128 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 59 protein from the host cell. Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Pilin motifs, containing conserved lysine (K) residues have been identified in the GBS 59 polypeptides. The pilin motif sequences are underlined in each of SEQ ID NOs: 126 and 128, below. Conserved lysine (K) residues are marked in bold. The conserved lysine (K) residues are located at amino acid residues 202 and 212 and amino acid residues 489 and 495 of SEQ ID NO: 126 and at amino acid residues 188 and 198 of SEQ ID NO: 128. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of GBS 59. Preferred fragments of GBS 59 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence. SEQ ID NO: 126
MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKGGAISDLAG YFGEGSKEIEGAFFALALKEDKSGKVQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
DTDYELSIDARGFTI1KFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII DNPESNDIKLSYGNKPGKDLTELPV
ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKTNEQGDRLAGAQFVVKNSAGKYLALK ADQSEGQKTLAAKKIALDEAIAAYNKLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNIDYVANSNQKDATRVENKKVTIPQTG GIGTILFTIIGLSIMLGAVVIMKRRQSKEA
SEQ ID NO: 128 MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFDNFTEGTKGKNDSDYVGKQINDLKSY FGSTDAKEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNFADKDLDYTDNRKDKGVVSATVGDKK EYIVGTKILKGSDYKKLVWTDSMTKGLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEIKVIKDWAVDGTITDANVAVKAIFTL QEKQTDGTWVNVASHEATKPSRFEHTFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAVKTAKLALDEAVKAYNDLTKEKQEGQ EGKTALATVDQKQKAYNDAFVKANYSYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKKRQSEEA An E box containing a conserved glutamic residue has also been identified in each of the GBS
59 polypeptides. The E box motif is underlined in each of SEQ ID NOs: 126 and 128 below. The conserved glutamic acid (E) is marked in bold at amino acid residue 621 in SEQ ID NO: 126 and at amino acid residue 588 in SEQ ID NO: 128. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of GBS 59. Preferred fragments of GBS 59 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. ClEi,/E 7i&3cl
MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKGGAISDLAG YFGEGSKEIEGAFFALALKEDKSGKVQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAIIDNPESNDIKLSYGNKPGKDLTELPV
ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKTNEQGDRLAGAQFVVKNSAGKYLALK ADQSEGQKTLAAKKIALDEAIAAYNKLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNIPYVANSNQKDATRVENKKVTIPQTG GIGTILFTIIGLSIMLGAVVIMKRRQSKEA
SEQ ID NO: 128
MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFDNFTEGTKGKNDSDYVGKQINDLKSY FGSTDAKEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNFADKDLDYTDNRKDKGVVSATVGDKK
EYIVGTKILKGSDYKKLVWTDSMTKGLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEIKVIKDWAVDGTITDANVAVKΆIFTL
QEKQTDGTWVNVASHEATKPSRFEHTFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAVKTAKLALDEAVKAYNDLTKEKQEGQ
EGKTALATVDQKQKAYNDAFVKANYSYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKKRQSEEA
Female mice were immunized with either SAG 1407 (SEQ ID NO: 126) or BO 1575 (SEQ ID NO: 128) in an active maternal immunization assay. Pups bred from the immunized female mice survived GBS challenge better than control (PBS) treated mice. Results of the active maternal immunization assay using the GBS 59 immunogenic compositions are shown in Table 17, below. TABLE 17: Active maternal immunization assay for GBS 59
Figure imgf000142_0001
* immunized with BO 1575 **immunized with SAG1407
Opsonophagocytosis assays also demonstrated that antibodies against BO1575 are opsonic for GBS serotype V, strain CJB 111. See Figure 67. GBS 52
Examples of polynucleotide and amino acid sequences for GBS 52 are set forth below. SEQ ID NO: 20 and 21 represent GBS 52 sequences from GBS serotype V, strain isolate 2603.
SEQ ID NO: 20
ATGAAACAAACATTAAAACTTATGTTTTCTTTTCTGTTGATGTTAGGGACTATGTTTGGAATTAGCCAAACTGTT
GAGATTGCCCCTAAAGAAGGGACTCCAATTGAAGGAGTACTCTATCAGTTGTACCAATTAAAATCAACTGAAGAT GGCGATTTGTTGGCACATTGGAATTCCCTAACTATCACAGAATTGAAAAAACAGGCGCAGCAGGTTTTTGAAGCC ACTACTAATCAACAAGGAAAGGCTACATTTAACCAACTACCAGATGGAATTTATTATGGTCTGGCGGTTAAAGCC GGTGAAAAAAΆTCGTAATGTCTCAGCTTTCTTGGTTGACTTGTCTGAGGATAAAGTGATTTATCCTAAAATCATC
GCAAAACATTTAGAAACAGATTCATCAGGGCATATCAGAATTTCCGGGCTCATCCATGGGGACTATGTCTTAAAA GAAATCGAGACACAGTCAGGATATCAGATCGGACAGGCAGAGACTGCTGTGACTATTGAΆAAATCAAAAACAGTA CGGAAACATCAAAATAAGGAT SEQ ID NO: 21
MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVLYQLYQLKSTED GDLLAHWNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPIRVKNGVHSQDIDAAKHLETDSSGHIRISGLIHGDYVLK EIETQSGYQIGQAETAVTIEKSKTVTVTIENKKVPTPKVPSRGGLIPKΓGEQQAMALVIIGGILIALALRLLSKH RKHQNKD
GBS 52 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 124 IPKTG (shown in italics in SEQ ID NO: 21, above). In some recombinant host cell systems, it may¬ be preferable to remove this motif to facilitate secretion of a recombinant GBS 52 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in GBS 52. The pilin motif sequence is underlined in SEQ ID NO: 21, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 148 and 160. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of GBS 52 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 21
MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVLYQLYQLKSTED GDLLAHWNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPIRVKNGVHSQDIDAAKHLETDSSGHIRISGLIHGDYVLK EIETQSGYQIGQAETAVTIEKSKTVTVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH RKHQNKD
An E box containing a conserved glutamic residue has been identified in GBS 52. The E-box motif is underlined in SEQ ID NO: 21, below. The conserved glutamic acid (E), at amino acid residue 226, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of GBS 52. Preferred fragments of GBS 52 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 21
MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVLYQLYQLKSTED GDLLAHWNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII
WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPIRVKNGVHSQDIDAAKHLETDSSGHIRISGLIHGDYVLK
EIETQSGYQIGQAETAVTIEKSKTVTVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH
RKHQNKD SAG0647 Ij-I, I ^xaiηjo|^;qjf'(p£|^αjj;teoti(te!atijdi!3piino acid sequences for SAG0647 are set forth below.
SEQ ID NO: 22 and 23 represent SAG0647 sequences from GBS serotype V, strain isolate 2603. SEQ ID NO: 22 TTCCTTGTTTTGGCATTTCCCATCGTTAGTCAGGTCATGTACTTTCAAGCCTCTCACGCCAATATTAATGCTTTT
GTCGTTGAGTACGCCCGCATGCTTGAAGTCAAAGAACAAATAGGTCATGTGATTATTCCAAGAATTAATCAGGAT
Figure imgf000144_0001
CACAATAATTCGAAATAA
SEQ ID NO: 23
MGQKSKISLATNIRIWIFRLIFLAGFLVLAFPIVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNAS IAGAKTNGEYPALKDPYSAEQKQAGVVEYARMLEVKEQIGHVIIPRINQDIPIYAGSAEENLQRGVGHLEGTSLP VGGESTHAVLTAHRGLPTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTC TPYMINSHRLLVRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVVVGLILLSLLIWFKKTKQKKRRKNEKAASQNS HNNSK SAG0648
Examples of polynucleotide and amino acid sequences for SAG0648 are set forth below. SEQ ID NO: 24 and 25 represent SAG0648 sequences from GBS serotype V, strain isolate 2603.
Figure imgf000144_0002
ATGATGAGAAGATGGATGCAACATCGTCAATAA
SEQ ID NO: 25
MGSLILLFPIVSQVSYYLASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLIDPFTSKQKEGLREYA RMLEVHEQIGHVAIPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKK GQIFYVTNIKETLAYKVVSIKVVDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEAEKHKEQ TVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ
GBS 150
Examples of polynucleotide and amino acid sequences for GBS 150 are set forth below. SEQ ID NO: 26 and 27 represent GBS 150 sequences from GBS serotype V, strain isolate 2603.
SEQ ID NO: 26
ATGAAAAAGATTAGAAAAAGTTTAGGACTTCTACTATGTTGCTTTTTAGGATTGGTACAATTAGCGTTTTTTTCG GTAGCCAGTGTAAATGCTGATACCCCTAATCAACTAACAATCACACAGATAGGACTTCAGCCAAATACTACAGAG GAGGGGATTTCTTATCGTTTATGGACTGTGACTGACAACTTAAAΆGTTGATTTATTGAGCCAAATGACAGATAGC GAATTGAACCAGAAGTATAAGAGTATCTTGACTTCTCCTACTGATACTAATGGTCAGACAAAGATAGCACTCCCA AATGGTTCGTACTTTGGTCGTGCTTATAAΆGCTGATCAAAGCGTTTCAACAATAGTACCTTTTTATATTGAATTA CCAGATGATAAGTTATCAAATCAATTACAGATAAATCCTAAGCGAAAAGTTGAAACAGGCCGATTAAAACTTATT
Figure imgf000145_0001
AAATCTGAAAGAAACGATACAGTA
SEQ ID NO: 27 MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGISYRLWTVTDNLKVDLLSQMTDS ELNQKYKSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLI KYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGEIEVEGLLPGKYIFREAKALTGYRI SMKDAVVAVVANKTQEVEVENEKETPPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNS KSERNDTV
GBS 150 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 130
LPKTG (shown in italics in SEQ ID NO: 27 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 150 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
As discussed above, a pilin motif, containing a conserved lysine (K) residue has been identified in GBS 150. The pilin motif sequence is underlined in SEQ ID NO: 27, below. Conserved lysine (K) residues are marked in bold, at amino acid residues 139 and 148. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of GBS 150. Preferred fragments of GBS 150 include a conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 27 MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGISYRLWTVTDNLKVDLLSQMTDS ELNQKYKSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQI1QINPKRKVETGRLKLI KYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGEIEVEGLLPGKYIFREAKALTGYRI SMKDAVVAVVANKTQEVEVENEKETPPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNS KSERNDTV
An E box containing a conserved glutamic residue has also been identified in GBS 150. The
E box motif is underlined in SEQ ID NO: 27 below. The conserved glutamic acid (E), at amino acid residue 216, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of GBS 150. Preferred fragments of GBS 150 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 27
MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGIΞYRLWTVTDNLKVDLLSQMTDS ELNQKYKSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLI KYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGEIEVEGLLPGKYIFREAKALTGYRI SMKDAVVAVVANKTQEVEVENEKETPPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNS KSERNDTV
SAG1405 pi J] mpxat]|^gSj;ojfj[E«Sjyfim3|e'9ϊig! srjd!;|jmino acid sequences for SAG1405 are set forth below.
SEQ ID NO: 28 and 29 represent SAG1405 sequences from GBS serotype V, strain isolate 2603.
Figure imgf000146_0001
GCTTATAATAGAACACTGGACCCAAGCCGCCTATCAGATCCCTATACTGAAAAAGAAAAAAAAGGTATTGCTGAA
TATGCGGGGACTACCAGTAGTGTTCTTGAAAAAGGAGCAGGACACCTTGAAGGAACCTCCTTGCCAATTGGTGGA AAAAGTTCACATACTGTTATCACAGCTCATCGCGGCTTACCTAAAGCTAAGTTATTTACAGATTTAGATAAACTT
Figure imgf000146_0002
TTAATGAAAGAATTGCAAACACACTATAAACTTTATTTCCTCTTATCAATCCTAGTTATTCTTATATTAGTCGCT TTACTATTATATTTAAAACGAAAATTTAAAGAGAGAAAGAGAAAGGGAAATCAAAAATGA
SEQ ID NO: 29
MGGKFQKNLKKSVVLNRWMNVGLILLFLVGLLITSYPFISNWYYNIKANNQVTNFDNQTQKLNTKEINRRFELAK AYNRTLDPSRLSDPYTEKEKKGIAEYAHMLEIAEMIGYIDIPSIKQKLPIYAGTTSSVLEKGAGHLEGTSLPIGG KSSHTVITAHRGLPKAKLFTDLDKLKKGKIFYIHNIKEVLAYKVDQISVVKPDNFSKLLVVKGKDYATLLTCTPY SINSHRLLVRGHRIKYVPPVKEKNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
SAG1406
Examples of polynucleotide and amino acid sequences for SAG 1405 are set forth below. SEQ ID NO: 30 and 31 represent SAG1405 sequences from GBS serotype V, strain isolate 2603.
Figure imgf000146_0003
GGTTCAAGTCAAGAAGTTCTATCTAAAGGAGCAGGGCATTTAGAAGGTACCTCTCTTCCAATTGGGGGCAATAGT
Figure imgf000146_0004
AAACGTCAACGTCAAAAAAATCGTTTAGCAAGTGTTAGAAAAGGAATTGAATCATAA SEQ ID NO: 31
MKTKKIIKKTKKKKKSNLPFIILFLIGLSILLYPVVSRFYYTIESNNQTQDFERAAKKLSQKEINRRMALAQAYN DSLNNVHLEDPYEKKRIQKGVAEYARMLEVSEKIGTISVPKIGQKLPIFAGSSQEVLSKGAGHLEGTSLPIGGNS THTVITAHSGIPDKELFSNLKKLKKGDKFYIQNIKETIAYQVDQIKVVTPDNFSDLLVVPGHDYATLLTCTPIMI NTHRLLVRGHRIPYKGPIDEKLIKDGHLNTIYRYLFYISLVIIAWLLWLIKRQRQKNRLASVRKGIES
01520
An example of an amino acid sequence for 01520 is set forth below. SEQ ID NO: 32 represents a 01520 sequence from GBS serotype III, strain isolate COHl.
SEQ ID NO: 32 MIRRYSANFLAILGIILVSSGIYWGWYNINQAHQADLTSQHIVKVLDKSITHQVKGSENGELPVKKLDKTDYLGT LDIPNLKLHLPVAANYSFEQLSKTPTRYYGSYLTNNMVICAHNFPYHFDALKNVDMGTDVYFTTTTGQIYHYKIS NREIIEPTAIEKVYKTATSDNDWDLSLFTCTKAGVARVLVRCQLIDVKN
01521 I,--,, ,i An,,e|jnjml£'!p£^,a^np'|oid:|eqwence for 01521 is set forth below. SEQ ID NO: 33 represents a 01521 sequence from GBS serotype III, strain isolate COHl. SEQ ID NO: 33
MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGNLLVYQVAQAKDVDGNQVFTLTTPFQ GIGIKDDDLTQVNLDSNQAKYVNLLTKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFIL SIPKDGKYDITAFEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWPIPILIMSGLLCLIIALKWRRRRD
01521 contains an amino acid motif indicative of a cell wall anchor: SEQ ED NO: 132 LPFTG (shown in italics in SEQ ID NO: 33 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 01521 protein from the host cell. Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Two pilin motifs, containing conserved lysine (K) residues have been identified in 01521. The pilin motif sequences are underlined in SEQ ID NO: 33, below. Conserved lysine (K) residues are marked in bold, at amino acid residues 154 and 165 and at amino acid residues 174 and 188. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of 01521. Preferred fragments of 01521 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence. SEQ ID NO: 33
MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGNLLVYQVAQAKDVDGNQVFTLTTPFQ GIGIKDDDLTQVNLDSNQAKYVNLLTKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFIL SIPKDGKYDITAFEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWPIPILIMSGLLCLIIALKWRRRRD
An E box containing a conserved glutamic residue has also been identified in 01521. The E box motif is underlined in SEQ ID NO: 33 below. The conserved glutamic acid (E), at amino acid residue 177, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of 01521. Preferred fragments of 01521 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. SEQ ID NO: 33
MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGNLLVYQVAQAKDVDGNQVFTLTTPFQ GIGIKDDDLTQVNLDSNQAKYVNLLTKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFIL SIPKDGKYDITAFEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWPIPILIMSGLLCLIIALKWRRRRD
01522 An example of an amino acid sequence for 01522 is set forth below. SEQ ID NO: 34 represents a 01522 sequence from GBS serotype III, strain isolate COHl.
SEQ ID NO: 34
MAYPSLANYWNSFHQSRAIMDYQDRVTHMDENDYKKIINRAKEYNKQFKTSGMKWHMTSQERLDYNSQLAIDKTG NMGYISIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWTVS ILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALVVAEAIQIEPIY IAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
01523 pi' C
Figure imgf000148_0001
for 01523 is set forth below. SEQ ID NO: 35 represents a 01523 sequence from GBS serotype III, strain isolate COHl.
SEQ ED NO: 35
MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKDGASYLIPQ GKEAEYKASTDFNSLFTTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYY
VSSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNYHGTEKVYQYVIKD
TMPSASVVDLNEGSYEVTITDGSGNITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFY
KGINTITVTYTGVLKSGAKPGSADLPENTNIATINPNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLK
NATGQFLNFNDTNNVEWGTEANATEYTTGADGIITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTN SDNLLVNPTVENNKGTELPSTGGIGTTIFYIIGAILVIGAGIVLVARRRLRS
01523 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO: 35 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 01523 protein from the host cell. Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
An E box containing a conserved glutamic residue has also been identified in 01523. The E box motif is underlined in SEQ ID NO: 35 below. The conserved glutamic acid (E), at amino acid residue 423, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of 01523. Preferred fragments of 01523 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. SEQ ID NO: 35
MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKDGASYLIPQ GKEAEYKASTDFNSLFTTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYY VSSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNYHGTEKVYQYVIKD TMPSASVVDLNEGSYEVTITDGSGNITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFY KGINTITVTYTGVLKSGAKPGSADLPENTNIATINPNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLK
NATGQFLNFNDTNNVEWGTEANATEYTTGADGIITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTN SDNLLVNPTVENNKGTELPSTGGIGTTIFYIIGAILVIGAGIVLVARRRLRS
01524 An example of an amino acid sequence for 01524 is set forth below. SEQ ID NO: 36 represents a 01524 sequence from GBS serotype III, strain isolate COHl.
SEQ ID NO: 36
MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEETKTDDVGITLENKNSSQVTSSTSSS QSSVEQSKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKK ALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDENLKVVHFKDDGQTEVLKSKDTAETK NTSSDVAFKTDSFSIYAIVQEDNTEVPRLTYHFQNNDGTDYDFLTASGMQVHHQIIKDGESLGEVGIPTIKAGEH FNGWYTYDPTTGKYGDPVKFGEPITVTETKEICVRPFMSKVATVTLYDDSAGKSILERYQVPLDSSGNGTADLSS FKVSPPTSTLLFVGWSKTQNGAPLSESEIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPA STIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTKDTTLYAHWSPAQTTYTINYWQQSATDNKNATDAQKTYEY AGQVTRSGLSLSNQTLTQQDINDKLPTGFKVNNTRTETSVMIKDDGSSVVNVYYDRKLITIKFAKYGGYSLPEYY YSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSYLGEFILPNDTVDSDVIKLFPKGNIV QTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAGVKVPISDEYYDRY GAYHKDYLNLVVWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYAPDTTTVQPKPSRPGYVWDGKWYKDQ AQTQVFDFNTTMPPHDVKVYAGWQKVTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYIQDPSGTYYY KYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPDGSIRPYNFSGAVTQDINLRAIWRKA ψ fΛ Ir . " 4HfSH- O'S^Misei^ie'β'
DΪDAHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNVVTQVETPRMELNSTTTIPENQYFTRTGY NLIGWHHDKDLADTGRVEFTAGQSIGIDNNPDATNTLYAVWQPKEYTVRVSKTVVGLDEDKTKDFLFNPSETLQQ ENFPLRDGQTKEFKVPYGTSISIDEQAYDEFKVSESITEKNLATGEΆDKTYDATGLQSLTVSGDVDISFTNTRIK QKVRLQKVNVENDNNFLAGAVFDIYESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPG YLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGTEIPSTGGIGTHIYILVGLALALPSG LILYYRKKI
01524 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO: 36 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 01524 protein from the host cell. Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. Three pilin motifs, containing conserved lysine (K) residues have been identified in 01524.
The pilin motif sequences are underlined in SEQ ID NO: 36, below. Conserved lysine (K) residues are marked in bold, at amino acid residues 128 and 138, amino acid residues 671 and 682, and amino acid residues 809 and 820. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of 01524. Preferred fragments of 01524 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ED NO: 36
MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEETKTDDVGITLENKNSSQVTSSTSSS QSSVEQSKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKK ALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDENLKWHFKDDGQTEVLKSKDTAETK NTSSDVAFKTDSFSIYAIVQEDNTEVPRLTYHFQNNDGTDYDFLTASGMQVHHQIIKDGESLGEVGIPTIKAGEH FNGWYTYDPTTGKYGDPVKFGEPITVTETKEICVRPFMSKVATVTLYDDSAGKSILERYQVPLDSSGNGTADLSS FKVSPPTSTLLFVGWSKTQNGAPLSESEIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPA STIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTKDTTLYAHWSPAQTTYTINYWQQSATDNKNATDAQKTYEY AGQVTRSGLSLSNQTLTQQDINDKLPTGFKVNNTRTETSVMIKDDGSSVVNVYYDRKLITIKFAKYGGYSLPEYY YSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSYLGEFILPNDTVDSDVIKLFPKGNIV QTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAGVKVPISDEYYDRY GAYHKDYLNLWWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYAPDTTTVQPKPSRPGYVWDGKWYKDQ AQTQVFDFNTTMPPHDVKVYAGWQKVTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYIQDPΞGTYYY KYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPDGSIRPYNFSGAVTQDINLRAIWRKA GDYHIIYSNDAVGTDGKPALDASGQQLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSI DIDAHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNVVTQVETPRMELNSTTTIPENQYFTRTGY NLIGWHHDKDLADTGRVEFTAGQΞIGIDNNPDATNTLYAVWQPKEYTVRVSKTVVGLDEDKTKDFLFNPSETLQQ ENFPLRDGQTKEFKVPYGTSISIDEQAYDEFKVSESITEKNLATGEADKTYDATGLQSLTVSGDVDISFTNTRIK QKVRLQKVNVENDNNFLAGAVFDIYESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPG YLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGTELPSTGGIGTHIYILVGLALALPSG LILYYRKKI
An E box containing a conserved glutamic residue has also been identified in 01524. The E box motif is underlined in SEQ ID NO: 36 below. The conserved glutamic acid (E), at amino acid residue 1344, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of 01524. Preferred frjEgr$L£flf&' pf f|S$jfi$ifIμde' fEfe p&gfeSdi^utamic acid residue. Preferably, fragments include the E box motif. SEQ ID NO: 36
MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEETKTDDVGITLENKNSSQVTSSTSSS QSSVEQSKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKK
ALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDENLKVVHFKDDGQTEVLKSKDTAETK NTSSDVAFKTDSFSIYAIVQEDNTEVPRLTYHFQNNDGTDYDFLTASGMQVHHQIIKDGESLGEVGIPTIKAGEH FNGWYTYDPTTGKYGDPVKFGEPITVTETKEICVRPFMSKVATVTLYDDSAGKSILERYQVPLDSSGNGTADLSS FKVSPPTSTLLFVGWSKTQNGAPLSESEIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPA STIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTKDTTLYAHWSPAQTTYTINYWQQSATDNKNATDAQKTYEY AGQVTRSGLSLSNQTLTQQDINDKLPTGFKVNNTRTETSVMIKDDGSSVVNVYYDRKLITIKFAKYGGYSLPEYY YSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSYLGEFILPNDTVDSDVIKLFPKGNIV QTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAGVKVPISDEYYDRY GAYHKDYLNLVVWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYAPDTTTVQPKPSRPGYVWDGKWYKDQ AQTQVFDFNTTMPPHDVKVYAGWQKVTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYIQDPSGTYYY KYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPDGSIRPYNFSGAVTQDINLRAIWRKA GDYHIIYSNDAVGTDGKPALDASGQQLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSI DIDAHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNVVTQVETPRMELNSTTTIPENQYFTRTGY NLIGWHHDKDLADTGRVEFTAGQSIGIDNNPDATNTLYAVWQPKEYTVRVSKTVVGLDEDKTKDFLFNPSETLQQ ENFPLRDGQTKEFKVPYGTSISIDEQAYDEFKVSESITEKNLATGEADKTYDATGLQSLTVΞGDVDISFTNTRIK QKVRLQKVNVENDNNFLAGAVFDIYESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPG YLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGTELPSTGGIGTHIYILVGLALALPSG LILYYRKKI 01525
An example of an amino acid sequence for 01525 is set forth below. SEQ ID NO: 37 represents a 01525 sequence from GBS serotype III, strain isolate COHl. SEQ ID NO: 37
MKRQISSDKLSQELDRVTYQKRFWSVIKNTIYILMAVASIAILIAVLWLPVLRIYGHSMNKTLSAGDVVFTVKGS NFKTGDVVAFYYNNKVLVKRVIAESGDWVNIDSQGDVYVNQHKLKEPYVIHKALGNSNIKYPYQVPDKKIFVLGD NRKTSIDSRSTSVGDVSEEQIVGKISFRIWPLGKISSIN
GBS 322
GBS 322 refers to a surface immunogenic protein, also referred to as "sip". Nucleotide and amino acid sequences of GBS 322 sequenced from serotype V isolated strain 2603 WR are set forth in Ref. 3 as SEQ ID 8539 and SEQ ID 8540. These sequences are set forth below as SEQ ID NOS 38 and 39:
Figure imgf000150_0001
ACCAATCAAGTTTCTGTTGCAGACCAAAAAGTTTCTCTCAATACAATTTCGGAAGGTATGACACCAGAAGCAGCA ACAACGATTGTTTCGCCAATGAAGACATATTCTTCTGCGCCAGCTTTGAAATCAAAAGAAGTATTAGCACAAGAG
Figure imgf000150_0002
GTAGTCACTCCTAAAGTAGAAACTGGTGCATCACCAGAGCATGTATCAGCTCCAGCAGTTCCTGTGACTACGACT TCACCAGCTACAGACAGTAAGTTACAAGCGACTGAAGTTAAGAGCGTTCCGGTAGCACAAAAAGCTCCAACAGCA
GGTGATCATGGTAAAGGTTTAGCAGTTGACTTTATTGTAGGTACTAATCAAGCACTTGGTAATAAAGTTGCACAG
TACTCTACACAAAATATGGCAGCAAATAACATTTCATATGTTATCTGGCAACAAAAGTTTTACTCAAATACAAAC AGTATTTATGGACCTGCTAATACTTGGAATGCAATGCCAGATCGTGGTGGCGTTACTGCCAACCACTATGACCAC T1CMtGTTCTVATAfAATTTTTATTA
SEQ ID NO. 39 MNKKVLIiTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAK INNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAA TTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVA AETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKΞVPVAQKAPTA TPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQ YSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK
GBS 322 contains an N-terminal leader or signal sequence region which is indicated by the underlined sequence near the beginning of SEQ ID NO: 39. In one embodiment, one or more amino acids from the leader or signal sequence region of GBS 322 are removed. An example of such a GBS 322 fragment is set forth below as SEQ ID NO: 40. SEQ ID NO: 40
DLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAA GQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPA PVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHV SAPAVPVTTTSPATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVN EFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDR GGVTANHYDHVHVSFNK
Additional preferred fragments of GBS 322 comprise the immunogenic epitopes identified in WO 03/068813, each of which are specifically incorporated by reference herein.
There may be an upper limit to the number of GBS proteins which will be in the compositions of the invention. Preferably, the number of GBS proteins in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3. Still more preferably, the number of GBS proteins in a composition of the invention is less than 6, less than 5, or less than 4. Still more preferably, the number of GBS proteins in a composition of the invention is 3.
The GBS proteins and polynucleotides used in the invention are preferably isolated, i.e., separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended puipose. Group A Streptococcus Adhesin Island Sequences
The GAS AI polypeptides of the invention can, of course, be prepared by various means {e.g. recombinant expression, purification from GAS, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form.
The GAS AI proteins of the invention may include polypeptide sequences having sequence identity to the identified GAS proteins. The degree of sequence identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%,
Figure imgf000152_0001
96%, 97%, 98%, 99%, 99.5% or more). Polypeptides having sequence identity include homologs, orthologs, allelic variants and functional mutants of the identified GBS proteins. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith- Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affinity gap search with parameters gap open penalty=12 and gap extension penalty=l.
The GAS adhesin island polynucleotide sequences may include polynucleotide sequences having sequence identity to the identified GAS adhesin island polynucleotide sequences. The degree of sequence identity may vary depending on the polynucleotide sequence in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more).
The GAS adhesin island polynucleotide sequences of the invention may include polynucleotide fragments of the identified adhesin island sequences. The length of the fragment may vary depending on the polynucleotide sequence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, {e.g. at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
The GAS adhesin island amino acid sequences of the invention may include polypeptide fragments of the identified GAS proteins. The length of the fragment may vary depending on the amino acid sequence of the specific GAS antigen, but the fragment is preferably at least 7 consecutive amino acids, {e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferably the fragment comprises one or more epitopes from the sequence. Other preferred fragments include (1) the N-terminal signal peptides of each identified GAS protein, (2) the identified GAS protein without their N-terminal signal peptides, and (3) each identified GAS protein wherein up to 10 amino acid residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the N- terminus and/or the C-terminus e.g. the N-terminal amino acid residue may be deleted. Other fragments omit one or more domains of the protein {e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain).
GAS AI-I sequences ■ As discussed above, a GAS AI-I sequence is present in an M6 strain isolate (MGAS 10394).
Examples of GAS AI-I sequences from M6 strain isolate MGAS 10394 are set forth below.
M6_SpyO156: SpyO156 is arofA transcriptional regulator. An example of an amino acid sequence for M6_SpyO156 is set forth in SEQ ID NO: 41.
SEQ ID NO: 41 MIEKYLESSIESKCQLVVLFFKTSYLPITEVAEKTGLTFLQLNHYCEELNAFFPDSLSMTIQKRMISCQFTHPFK ETYLYQLYASSNVLQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALIPLLRNFELKLSKNKIVGEEYRIR YLIALLYSKFGIKVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRHQFSVTIPQTRIFQQL KKLFIYDSLKKSSRDIIETYCQLNFSAGDLDYLYLIYITANNSFASLQWTPEHIRQCCQLFEENDTFRLLLKPII TLLPNLKEQKPSLVKALMFFSKSFLFNLQHFIPETNLFVSPYYKGNQKLYTSLKLIVEEWLAKLPGKRYLNHKHF HLFCHYVEQILRNIQPPLVVVFVASNFINAHLLTDSFPRYFSDKSIDFHSYIAR p C T M/6_S II Jρy S01 O57: S M /6_ SSpy 7O11E5U73 is a 1Q fiibronectin binding protein. It contains a sortase substrate motif LPXTG (SEQ ID NO: 122), shown in italics in the amino acid sequence SEQ ID NO: 42 . SEQ ID NO: 42 MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACAGAIGFGQVAYAADEKTVPSHSSPNP EFPWYGYDAYGKEYPGYNIWTRYHDLRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHT TKLGKEELEQRLLSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDNSQYQFETLWESEAKEGKISRSQVTL MREALKKLIDPNLEATAVNKIPSGYRLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDGTPIPEDP KHPDDNLEPTLPPVMLDGEEVPEVPSESLEPALPPLMPELDGQEVPEKPSIDLPIEVPRYEFNNKDQSPLAGESG ETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDT KEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTK EPEVLMGGQSESVEFTKDTQTGMSGFSETATVVEDTRPKLVFHFDNNEPKVEENREKPTKNITPIiPATGDIENV LAFLGILILSVLSIFSLLKNKQSNKKV
M6_SpyO157 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ ID NO: 42, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant M6_SpyO157 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in M6_SpyO157. The pilin motif sequence is underlined in SEQ ID NO: 42, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 277, 287, and 301. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of M6_Spy0157 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 42
MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACAGAIGFGQVAYAADEKTVPSHSSPNP EFPWYGYDAYGKEYPGYNIWTRYHDLRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHT TKLGKEELEQRLLSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDNSQYQFETLWESEAKEGKISRSQVTL MREALKKLIDPNLEATAVNKIPSGYRLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDGTPIPEDP KHPDDNLEPTLPPVMLDGEEVPEVPSESLEPALPPLMPELDGQEVPEKPSIDLPIEVPRYEFNNKDQSPLAGESG ETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDT KEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTK EPEVLMGGQSESVEFTKDTQTGMSGFSETATVVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENV LAFLGILILSVLSIFSLLKNKQSNKKV
A repeated series of four E boxes containing a conserved glutamic residue have been identified in M6_SρyO157. The E-box motifs are underlined in SEQ ID NO: 42, below. The conserved glutamic acid (E) residues, at amino acid residues 415, 452, 489, and 526 are marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of M6_SpyO157. Preferred fragments of M6_SpyO157 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 42 MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACAGAIGFGQVAYAADEKTVPSHSSPNP EFPWYGYDAYGKE YPGYNIWTRYHDLRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKS FVDYAHT KHPDDNLEPTLPPVMLDGEEVPEVPSESLEPALPPLMPELDGQEVPEKPSIDLPIEVPRYEFNNKDQSPLΆGESG ETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDT KEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTK EPEVLMGGQSESVEFTKDTQTGMSGFSETATVVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENV LAFLGILILSVLSIFSLLKNKQSNKKV
M6_Spy0158: M6_SpyO158 is areverse transcriptase. An example ofSpyO158 is shown in theamino acid sequence SEQ IDNO 43. SEQ ID NO: 43
MSLRHQNKKGIRKEGWKSRPQSRWSDHCQLVAQKSVLKQAISKTVLAERGLFSCLDDYLERHALKVN
M6_SpyO159: M6_SρyO159 is a collagen adhesion protein. It contains a sortase substrate motif LPXSG, shown in italics in the amino acid sequence SEQ ID NO: 44. SEQ ID NO: 44
MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQTKVIPQEIVTQTETQGTQVVATKQK LESENSSLKVALKRESGFEHNATIDASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRN DITTTLTFKNGLSLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADGKVKNLVFTGRLGKQVIIVSTTRLKE EQTISLDSYGELVIDGAVGLSQKDRPPYSKPITVNILKPKLSSIESSLDSKDFEIVKTIDNLYTWDDQFYLLDFI SKQYEVLKTDYQSAKDSTPQTRDILFGEYTVEPLVMNKGHNNTINIYIRSTRPLGLKPIGAAPALIQPRSFRSLT PRSTRMKRSAPVEKFEGELEHHKRIDYLGDNQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVVVDKSG SMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSSYQGESFNRGQIHYRYRGIVSVSDGIRRDDAVKNSLLGVN GLLQRFVNINPENKLSVIGFQGSADYHAGKWYPDQSPRGGFYQPNLNNSRDAELLKGWSTNSLLDPNTLTALHNN GTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTFYFGEDGYRSGNGSSNDRNNVTRSQEGSKLAIDEFKARY PNLSIYSLGVSKDINSDTASSSPVVLKYLSGEEHYYGITDTAELEKTLNKIVEDSKLSQLGISDSLSQYVDYYDK QPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKVTLTFKSDYKVDDEYTYTLSFNVKAS DEAYEKYKDNEGRYSEMGDSDTDYGTNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKTVPITFTKVD ADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYETKAKLGYTLPENPWEVAVANNGDIK VKHPIEGELKSKDGSYMIKNYKIYQiPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY
M6_SpyO159 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:
181 LPSSG (shown in italics in SEQ ID NO: 44, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant M6_SpyO159 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in M6_SpyO159. The pilin motif sequence is underlined in SEQ ID NO: 44, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 265 and 276. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of M6_SpyO159 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 44 MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQTKVIPQEIVTQTETQGTQVVATKQK LESENSSLKVALKRESGFEHNATIDASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRN DITTTLTFKNGLSLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADGKVKNLVFTGRLGKQVIIVSTTRLKE EQTISLDSYGELVIDGAVGLSQKDRPPYSKPITVNILKPKLSSIESSLDSKDFEIVKTIDNLYTWDDQFYLLDFI SKQYEVLKTDYQSAKDSTPQTRDILFGEYTVEPLVMNKGHNNTINIYIRSTRPLGLKPIGAAPALIQPRSFRSLT PRSTRMKRSAPVEKFEGELEHHKRIDYLGDNQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVVVDKSG
Figure imgf000155_0001
GTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTFYFGEDGYRSGNGSSNDRNNVTRSQEGSKLAIDEFKARY PNLSIYSLGVSKDINSDTASSSPVVLKYLSGEEHYYGITDTAELEKTLNKIVEDSKLSQLGISDSLSQYVDYYDK QPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKVTLTFKSDYKVDDEYTYTLSFNVKAS DEAYEKYKDNEGRYSEMGDSDTDYGTNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKTVPITFTKVD ADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYETKAKLGYTLPENPWEVAVANNGDIK VKHPIEGELKSKDGSYMIKNYKIYQLPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY An E box containing a conserved glutamic residue has been identified in M6_Spy0159. The
E-box motif is underlined in SEQ ID NO: 44, below. The conserved glutamic acid (E), at amino acid residue 950, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of M6_SpyO159. Preferred fragments of M6_SpyO159 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 44
MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQTKVIPQEIVTQTETQGTQVVATKQK LESENSSLKVALKRESGFEHNATIDASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRN DITTTLTFKNGLSLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADGKVKNLVFTGRLGKQVIIVSTTRLKE EQTISLDSYGELVIDGAVGLSQKDRPPYSKPITVNILKPKLSSIESSLDSKDFEIVKTIDNLYTWDDQFYLLDFI SKQYEVLKTDYQSAKDSTPQTRDILFGEYTVEPLVMNKGHNNTINIYIRSTRPLGLKPIGAAPALIQPRSFRSLT PRSTRMKRSAPVEKFEGELEHHKRIDYLGDNQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVVVDKSG SMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSSYQGESFNRGQIHYRYRGIVSVSDGIRRDDAVKNSLLGVN GLLQRFVNINPENKLSVIGFQGSADYHAGKWYPDQSPRGGFYQPNLNNSRDAELLKGWSTNSLLDPNTLTALHNN GTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTFYFGEDGYRSGNGSSNDRNNVTRSQEGSKLAIDEFKARY PNLSIYSLGVSKDINSDTASSSPVVLKYLSGEEHYYGITDTAELEKTLNKIVEDSKLSQLGISDSLSQYVDYYDK QPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKVTLTFKSDYKVDDEYTYTLSFNVKΆS DEAYEKYKDNEGRYSEMGDSDTDYGTNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKTVPITFTKVD ADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYETKAKLGYTLPENPWEVAVANNGDIK VKHPIEGELKSKDGSYMIKNYKIYQLPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY
M6_Spy0160: M6_SpyO16O is a fimbrial structural subunit. It contains a sortase substrate motif LPXTG (SEQ ID NO: 122), shown in italics in amino acid sequence SEQ ID NO: 45.
SEQ ID NO: 45 MTNRRETVREKILITAKKLMLACLAILAVVGLGMTRVSALSKDDTAQLKITNIEGGPTVTLYKIGEGVYNTNGDS FINFKYAEGVSLTETGPTSQEITTIANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGATDGRTYNPI LLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVNKKTTSLGSVLSYSLTFELPSYTKEA
AVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKEDSKIVYTYQIAFRKVDSVSKTPLIGA IFGVYDTSNKLIDIVTTNKNGYAISTQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTS DKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKALTDGTTFSKSNEGSGTVLLETDIPNTKLGEIPS ΓGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA
M6_SpyO16O contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO: 45, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant M6_Spy0160 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. ,,,„,, ff... An p ba^;gQqtaijimg;»ccKn|eirye^'.i§}utamic residue has been identified in M6_Spy0160. The E-box motif is underlined in SEQ ID NO: 45, below. The conserved glutamic acid (E), at amino acid residue 412, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of M6_SpyO16O. Preferred fragments of M6_SpyO16O include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 45
MTNRRETVREKILITAKKLMLACLAILAVVGLGMTRVSALSKDDTAQLKITNIEGGPTVTLYKIGEGVYNTNGDS FINFKYAEGVSLTETGPTSQEITTIANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGATDGRTYNPI
LLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVNKKTTSLGSVLSYSLTFELPSYTKEA
AVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKEDSKIVYTYQIAFRKVDSVSKTPLIGA IFGVYDTSHKLIDIVTTNKNGYAISTQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTS DKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKALTDGTTFSKSNEGSGTVLLETDIPNTKLGELPS TGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA
M6_SpyO161 is a srtB type sortase. An example of an amino acid sequence of M6_Spy-161 is shown in SEQ ID NO: 46. SEQ ID NO: 46
MTERLKNLGILLLFLLGTAIFLYPTLSSQWNAYRDRQLLSTYHKQVIQKKPSEMEEVWQKAKAYNARLGIQPVPD AFSFRDGIHDKNYESLLQIENNDIMGYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHR GLPSAEMFTNLNLVKKGDTFYFRVLNKVLAYKVDQILIVEPDQATSLSGVMGKDYATLVTCTPYGVNTKRLLVRG HRIAYHYKKYQQAKKAMKLVDKSRMWAEVVCAAFGVVIAIILVFMYSRVSAKKSK
As discussed above, applicants have also determined the nucleotide and encoded amino acid sequence of fimbrial structural subunits in several other GAS AI-I strains of bacteria. Examples of sequences of these fimbrial structural subunits are set forth below.
M6 strain isolate CDC SS 410 is a GAS AI-I strain of bacteria. CDC SS 410_fimbrial is thought to be a fimbrial structural subunit of M6 strain isolate CDC SS 410. An example of a nucleotide sequence encoding the CDC SS 410_fimbrial protein (SEQ ID NO: 267) and a CDC SS 410_fimbrial protein amino acid sequence (SEQ ID NO: 268) are set forth below. SEQ ID NO: 267 aaagatgatactgcacaactaaagataacaaatattgaaggtgggccaacagtaacactt tataaaataggagaaggtgtttacaacactaatggtgattcttttattaactttaaatat gctgagggggtttctttaactgaaacaggacctacatcacaagaaattactactattgca aatggtattaatacgggtaaaataaagccttttagtactgaaaacgttagtatttctaat ggaacagcaacttataatgcgagaggtgcatctgtttatattgcattattaacaggtgcg acagatggccgtacctaoaatcctattttattagctgcatcttataatggtgagggaaat ttagttactaaaaatattgattccaaatctaattatttatatggacaaacaagtgttgca aaatcatcattaccatctattacaaagaaagtaaccgggacaatagatgacgtgaataaa aagactacctcgttaggaagtgtattgtcttattcgctgacatttgaattaccaagttat accaaagaagcagtcaataaaacagtatatgtttctgataatatgtcggaaggtcttact tttaactttaatagtcttacagtagaatggaaaggtaagatggctaatattactgaagat ggttcagtaatggtagaaaatacaaaaatcggaatagctaaggaggttaataacggtttt aatttaagttttatttatgatagtttagaatctatatcaccaaatataagttataaagct gttgtaaacaataaagctattgttggtgaagagggtaatcctaataaagctgaattcttc tattcaaataatccaacaaaaggtaatacatacgataatttagataagaagcctgataaa gggaatggtattacatccaaagaagattctaaaattgtttatacttatcaaatagcgttt agaaaagttgatagtgttagtaagaccccacttattggtgcaatttttggagtttatgat actagtaataaattaattgatattgttacaaccaataaaaatggatatgctatttcaaca aattcaaaaagtactacttatacatctgataaaaataaggcgacagataattcagagcaa gtaggatggttaaaaaatggtatattctattctatagatagtagacctacaggaaatgat gttaaagaggcttatattgaatctactaaggctttaactgatggaacaactttctcaaaa tcgaatgaaggttcaggtacagtattattagaaactgacatccctaacaccaagctaggt gaactc
SEQ ID NO: 268
KDDTAQLKITNIEGGPTVTLYKIGEGVYNTNGDSFINFKYAEGV SLTETGPTSQEITTIANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGATD GRTYNPILLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVNK KTTSLGSVLSYSLTFELPSYTKEAVNKTVYVSDNMSEGLTFNFNSLTVEWKGKMANIT EDGSVMVENTKIGIAKEVNNGFNLSFIYDSLESISPNISYKAVVNNKAIVGEEGNPNK AEFFYSNNPTKGNTYDNLDKKPDKGNGITSKEDSKIVYTYQIAFRKVDSVSKTPLIGA IFGVYDTSNKLIDIVTTNKNGYAISTQVSSGKYKIKELKAPKGYSLNTETYEITANWV TATVKTSANSKSTTYTSDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTK ALTDGTTFSKSNEGSGTVLLETDIPNTKLGEL
M6 strain isolate ISS 3650 is a GAS AI-I strain of bacteria. ISS3650_fimbrial is thought to be a fimbrial structural subunit of M6 strain isolate ISS 3650. An example of a nucleotide sequence encoding the ISS3650_fimbrial protein (SEQ ID NO: 269) and an ISS3650_fimbrial protein amino acid sequence (SEQ ID NO: 270) are set forth below. SEQ ID NO: 269 gaatggaaaggtaagatggctaatattactgaagatggttcagtaatggtagaaaataca aaaatcggaatagctaaggaggttaataacggttttaatttaagttttatttatgatagt ttagaatctatatcaccaaatataagttataaagctgttgtaaacaataaagctattgtt ggtgaagagggtaatcctaataaagctgaattcttctattcaaataatccaacaaaaggt aatacatacgataatttagataagaagcctgataaagggaatggtattacatccaaagaa gattctaaaattgtttatacttatcaaatagcgtttagaaaagttgatagtgttagtaag accccacttattggtgcaatttttggagtttatgatactagtaataaattaattgatatt gttacaaccaataaaaatggatatgctatttcaacacaagtatcttcaggaaaatataaa attaaggaattaaaagctcctaaaggttattcattgaatacagaaacttatgaaattacg gcaaattgggtaactgctacagtcaagacaagtgctaattcaaaaagtactacttataca tctgataaaaataaggcgacagataattcagagcaagtaggatggttaaaaaatggtata ttctattctatagatagtagacctacaggaaatgatgttaaagaggcttatattgaatct actaaggctttaactgatggaacaactttctcaaaatcgaatgaaggttcaggtacagta ttattagaaactgacatcc
SEQ ID NO: 270
EWKGKMANITEDGSVMVENTKIGIAKEVNNGFNLSFIYDSLESI SPNISYKAVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKEDS KIVYTYQIAFRKVDSVSKTPLIGAIFGVYDTSNKLIDIVTTNKNGYAISTQVSSGKYK IKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTSDKNKATDNSEQVGWLKN GIFYSIDSRPTGNDVKEAYIESTKALTDGTTFSKSNEGSGTVLLETDI
M23 strain isolate DSM2071 is a GAS AI-I strain of bacteria. DSM2071_Fimbrial is thought to be a fimbrial structural subunit of M23 strain DSM2071. An example of a nucleotide sequence encoding the DSM2071_fimbrial protein (SEQ ID NO: 251) and a DSM2071_fimbrial protein amino acid sequence (SEQ ID NO: 252) are set forth below. SEQ ID NO: 251 atgagagagaaaatattaatagcagcaaaaaaactaatgctagcttgtttagctatctta gctgtagtagggcttggaatgacaagagtatcagctttatcaaaagatgataaggcggag ttgaagataacaaatatcgaaggtaaaccgaccgtgacactgtataaaattggtgatgga aaatacagtgagcgaggggattcttttattggatttgagttaaagcaaggtgtggagcta aataaggcaaaacctacatctcaagaaataaataaaatcgctaatggtattaataaaggt agtgttaaggctgaagtagttaatataaaagaacatgctagtacaacttatagttataca aippi'etp^t'^^ll^Qgta'c^te^giJαif^Jjjtgactggagctactgatggacgtgcctat aatcctatcttactgacagcttctta'caatgaggaaaatccacttaagggagggcagatt gacgcaactagtcattatctttttggagaagaagcagttgctaaatctagccaaccaaca attagcaagtcaattacaaaatccacaaaagatggtgataaagatacagcatctgtaggt gaaaaagttgattacaaattaactgttcagttaccaagttattcgaaagatgctatcaat aaaacggtgtttatcactgacaaattgtctcagggacttactttccttccaaaaagttta aagattatctggaatggtcaaacgttaacaaaggtgaatgaagaatttaaagctggagat aaggtaattgctcaacttaaggttgaaaataatggatttaatctgaactttaattatgat aaccttgataatcatgccccagaagttaactatagtgctctactaaatgaaaacgcagtt gttggtaaaggtggtaatgacaataatgtagactattactattcaaataatccgaataaa ggagagacccataaaacaactgagaagcctaaagagggtgaaggtactggtatcactaaa aagacggataaaaaaaccgtctacacctatcgtgtagcctttaagaaaacaggcaaagat catgccccactagctggtgctgttttcggtatctattcagataaggaagcgaaacaatta gtcgatattgttgtgacaaatgcacagggttatgcagcatcaagcgaagttgggaaaggg acttattacattaaagaaattaaatcccctaagggttactctttaaatacaaatatttat gaagtggaaacttcatgggaaaaagctacaacgacttctacaactaatcgtttagagaca atttatacaacagatgataatcaaaagtctccaggaactaatacagttggttggttggaa gatggtgtcttttacaaagaaaatccaggtggtgatgctaaacttgcctatatcaaacaa tcaacagaggagacttctacaactatagaagtcaaagaaaatcaagctgaaggttcaggt acggtattattagaaactgaaattcctaacaccaaattaggtgaattaccttcgacaggt agcattggtacttacctctttaaagctattggttcggctgctatgatcggtgcaattggt atttatattgttaaacgtcgtaaagcttaa
SEQ ID NO: 252 MREKILIAAKKLMLACLAILAVVGLGMTRVSALSKDDKAELKIT
NIEGKPTVTLYKIGDGKYSERGDSFIGFELKQGVELNKAKPTSQEINKIANGINKGSV KAEVVNIKEHASTTYSYTTTGAGIYLAILTGATDGRAYNPILLTASYNEENPLKGGQI DATSHYLFGEEAVAKSSQPTISKSITKSTKDGDKDTASVGEKVDYKLTVQLPSYSKDA INKTVFITDKLSQGLTFLPKSLKIIWNGQTLTKVNEEFKAGDKVIAQLKVENNGFNLN FNYDNLDNHAPEVNYSALLNENAVVGKGGNDNNVDYYYSNNPNKGETHKTTEKPKEGE GTGITKKTDKKTVYTYRVAFKKTGKDHAPLAGAVFGIYSDKEAKQLVDIVVTNAQGYA ASSEVGKGTYYIKEIKSPKGYSLNTNIYEVETSWEKATTTSTTNRLETIYTTDDNQKS PGTNTVGWLEDGVFYKENPGGDAKLAYIKQSTEETSTTIEVKENQAEGSGTVLLETEI PNTKLGELPSTGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA GAS AI-2 sequences
As discussed above, a GAS AI-2 sequence is present in an Ml strain isolate (SF370). Examples of GAS AI-2 sequences from Ml strain isolate SF370 are set forth below.
SpyO124 is a rofA transcriptional regulator. An example of an amino acid sequence for SρyO124 is set forth in SEQ ID NO:47. SEQ ID NO: 47
MIEKYLESSIESKCQLIVLFFKTSYLPITEVAEKTGLTFLQLNHYCEELNAFFPGSLSMTIQKRMISCQFTHPFK ETYLYQLYASSNVLQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALIPLLRNFELKLSKNKIVGEEYRIR YLIALLYSKFGIKVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRHQFSVTIPQTRIFQQL KKLFVYDSLKKSSHDIIETYCQLNFSAGDLDYLYLIYITANNSFASLQWTPEHIRQYCQLFEENDTFRLLLNPII TLLPNLKEQKAΞLVKALMFFSKSFLFNLQHFIPETNLFVSPYYKGNQKLYTSLKLIVEEWMAKLPGKRDLNHKHF HLFCHYVEQSLRNIQPPLVVVFVASNFINAHLLTDSFPRYFSDKSIDFHSYYLLQDNVYQIPDLKPDLVITHSQL IPFVHHELTKGIAVAEISFDESILSIQELMYQVKEEKFQADLTKQLT
GAS 015 is also referred to as Cpa. It contains a sortase substrate motif WXTG (SEQ ID NO: 135), shown in italics in SEQ ID NO: 48.
SEQ ID NO: 48
LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFALVTSMVGAKTVFGLVESSTPNAINPDSSSEY RWYGYESYVRGHPYYKQFRVAHDLRVNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPR ITGDELNQKLRAVMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAPISNPDESFKRESESNLVSTSQLSLM RQALKQLIDPNLATKMPKQVPDDFQLSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLI RKYAIGDYSKLLEGATLQLTGDNVNSFQARVFSSNDIGERIELSDGTYTLTELNSPAGYSIAEPITFKVEAGKVY PDFTTGEVKYTHIAGRDLF'KYTVKPRDTDPDTFLKHIKKVIEKGYREKGQAIEYSGLTETQLRAATQLAI YYFTD SAELDKDKLKDYHGFGDMNDSTLAVAKILVEYAQDSNPPQLTDLDFFIPNNNKYQSLIGTQWHPEDLVDIIRMED KKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKTDKTNLEFKDGKATINLKHGESLTLQGL PEGYSYLVKETDSEGYKVKVNSQEVANATVSKTGITSDETLAFENNKEP WPΓGVDQKINGYLALIVIAGISLGI
WGIHTIRIRKHD
GAS 015 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 182 WPTG (shown in italics in SEQ ID NO: 48, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GAS 015 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in GAS 015. The pilin motif sequence is underlined in SEQ ID NO: 48, below. Conserved lysine (K) residues are also marked in bold, at amino acid residue 243. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of GAS 015 include the conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 48
LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFALVTSMVGAKTVFGLVESSTPNAINPDSSSEY RWYGYESYVRGHPYYKQFRVAHDLRVNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPR ITGDELNQKLRAVMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAPISNPDESFKRESESNLVSTSQLSLM RQALKQLIDPNLATKMPKQVPDDFQLSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLI RKYAIGDYSKLLEGATLQLTGDNVNSFQΆRVFSSNDIGERIELSDGTYTLTELNSPAGYSIAEPITFKVEAGKVY TIIDGKQIENPNKEIVEPYSVEAYNDFEEFSVLTTQNYAKFYYAKNKNGSSQVVYCFNADLKSPPDSEDGGKTMT PDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKHIKKVIEKGYREKGQAIEYSGLTETQLRAATQLAIYYFTD SAELDKDKLKDYHGFGDMNDSTLAVAKILVEYAQDSNPPQLTDLDFFIPNNNKYQSLIGTQWHPEDLVDIIRMED KKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKTDKTNLEFKDGKATINLKHGESLTLQGL PEGYSYLVKETDSEGYKVKVNSQEVANATVSKTGITSDETLAFENNKEPVVPTGVDQKINGYLALIVIAGISLGI WGIHTIRIRKHD
An E box containing a conserved glutamic residue has been identified in GAS 015. The E- box motif is underlined in SEQ ID NO: 48, below. The conserved glutamic acid (E), at amino acid residue 352, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of GAS 015. Preferred fragments of GAS 015 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. SEQ ID NO: 48
LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFALVTSMVGAKTVFGLVESSTPNAINPDSSSEY RWYGYESYVRGHPYYKQFRVAHDLRVNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPR ITGDELNQKLRAVMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAPISNPDESFKRESESNLVSTSQLSLM RQALKQLIDPNLATKMPKQVPDDFQLSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLI RKYAIGDYSKLLEGATLQLTGDNVNSFQARVFSSNDIGERIELSDGTYTLTELNSPAGYSIAEPITFKVEAGKVY TIIDGKQIENPNKEIVEPYSVEAYNDFEEFSVLTTQNYAKFYYAKNKNGSSQVVYCFNADLKSPPDSEDGGKTMT PDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKHIKKVIEKGYREKGQAIEYSGLTETQLRAATQLAIYYFTD SΆELDKDKLKDYHGFGDMNDSTLAVAKILVEYAQDSNPPQLTDLDFFIPNNNKYQSLIGTQWHPEDLVDIIRMED KKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKTDKTNLEFKDGKATINLKHGESLTLQGL p
WGIHTIRIRKHD
SpyO127 is a LepA putative signal peptidase. An example of an amino acid sequence for SpyO127 is set forth in SEQ ID NO: 49. SEQ ID NO: 49
MIIKRNDMAPSVKAGDAILFYRLSQTYKVEEAVVYEDSKTSITKVGRIIAQAGDEVDLTEQGELKINGHIQNEGL TFIKSREANYPYRIADNSYLILNDYYSQESENYLQDAIAKDAIKGTINTLIRLRNH SpyO128 is thought to be a fibrial protein. It contains a sortase substrate motif EVXTG (SEQ
ID NO: 136) shown in italics in SEQ ID NO: 50. SEQ ID NO: 50
TPMTKVTYTNSDKGGSNTKTAEFDFSEVTFEKPGVYYYKVTEEKIDKVPGVSYDTTSYTVQVHVLWNEEQQKPVA TYIVGYKEGSKVPIQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKANQYYKASEKVMIEKTTKGGQAPVQT EASIDQLYHFTLKDGESIKVTNLPVGVDYWTEDDYKSEKYTTNVEVSPQDGAVKNIAGNSTEQETSTDKDMTIT FTNKKDFSVPTGVAMTVAPYIALGIVAVGGALYFVKKKNA
SpyO128 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 183 EVPTG (shown in italics in SEQ ID NO: 50, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyO128 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Two E boxes containing a conserved glutamic residue have been identified in SpyO128. The E-box motifs are underlined in SEQ ID NO: 50, below. The conserved glutamic acid (E) residues, at amino acid residues 271 and 290, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of SpyO128. Preferred fragments of SpyO128 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ID NO: 50
TPMTKVT YTNSDKGGSNTKTAEFDFSEVTFEKPGVYYYKVTEEKIDKVPGVSYDTTSYTVQVHVLWNEEQQKPVA TYIVGYKEGSKVPIQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKANQYYKASEKVMIEKTTKGGQAPVQT
EASIDQLYHFTLKDGESIKVTNL PVGVDYVVTE DD YKS E KYTTNVEVS PQDGAVKN I AGNSTEQETSTDKDMT I T
FTNKKDFEVPTGVAMTVAPYIALGIVAVGGALYFVKKKNA
SpyO129 is asrtCl type sortase. An example ofanamino acidsequence for SpyO129 is set forthin SEQ IDNO: 51. SEQ ID NO: 51
MIVRLIKLLDKLINVIVLCFFFLCLLIAALGIYDALTVYQGANATNYQQYKKKGVQFDDLLAINSDVMAWLTVKG THIDYPIVQGENNLEYINKSVEGEYSLSGSVFLDYRNKVTFEDKYSLIYAHHMAGNVMFGELPNFRKKSFFNKHK EFSIETKTKQKLKINIFACIQTDAFDSLLFNPIDVDISSKNEFLNHIKQKSVQYREILTTNESRFVALSTCEDMT TDGRiiviGQiE"
SρyO13O is referred to as a hypothetical protein. It contains a sortase substrate motif LPXTG (SEQ ID NO: 122), shown in italics in SEQ ID NO: 52. spg;jpDW:li 5jzS Dl Ξ /'" ES 7 E 31|;1
MKKSILRILAIGYLLMSFCLLDSVEAENLTASINIEVINQVDVATNKQSSDIDETFMFVIEALDKESPLPNSVTT SVKGNGKTSFEQLTFSEVGQYHYKIHQLLGKNSQYHYDETVYEVVIYVLYNEQSGALETNLVSNKLGETEKSELI FKQEYSEKTPEPHQPDTTEKEKPQKKRNGILPSTGEMVSYVSALGIVLVATITLYSIYKKLKTSK Spy0130 contains an amino acid motif indicative of a cell wall anchor: SEQ ED NO: 131
LPSTG (shown in italics in SEQ ID NO: 52, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyO13O protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Two E boxes containing conserved glutamic residues have been identified in SpyO13O. The E-box motifs are underlined in SEQ ID NO: 52, below. The conserved glutamic acid (E) residues, at amino acid residues 118 and 148, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of Spy0130. Preferred fragments of SpyO13O include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ID NO: 52
MKKSILRILAIGYLLMSFCLLDSVEAENLTASINIEVINQVDVATNKQSSDIDETFMFVIEALDKEΞPLPNSVTT SVKGNGKTSFEQLTFSEVGQYHYKIHQLLGKNSQYHYDETVYEVVIYVLYNEQSGALETNLVSNKLGETEKSELI FKQEYSEKTPEPHQPDTTEKEKPQKKRNGILPSTGEMVSYVSALGIVLVATITLYSIYKKLKTSK
SpyO131 is referred to as a conserved hypothetical protein. An example of an amino acid sequence of SpyO131 is set forth in SEQ ID NO: 53 SEQ ID NO: 53
MTRTNYQKKRMTCPVETEDITYRRKKIKGRRQAILAQFEPELVHHELIGDSCTCPDCHGTLTEIGSVVQRQELVF iPAQLKRiNHVQHAYKCQTCSDNSLSDKIIKAPVPKAPLΆHSLGSASIIAHTVHQKFTLKVPNYRQEEDWNKLGL
SISRKEIANWHIKSSQYYFEPLYDLLRDILLSQEVIHADETSYRVLESDTQLTYYWTFLSGKHEKKGITLYHHDK
RRSGL VTQEVLGDYSGYVHCDMHGAYRQLEHAKL VGCWAHVRRKFFEATPKQADKTSLGRKGLVYCDKLFALEAE WCELPPQERL VKRKEILTPLMTTFFDWCREQVVLSGSKLGLAIAYSLKHERTFRTVLEDGHIVLSNNMAERAIKS
LVMGRKNWLFSQSFEGAKAAAIIMSLLETAKRHGLNSEKYIS YLLDRLPNEETLAKREVLEAYLPWAKKVQTNCQ
SpyO133 is referred to as a conserved hypothetical protein. An example of an amino acid sequence of SpyO133 is set forth in SEQ ID NO: 54. SEQ ID NO: 54
MTIRLNDLGQVYLVCGKTDMRQGIDSLAYLVKSQHELDLFSGAVYLFCGGRRDRFKALYWDGQGFWLLYKRFENG KLAWPRNRDEVKCLTAVQVDWLMKGFFISPNIKISKSHDFY
SpyO135 is a SrtB type sortase. It is also referred to as a putative fibria-associated protein. An example of an amino acid sequence of SpyO135 is set forth in SEQ ID NO: 55. SEQ ID NO: 55
MECYRDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPDAFSFRDGIHDKNYESLLQIENNDIMGYVE
AYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKKYQQAKKAMKLVDKSRMWAEV VCAAFGVVIAIILVFMYSRVSAKKSK
GASAI-3 sequences F8
Figure imgf000162_0001
E!&AJ:;M-ite!lsequence is present in a M3, M18 and M5 strain isolates.
Examples of GAS AI-3 sequences from M3 strain isolate MGAS315 are set forth below.
SpyM30097 is as a negative transcriptional regulator (Nra). An example of an amino acid sequence of SpyM30097 is set forth in SEQ ID NO: 56. SEQ ID NO: 56
MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKG KIICCFVKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALSWKRHQFAV SIPQTRIFRQLKKLFIYDCLTRSSRQVIENAFSLTFSQGDLEYLFLIYITTNNSFASLQWTPQHIETCCHIFEKN DTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFSLPTYTGNSNLYKALKNIVNQWLAQL PGKRHLNEKHLQLFCSHIEQILKNKQPALTVVLISSNFINAKLLTDTIPRYFSDKGIHFYSFYLLRDDIYQIPSL KPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLIYQLKDKKYQDFLNEQLQ
SpyM30098 is thought to be a collagen binding protein (Cpb). It contains a sortase substrate motif VPXTG (SEQ ID NO: 137) shown in italics in SEQ ID NO: 57.
SEQ m NO: 57
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPD YSPLKTYHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL YNGYPNDRNGIMKGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVE SLPNQVPANYQLSIFQSSDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSV TAFNDFEEIGYLSDFNNYGKFYYAKNTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDK LDDATLAVVHELITYAEDVTLPMTQNLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNΞGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETGASDY
EVSVNGKNAPDGKATKASVKEDETVAFENRKDL VPPTGLTTDGAIYLWLLLLVPFGLL VWLFGRKGTKK
SpyM30098 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID NO: 57, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30098 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM30098. The pilin motif sequence is underlined in SEQ ID NO: 57, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 262 and 270. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyM30098 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 57
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPD YSPLKTYHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL YNGYPNDRNGIMKGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVE SLPNQVPANYQLSIFQSSDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSV TAFNDFEEIGYLSDFNNYGKFYYAKNTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDK LDDATLAVVHELITYAEDVTLPMTQNLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT
Figure imgf000163_0001
EVSVNGKNAPDGKATKASVKEDETVAFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
An E box containing a conserved glutamic residue has been identified in SpyM30098. The E- box motif is underlined in SEQ ID NO: 57, below. The conserved glutamic acid (E), at amino acid residue 330, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of SpyM30098. Preferred fragments of SpyM30098 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. SEQ ID NO: 57
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPD YSPLKTYHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL YNGYPNDRNGIMKGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVE SLPNQVPANYQLSIFQSSDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSV TAFNDFEEIGYLSDFNNYGKFYYAKNTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDK LDDATLAVVHELITYAEDVTLPMTQNLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETGASDY EVSVNGKNAPDGKATKASVKEDETVAFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
SpyM30099 is referred to as LepA. An example of an amino acid sequence of SpyM30099 is set forth in SEQ ID NO: 58. SEQ ID NO: 58 MTNYLNRLNENPLLKAFIRLVLKISIIGFLGYILFQYVFGVMIVNTNQMSPAVSAGDGVLYYRLTDRYHINDVVV YEVDDTLKVGRIAAQAGDEVNFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR YYGALPINQIKGKISTLLRVRGI
SpyM30100 is thought to be a fimbrial protein. An example of an amino acid sequence of SpyM30100 is set forth in SEQ ID NO: 59. SEQ ID NO: 59
MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTK DGLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYV GNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQ KGNEKFEVKIGTPYKFKLKNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESA DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
SpyM30100 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 140
QVPTG (shown in italics in SEQ ID NO: 59, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30100 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in SpyM30100. The pilin motif sequences are underlined in SEQ ID NO: 59, below.
Conserved lysine (K) residues are also marked in bold, at amino acid residues 57 and 63 and at amino acid residues 161 and 166. The pilin sequences, in particular the conserved lysine residues, are thiωjh't tS be
Figure imgf000164_0001
structures. Preferred fragments of SpyM30100 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO: 59 MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTK DGLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYV GNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQ KGNEKFEVKIGTPYKFKLKNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESA DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA Two E boxes, each containing a conserved glutamic residue, have been identified in
SpyM30100. The E-box motifs are underlined in SEQ ID NO: 59, below. The conserved glutamic acid (E) residues, at amino acid residues 232 and 264, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of SpyM30100. Preferred fragments of SpyM30100 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 59
MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTK DGLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYV GNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQ KGNEKFEVKIGTPYKFKLKNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESA DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
SpyM30101 is a SrtC2 type sortase. An exampleofanamino acid sequence ofSpyM30101 is setforthin SEQ IDNO: 60. SEQ ED NO: 60
MTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNIPG THIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFSKHN KAIIETKERKKLTVTIFACLKTDAFNQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTCENF STDNRVIVVGTIQE
SpyM30102 is referred to as a hypothetical protein. An example of an amino acid sequence of SρyM30102 is set forth in SEQ ID NO: 61. SEQ ID NO: 61
MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQY TYRVYQKPSQNKDYQADTTVFDVL VYVTYDEDGTLVAKVI SRRAGDEEKSAITFKPKWL VKPI PPRQPNI PKTPL PiAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
SpyM30102 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID NO: 61, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30102 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM30102. The pilin motif sequence is underlined in SEQ ID NO: 61, below. The conserved lysine (K) residue is also marked in bold, at amino acid residue 132. The pilin sequence, in paffiicOarll'the (jiøiilii-liiciiiysi'iiieil-eSdSfesiiffi thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyM30102 include the conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 61 MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQY TYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPL PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
Two E boxes containing conserved glutamic residues have been identified in SpyM30102. The E-box motifs are underlined in SEQ ID NO: 61, below. The conserved glutamic acid (E) residues, at amino acid residues 52 and 122, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus- like structures of SpyM30102. Preferred fragments of SpyM30102 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence. SEQ ID NO: 61 MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQY TYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPL PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
SpyM30103 is referred to as a putative multiple sugar metabolism regulator. An example of an amino acid sequence for SpyM3103 is set forth in SEQ ID NO: 62.
SEQ ID NO: 62
MVRFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAIPQAAQDVIFYEGLFEESFMIFPLC HYIIAIGPFYPYSLNKDYQEQLANNCLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT IHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRIPLSSITSSSISALRA EKNLTVIYLTRLLEFSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS VLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDT THFHKIFKKYTGISSKDYLAKYRDNI
SρyM30104 is thought to be a F2 like fibronectic binding protein. An example of an amino acid sequence for SpyM30104 is set forth in SEQ ID NO: 63.
SEQ ID NO: 63
MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPAD RSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKI WVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLE PKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEF GKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSS GKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGS GQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTEIEDSKSSDVIIGGQGE VVDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHFDNKEP ESNSEIPKKDKSKSNTSZPΛΓGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLSSC
SpyM30104 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ ID NO: 63, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SρyM30104 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. I"1' i^ifiVo^lliyaBlSiidisdiitffeslciiibicilliiibontaiiiing conserved lysine (K) residues have also been identified in SpyM30104. The pilin motif sequences are underlined in SEQ ID NO: 63, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 156 and 227. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyM30104 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO: 63
MSSSDEETLKQYΆSKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPAD RSYTNRMVMSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTMLQAVISVEPVIESLPWTSI1KPIAQKDITAKKI WVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLE PKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGEMQMKQIEGEDSKPIDEVTENNLIEF GKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRBSS GKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGS GQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTEIEDSKSSDVIIGGQGE VVDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHFDNKEP ESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLSSC
An E box containing a conserved glutamic residue has been identified in SρyM30104. The E- box motif is underlined in SEQ ID NO: 63, below. The conserved glutamic acid (E), at amino acid residue 402, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of SpyM30104. Preferred fragments of SpyM30104 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 63
MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPAD RSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKI WVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLE PKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEF GKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSS GKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGS GQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTEIEDSKSSDVIIGGQGE VVDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHFDNKEP ESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLSSC
Examples of GAS AI-3 sequences from M3 strain isolate SSI-I are set forth below.
Sps0099 is a negative transcriptional regulator (Nra). An example of an amino acid sequence for Sps0099 is set forth in SEQ ID NO: 64. SEQ ID NO: 64 MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKG KIICCFVKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALSWKRHQFAV SIPQTRIFRQLKKLFIYDCLTRSSRQVIENAFSLTFSQGDLEYLFLIYITTNNSFASLQWTPQHIETCCHIFEKN DTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFSLPTYTGNSNLYKALKNIVNQWLAQL PGKRHLNEKHLQLFCSHIEQILKNKQPALTVVLISSNFINAKLLTDTIPRYFSDKGIHFYSFYLLRDDIYQIPSL KPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLIYQLKDKKYQDFLNEQLQ
SpsOlOO is thought to be a collagen binding protein (Cbp). It contains a sortase substrate motif VPXTG shown in italics in SEQ ID NO: 65. SEQ ID NO: 65 MQ^iKTiydJiWkE.lST€cϊ-ΪΪ^HiϊWiltl-IGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGyPD YSPLKTYHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL YNGYPNDRNGIMKGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVE SLPNQVPANYQLSIFQSSDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSV TAFNDFEEIGYLSDFNNYGKFYYAKNTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDK LDDATLAVVHELITYAEDVTLPMTQNLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETGASDY EVSVNGKNAPDGKATKASVKEDETVAFENRKDL^PPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
SpsOlOl is referred to as a LepA protein. An example of an amino acid sequence of SpsOlOl is set forth as SEQ ID NO: 66 SEQ ID NO: 66 MTNYLNRLNENPLLKAFIRLVLKISIIGFLGYILFQYVFGVMIVNTNQMSPAVSAGDGVLYYRLTDRYHINDVVV YEVDDTLKVGRIAAQAGDEVNFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR YYGALPINQIKGKISTLLRVRGI
Sps0102 is thought to be a fimbrial protein. It contains a sortase substrate motif QVXTG shown in italics in SEQ ID NO: 67.
SEQ ID NO: 67
MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTA SGKTKDGLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWT VDVYVGNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQ IVSLQKGNEKFEVKIGTPYKFKLKNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQK TDESADEIVVTNKRDTQVPΓGVVGTLAPFAVLSIVAIGGVIYITKRKKA
Sps0103 is a SrtC2 typesortase. An exampleofSps0103 is set forthin SEQ IDNO: 68. SEQ ID NO: 68 MVMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNI PGTHIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFSK HNKAIIETKERKKLTVTIFACLKTDAFNQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTCE NFSTDNRVIVVGTIQE Sps0104 is referred to as a hypothetical protein. It contains a sortase substrate motif LPXAG shown in italics in SEQ ID NO: 69. SEQ ID NO: 69
MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTF TTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPN IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
Sρs0105 is referred to as a putative multiple sugar metabolism regulator. An example of Sps0105 is set forth in SEQ ID NO: 70.
SEQ ID NO: 70 MALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTIHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHI MDLVKLGNPQLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFVENTDVAKHYΞLVKYYMALNEEAS DLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNSVLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSL
QHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI
Sps0106 is thought to be a F2 like fibronectic binding protein. It contains a sortase substrate
LPXTG (SEQ ID NO: 122) shown in italics in SEQ ID NO: 71. SEQ ID NO: 71 H C
MTlKMSY.
HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL
FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHI
DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEV
ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GEVVDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHFDNK EPESNSEIPKKDKSKSNTSIPARGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLSSC
Examples of GAS AI-3 sequences from M5 isolate Manfredo are set forth below. Orf 77 encodes a negative transcription regulator (Nra). An example of the nucleotide sequence encoding Nra (SEQ ID NO: 88) and an Nra amino acid sequence (SEQ ID NO: 89) are set forth below.
SEQ ID NO: 88
ATGCCTTATGTCAAAAAGAAAAAGGATAGTTTCTTAGTAGAAACATATCTTGAACAGTCTATTAGAGATAAAAGT
Figure imgf000168_0001
GACACATTTCGGTTATTGTTAGAGCCCATTCTTAAACGTTTACCGCAATTAAACCATTCTAAACAAGACCTTATT
Figure imgf000168_0002
AAATATCAAGATTTTCTAAACGAGCAATTACAA
SEQ ID NO: 89 MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKG KIICCFVKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALSWKRHQFAV SIPQTRIFRQLKKLFIYDCLTRSSRQVIENAFSLMFSQGDLDYLFLIYITTNNSFASLQWTPQHIETCCHIFEKN DTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFSLPTYTGNSNLYKALKNIVNQWLAQL PGKRHLNEKHLQLFCSHIEQILKNKQPALTWLISSNFINAKLLTDTIPRYFSDKGIHFYSFYLLRDDIYQIPSL KPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLIYQLKDKKYQDFLNEQLQ
Orf 78 is thought to be a collagen binding protein (Cbp). An example of the nucleotide sequence encoding Cbp (SEQ ID NO: 90) and a Cbp amino acid sequence (SEQ ID NO: 91) are set forth below.
SEQ ID NO: 90
TTGCAAAAGAGGGATAAAACCAATTATGGAAGCGCTAACAACAAACGACGACAAACGACGATCGGATTACTGAAA
ACTGAAACTAAAAAAACGTCAGTCATTATTAGAAAATATGCTGAAGGTGACTACTCTAAACTTCTAGAGGGAGCA ACTTTGCGTTTAACAGGGGAAGATATCCCAGATTTTCAAGAAAAAGTCTTCCAAAGTAATGGAACAGGAGAAAAG
AACATt
Figure imgf000169_0001
CTAGGTTCTCCATATACTATAGAGGCATACAATGATTTTGATGAATTTGGCTTACTGTCAACACAAAATTATGCG
CCTGACTCGGAAGATCATGGTGCTACAATAAATCCTGACTTTACGACTGGTGATATTAGGTACAGTCATATTGCT
Figure imgf000169_0002
AAGGATTATATAGTAACTGTTGATAACAAAGTTAGTCAAGAAGCTCAATCAGCAAGTGAGAATGTCACAGCAGAC TGGTTATTACTACTTGTTCCATTTGGGTTATTGGTTTGGCTATTTGGTCGTAAAGGGTTAAAAAATGAC
SEQ ID NO: 91
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEKSTETKKTSVIIRKYAEGDYSKLLEGA TLRLTGEDIPDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKITEPIKFRVVNKKVFIVQKDGSQVENPNKE LGSPYTIEAYNDFDEFGLLSTQNYAKFYYGKNYDGSSQIVYCFNANLKSPPDSEDHGATINPDFTTGDIRYSHIA GSDLIKYANTARDEDPQLFLKHVKKVIENGYHKKGQAIPYNGLTEAQFRAATQLAIYYFTDSVDLTKDRLKDFHG FGDMNDQTLGVAKKIVEYALSDEDSKLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTV QKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEA KDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGLKND
Orf 78 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID NO: 91, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 78 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Three E boxes containing conserved glutamic residues have been identified in Orf 78. The E- box motifs are underlined in SEQ ID NO: 91, below. The conserved glutamic acid (E) residues, at amino acid residues 112, 395, and 447, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus- like structures of Orf 78. Preferred fragments of Orf 78 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ID NO: 91
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEKSTETKKTSVIIRKYAEGDYSKLLEGA TLRLTGEDIPDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKITEPIKFRVVNKKVFIVQKDGSQVENPNKE LGSPYTIEAYNDFDEFGLLSTQNYAKFYYGKNYDGSSQIVYCFNANLKSPPDSEDHGATINPDFTTGDIRYSHIA GSDLIKYANTARDEDPQLFLKHVKKVIENGYHKKGQAIPYNGLTEAQFRAATQLAIYYFTDSVDLTKDRLKDFHG FGDMNDQTLGVAKKIVEYALSDEDSKLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTV QKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEA KDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGLKND
Orf 79 is thought to be a LepA signal peptidase I. An example of the nucleotide sequence encoding a LepA signal peptidase I (SEQ ID NO: 92) and a LepA signal peptidase I amino acid sequence (SEQ ID NO: 93) are set forth below. JP1Cl' •^^SDl!i::;i;/iΞ7ΪΞ!3!9
ATGACTAATTACCTAAATCGTTTAAATGAGAATTCACTATTTAAAGCTTTCATACGGTTAGTACTTAAGATTTCT ATTATTGGGTTTCTAGGTTACATTCTATTTCAGTATGTTTTTGGTGTTATGATTATTAACACTAATGATATGAGT
CCTGCTTTAΆGTGCAGGTGACGGTGTTTTATATTATCGTTTGACTGATCGCTATCATATTAATGATGTGGTGGTC TATGAGGTTGATAACACTTTGAAAGTTGGTCGAATTGTCGCTCAAGCTGGCGATGAGGTTAGTTTTACGCAAGAA
GGAGGACTGTTGATTAATGGGCATCCACCAGAAAAAGAGGTCCCTTACCTGACGTATCCTCACTCAAGTGGCCCA
TATTATGGGGCGTTACCCGTCAATCAAATAAAAGGGAAAATCTCAACTCTATTAAGAGTGAGAGGAATT SEQ ID NO: 93
MTNYLNRLNENSLFKAFIRLVLKISIIGFLGYILFQYVFGVMIINTNDMSPALSAGDGVLYYRLTDRYHINDVVV YEVDNTLKVGRIVAQAGDEVSFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGKYFILNDYREERLDSR YYGALPVNQIKGKISTLLRVRGI Orf 80 is thought to to be a fimbrial protein. An example of the nucleotide sequence encoding the fimbrial protein (SEQ ID NO: 94) and a fimbrial protein amino acid sequence (SEQ ID NO: 95) are set forth below. SEQ ID NO: 94
TTGGAGAGAGAAAAAATGAAAAAAAACAAATTATTACTTGCTACTGCAATCTTAGCAACTGCTTTAGGAACAGCT ACTTATGATGATGAAGAGGTGTTAATGCCCGAAACCGCCTTTACTTTTACTATAGAGCCTGATATGACTGCAAGT
Figure imgf000170_0001
CACGGTAAGAGTGTCATGTTATCGAAATTACCAATTGGTATCAATTACTATCTTAGTGAAGACGAAGCGAATAAA
Figure imgf000170_0002
AAAGCT SEQ ID NO: 95
MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVVTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTAS GKEGSLDIKNGIVEGLDKQVTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTV DVYVGNKANNEEGFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQITGNAGDRKKSFNFTLTLQPSEYYKTG SVVKIEQDGSKKDVTIGTPYKFTLGHGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQ NQKTDESADEIVVTNKRDTQVPΓGVVGTLAPFAVLSIVAIGGVIYITKRKKA
Orf 82 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO: 95, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 82 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
An E box containing a conserved glutamic residue has been identified iri Orf 80. The E-box motif is underlined in SEQ ID NO: 95, below. The conserved glutamic acid (E), at amino acid residue 270, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is
Figure imgf000171_0001
of Orf 80. Preferred fragments of Orf 80 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ID NO: 95 MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVVTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTAS GKEGSLDIKNGIVEGLDKQVTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTV DVYVGNKANNEEGFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQITGNAGDRKKSFNFTLTLQPSEYYKTG SVVKIEQDGSKKDVTIGTPYKFTLGHGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQ NQKTDESADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
Orf 81 is thought to to be a SrtC2 type sortase. An example of the nucleotide sequence encoding the SrtC2 sortase (SEQ ID NO: 96) and a SrtC2 sortase amino acid sequence (SEQ ID NO: 97) are set forth below. SEQ ID NO: 96
Figure imgf000171_0002
TTTTCAACGTGTGAAAATTTTTCTACTGACAATCGTGTTATCGTTGTCGGTACTATTCAAGAA
SEQ ID NO: 97
MISQRMMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVI GWLNIPGTHIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKK DFFNKHNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQKQLVDYISKRSKQFKPVKLKHHTKFVA FSTCENFSTDNRVIVVGTIQE
Orf 82 is referred to as a hypothetical protein. It contains a sortase substrate motif LPXAG shown in italics in SEQ ID NO: 99. An example of the nucleotide sequence encoding the hypothetical protein (SEQ ID NO: 98) and a hypothetical protein amino acid sequence (SEQ ID NO: 99) are set forth below. SEQ ID NO: 98
Figure imgf000171_0003
SEQ ID NO: 99
MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGD STPFSVALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGT LVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPIPiAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL jf"11 C lrf^l,lfit£ϊiii;ian'aiiϊMώl'acϊdτriblif indicative of a cell wall anchor: SEQ ID NO: 185
LPLAG (shown in italics in SEQ ID NO: 99, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 82 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in Orf 82. The pilin motif sequence is underlined in SEQ ID NO: 99, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 173 and 188. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of Orf 82 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 99 MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGD STPFSVALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGT LVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
An E box containing a conserved glutamic residue has been identified in Orf 82. The E-box motif is underlined in SEQ ID NO: 99, below. The conserved glutamic acid (E), at amino acid residue 163, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of Orf 82. Preferred fragments of Orf 82 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. SEQ ID NO: 99 MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGD STPFSVALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGT LVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
Orf 83 is thought to to be a multiple sugar metabolism regulator protein. An example of a nucleotide sequence encoding the sugar metabolism regulator protein (SEQ ID NO: 100) and a sugar metabolism regulator protein amino acid sequence (SEQ ID NO: 101) are set forth below. SEQ DD NO: 100
Figure imgf000172_0001
TTTGAAGAGTCCTTTATGATTTTTCCTCTTTGTCACTACATTATTGCCATTGGACCTTTCTATCCTTATTCACTT AATAAAGACTATCAGGAACAATTAGCTAATAATTTTTTAAAACATTCTTCTCATCGTAGCAAAGAAGAGCTCTTG TCCTATATGGCACTTGTCCCACATTTTCCAATTAATAATGTGCGGAACCTTTTGATAGCTATTGACGCTTTTTTT
CATATTATGGATCTGGTAAAGCTAGGCAATCCACAATTGCTCAAGCAAGAAATCAATCGCATCCCCTTATCAAGT ATCACCTCATCTTCTATTTCTGCTCTAAGGGCGGAAAAGAACCTCACTGTTATCTATTTAACTAGGTTACTGGΆA TTCAGTTTTGTAGAAAATACTGACGTAGCAAAGCATTATAGCCTTGTCAAATACTACATGGCCTTAAATGAAGAA GCGAGTGACTTGCTCAAAGTTTTGAGAATTCGCTGTGCAGCTATCATCCATTTTTCCGAATCATTAACCAATAAA AGTATTTCTGATAAACGTCAAATGTACAATAGTGTGCTTCATTATGTCGATAGTCACCTGTATTCCAAATTAAAG GAτfcdkϊAWQCTyk®fc<5c(-i¥ft'!['-if OT^T'CCGAATCTCΆCTTACGTTCAGTCTTTAAAAAATACTCAAATGTT TCCTTACAACATTΆTATTCTAAGTACAAAAATCAAAGAAGCTCAACTACTCTTAAAACGAGGAATTCCTGTTGGA GAAGTGGCTAAAAGCTTATATTTTTATGACACTACCCATTTTCATAAAATCTTTAAAAAATACACGGGTATTTCT TCAAAAGACTATCTTGCTAAATACCGAGATAATATT
SEQ ID NO: 101
MIQLRMGAIYQMVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAIPQAAQDVIFYEGL FEESFMIFPLCHYIIAIGPFYPYSLNKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFF DTQFETTCQQTIHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRIPLSS
SISDKRQMYNSVLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVG EVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI
Orf 84 is thought to to be a F2-like fibronectin-binding protein. An example of a nucleotide sequence encoding the F2-like fibronectin-binding protein (SEQ ID NO: 102) and a F2-like fibronectin-binding protein amino acid sequence (SEQ ID NO: 103) are set forth below. SEQ ID NO: 102
Figure imgf000173_0001
TATGCCTCTAAGTATACAAGTAATAGGAGAGGAGATACTAGTGGTAATCTTAAAAAGCAAATTGCTAAGGTTCTG GATGCAATTTGGTATTTTACAGAAACGACAGTTCCGGCTGATAGAAGTTATACGAATCGCAACGTAAATAGTCAA
TTTGTGCCACAAGATACAAACTTACAGGCAGTAATTAGTGTAGAGCCTGTTATCGAAAGCCTTCCTTGGACATCG
Figure imgf000173_0002
AAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGA GCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCT
GACGAGCAGGGCCATTCTGGCTCAACTACTGAAATAGAAGATAGCAAGTCTTCAGACGTTATCATTGGTGGTCAG GGGCAGATTGTCGAGACAACAGAGGATACCCAAACTGGCATGCACGGGGATTCTGGTTGTAAAACGGAAGTCGAA GATACTAAACTAGTACAATCCTTCCACTTTGΆTAACAAGGAATCAGAAAGTAΆCTCTGAGATTCCTAAAAAAGAT AAGCCAAAGAGTAATACTAGTTTACCAGCAACTGGTGAGAAGCAACATAATATGTTCTTTTGGATGGTTACTTCT TGCTCACTTATTAGTAGTGTTTTTGTAATATCACTAAAAACTAAAAAACGCCTATCATCATGT
SEQ ID NO: 103
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGE HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHI DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEI ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKDKPKSNTSIPAI7GEKQHNMFFWMVTS CSLISSVFVISLKTKKRLSSC
Figure imgf000174_0001
181 LPATG (shown in italics in SEQ ID NO: 103, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 84 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in Orf 84. The pilin motif sequence is underlined in SEQ ID NO: 103, below. A conserved lysine (K) residue is also marked in bold, at amino acid residue 270. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of Orf 84 include the conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ BD NO: 103 MTQKNSYKLSFLLΞLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGE HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHI DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEI ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTS CSLISSVFVISLKTKKRLSSC An E box containing a conserved glutamic residue has been identified in Orf 84. The E-box motif is underlined in SEQ ID NO: 103, below. The conserved glutamic acid (E), at amino acid residue 516, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of Orf 84. Preferred fragments of Orf 84 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 103
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGE HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYI YSVKEVDKNGELLEPKDYIKKE DGLTVTNTYVKPTΞGHYDIEVTFGNGHI DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTIΞTWISDGQVKDFYLMPGKYTFVETAAP DGYEI ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GQIVETTEDTQTGMHGDSGCKTEVEDTKL VQSFHFDNKESESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTS CSLISSVFVISLKTKKRLSSC
Examples of GAS AI-3 sequences from Ml 8 strain isolate MGAS 8232 are set forth below. SρyM18_0125 is a negative transcriptional regulator (Nra). An example of SpyM18_0125 is set forth in SEQ ID NO: 72.
SEQ EO NO: 72 MP'WKKKKDSFwirtl.E^si^BK^El-VfflL'FKSPTIIFSHVAKQTGLTAVQLKYyCKELDDFFGNNLDITIKKG
KI ICCFVKPVKE FYLHQLYDTSTILKLLVFFIKNGTTSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALS SpyM18_0126 is thought to be a collagen binding protein (CBP). An example of
SpyM18_0126 is set forth in SEQ ID NO: 73. SEQ ro NO: 73
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSTETKKTSVIIRKYAEGDYSKLLEGA TLKLAQIEGSGFQEQSFESSTSGQKLQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKE VAEPYSVTAYNDFDDSGFINPKTFTPYGKFYYAKNANGTSQVVYCFNVDLHSPPDSLDKGETIDPDFNEGKEIKY THILGADLFSYANNPRASTNDELLSQVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHG FGALTTEALNATKEIVAYAEDRANLPNISNLDFYVPNSNKYQSLIGTQYHPESLVDIIRMEDKQAPIIPITHKLT ISKTVTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETG
SpyMl 8_0126 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:
184 VPPTG (shown in italics in SEQ ID NO: 73, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SρyM18_0126 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM 18_0126. The pilin motif sequence is underlined in SEQ ID NO: 73 , below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 172 and 179. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyMl 8_0126 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 73
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSTETKKTSVIIRKYAEGDYSKLLEGA TLKLAQIEGSGFQEQSFESSTSGQKLQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKE VAEPYSVTAYNDFDDSGFINPKTFTPYGKFYYAKNANGTSQVVYCFNVDLHSPPDSLDKGETIDPDFNEGKEIKY THILGADLFSYANNPRASTNDELLSQVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHG FGALTTEALNATKEIVAYAEDRANLPNISNLDFYVPNSNKYQSLIGTQYHPESLVDIIRMEDKQAPIIPITHKLT ISKTVTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETG
Three E boxes containing conserved glutamic residues have been identified in SpyM 18 0126. The E-box motifs are underlined in SEQ ID NO: 73, below. The conserved glutamic acid (E) residues, at amino acid residues 112, 257, and 415, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of SpyMl 8_0126. Preferred fragments of SpyM18_0126 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 73
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSTETKKTSVIIRKYAEGDYSKLLEGA TLKLAQIEGSGFQEQSFESSTSGQKLQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKE VA&lsvfft^JdyibSItliN^liiS'piilKM^AKNANGTSQVVYCFNVDLHSPPDSLDKGETIDPDFNEGKEIKY THILGADLFSYANNPRASTNDELLSQVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDHLADYHG FGALTTEALNATKEIVAYAEDRANLPNISNLDFYVPNSNKYQSLIGTQYHPESLVDI IRMEDKQAPII PITHKLT I SKTVTGTIADKKKEFNFEIHLKSS DGQAI SGTYPTNSGEI1TVTDGKATFTLKDGESLIVEGLPSGYSYEITETG ASDYEVSVNGKNAPDGKATKASVKEDETITFENRKDLVPPTGLTTDGAIYLWLLLLVLLGLWVWLIGRKGLKND
SpyM18_0127 is a LepA protein. An example of SpyM18_0127 is shown in SEQ ID NO: 74.
SEQ ID NO: 74 MTNYLNRLNENPLFKAFIRLVLKISIIGFLGYILFQYIFGVMIINTNVMSPALSAGDGILYYRLTDRYHINDVVV YEVDNTLKVGRIVAQAGDEVSFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR YYGALPINQIKGKISTLLRVRGI
SpyM18_0128 is thought to be a fimbria! protein. An example of SypM18_0128 is shown in SEQ ID NO: 75.
SEQ ID NO: 75
MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLVVKKTFPSYTDDKVLMPKADYTFKVEADDNAKGKTK DGLDIKPGVIDGLENTKTIHYGNSDKTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVY VVNREDGGFEAKYIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGEHQRSFSFTLLLTPNECFEKGQVVNI LQGGETKKVVIGEEYSFTLKDKESVTLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKST DEIVVTNKRDTQVPΓGVVGTLAPFAVLSIVAIGGVIYITKRKKA
SpyM18_0128 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO: 75, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM18_0128 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SρyM18_0128. The pilin motif sequence is underlined in SEQ ID NO: 75, below. A conserved lysine (K) residue is also marked in bold, at amino acid residue 57. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyM18_0128 include the conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO: 75
MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLVVKKTFPSYTDDKVLMPKADYTFKVEADDNAKGKTK DGLDIKPGVIDGLENTKTIHYGNSDKTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVY VVNREDGGFEAKYIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGEHQRSFSFTLLLTPNECFEKGQVVNI LQGGETKKVVIGEEYSFTLKDKESVTLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKST DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
An E box containing a conserved glutamic residue has been identified in SpyM18_0128. The E-box motif is underlined in SEQ ID NO: 75, below. The conserved glutamic acid (E), at amino acid residue 266, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of SρyM18_0128. P JMredifrέyLiS^tS^yMΪi^δiii^Mlide the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 75
MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLVVKKTFPSYTDDKVLMPKADYTFKVEADDNAKGKTK DGLDIKPGVIDGLENTKTIHYGNSDKTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVY VVNREDGGFEAKYIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGEHQRSFSFTLLLTPNECFEKGQVVNI LQGGETKKVVIGEEYSFTLKDKESVTLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKST DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
SpyM18_0129 is a SrtC2 type sortase. Anexample ofSρyM18_0129 is shown in SEQ ID NO: 76 SEQ ID NO: 76
MISQRMMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVI GWLNIPGTHMDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKK DFFNKHNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVA FSTCENFSTDNRVIVVGTIQE
SpyM18_0130 is referred to as a hypothetical protein. An example of SpyM18_0130 is shown in SEQ ID NO: 77. SEQ DD NO: 77
MRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSFSVALESIDAMKTIDEITIAGSGKAS FSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPI PPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL SpyM18_0130 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:
185 LPLAG (shown in italics in SEQ ID NO: 77, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM18_0130 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM18_0130. The pilin motif sequence is underlined in SEQ ID NO: 77, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 144, 159, and 169. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyM18_0130 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 77 MRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSFSVALESIDAMKTIDEITIAGSGKAS FSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPI PPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
An E box containing a conserved glutamic residue has been identified in SpyM18_0130. The E-box motif is underlined in SEQ ID NO: 77, below. The conserved glutamic acid (E), at amino acid residue 134, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thSiiJk't $> be SiApMay-firf'tϊϊ&fti'πiatiSti'iif oligomeric pilus-like structures of SpyM18_0130. Preferred fragments of SpyM18_O13O include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 77
MRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTS I S VENVLERAGDSTS FSVALES I DAMKT I DE ITIAGSGKAS FSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVL VYVTYDEDGTLVAKVΪSRRAGDEEKSAITFKPKRLVKPI PPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
, SpyM18_0131 is referred to as a putative multiple sugar metabolism regulator. An example of SpyM18_0131 is set forth in SEQ ID NO: 78.
SEQ ED NO: 78
MAIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAIPQAAQDVIFYEGLFEESFMIFPLC HYIIAIGPFYPYSLNKDYQEQLANNCLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT IHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRIPLSSITSSSISALRA EKNLTVIYLTRLLEFSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS VLHYVDSHLYSKLKVΞDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDT THFHKIFKKYTGISSKDYLAKYRDNI
SpyM 18_0132 is a F2 like fibronectic-binding protein. An example of SpyM 18_0132 is set forth in SEQ ID NO: 79.
SEQ ID NO: 79
MTQKNSYKLΞFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGE HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHI DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEI ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GQivETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKDKPKSNTSLPΛΓGEKQHNMFFWMVTS
CSLISSVFVISLKTKKRLSSC
SpyM18_0132 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ ID NO: 79, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM 18_0132 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM18_0132. The pilin motif sequence is underlined in SEQ ID NO: 79, below. A conserved lysine (K) residue is also marked in bold, at amino acid residue 270. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SρyM18_0132 include the conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 79 HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL
FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHI
DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEI
ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTS CSLISSVFVISLKTKKRLSSC
An E box containing a conserved glutamic residue has been identified in SpyM18_0132. The E-box motif is underlined in SEQ ID NO: 79, below. The conserved glutamic acid (E), at amino acid residue 516, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of SρyM18_0132. Preferred fragments of SpyM18_0132 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 79
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGE HKLEIKRVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDL FVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHI DITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEI ATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTS CSLISSVFVISLKTKKRLSSC
Examples of GAS AI-3 sequences from M49 strain isolate 591 are set forth below. SpyoM01000156 is a negative transcriptional regulator (Nra). An example of
SpyoM01000156 is set forth in SEQ ID NO: 243. SEQ ID NO: 243
MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDI TIKKGKIICCFVKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKL LREFGLRVSKNTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFY NMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYDCLTRSSRQVIENAFSLTFSQGDLDYLFLIYITTNNSF ASLQWTPQHIETCCHIFEKNDTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFS LPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNKQPALTVVLISSNFINAKLLT DTIPRYFSDKGIHFYSFYLLRDDIYQIPSLKPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASI QNLIYQLKDKKYQDFLNEQLQ
SpyoMO 1000155 is thought to be a collagen binding protein (CPA). An example of SpyoM01000155 is set forth in SEQ ID NO: 244. SEQ ID NO: 244
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNRQSSIQDYPWYGYDSYP KGYPDYSPLKTYHNLKVNLEGSKDYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDG QLQQNILRILYNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSNGINDQQLGLM RKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKY AEGDYSKLLEGATLKLSQIEGSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK VFIVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKNKDKSSQVVYCFNADLHSPPD SYDSGETINPDTSTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQF RAATQLAIYYFTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTNLDFFVPNNSK YQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLK TNiQfevlKDjfiysJltlMG'έ'TiilRϊ'EiitficSϊlsyTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT FENRKDL VTPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND
SpyoMO 1000155 contains an amino acid motif indicative of a cell wall anchor: SEQ ID
NO: 184 VPPTG (shown in italics in SEQ ID NO: 244, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000155 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in SpyoM01000155. The pilin motif sequence is underlined in SEQ ID NO: 244, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 71 and 261. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyoMO 1000155 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO: 244
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNRQSSIQDYPWYGYDSYP KGYPDYSPLKTYHNLKVNLEGSKDYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDG QLQQNILRILYNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSNGINDQQLGLM RKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKY AEGDYSKLLEGATLKLSQIEGSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK VFIVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKNKDKSSQVVYCFNADLHSPPD SYDSGETINPDTSTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQF RAATQLAIYYFTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTNLDFFVPNNSK YQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLK TNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDK-KVT FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND
Two E boxes containing conserved glutamic residues have been identified in SpyoM01000155. The E-box motifs are underlined in SEQ ID NO: 244, below. The conserved glutamic acid (E) residues, at amino acid residues 329 and 668, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of SpyoMO 1000155. Preferred fragments of SpyoMO 1000155 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 244
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNRQSSIQDYPWYGYDSYP KGYPDYSPLKTYHNLKVNLEGSKDYQAYCFNLTKHFPΞKSDSVRSQWYKKLEGTNENFIKLADKPRIEDG QLQQNILRILYNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSNGINDQQLGLM RKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKY AEGDYSKLLEGATLKLSQIEGSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK VFIVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKNKDKSSQVVYCFNADLHSPPD SYDSGETINPDTSTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQF RmTteAtY^OTDS^MCTLKTraW'GffiGYlHlG'HiESiyiDEKTLAVTKELITYAQNGSAPQLTNLDFFVPNNSK
YQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLK TNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND
SpyoMO1000154 is a LepAprotein. An example ofSpyoMO1000154 is shown inSEQ ID
NO: 245.
SEQ ID NO: 245
MTNYLNRLNENSLFKAFIRLVLKISIIGFLGY.ILFQYVFGVMIINTNDMSPALSAGDGVLYYRLADRSHI NDVVVYEVDNTLKVGRIAAQAGDEVNFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILN DYREERLDSRYYGALPINQIKGKISTLLRVRGI
SpyoM01000153 is thought to be a fimbrial protein. An example of SpyoMO 1000153 is shown in SEQ ID NO: 246. SEQ ID NO: 246
MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLVVKKTFPSYTDDNVLMPKADYSFKVEADDNA
DSQQWTVDVYVVNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGEHQRLFSFTLL LTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESVTLSQLPVGIEYKLTEEDVTKDGYKTSATLK DGEQSSTYELGKDHKTDKSADEIVVTNKRDTQVTTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
SpyoMO 1000153 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO: 246, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000153 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyoM01000153. The pilin motif sequence is underlined in SEQ ID NO: 246, below. A conserved lysine (K) residue is also marked in bold, at amino acid residue 57. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SpyoMO 1000153 include the conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 246
MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLVVKKTFPSYTDDNVLMPKADYSFKVEADDNA KGKTKDGLDIKPGVIDGLENTKTIRYSNSDKITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITY DSQQWTVDVYVVNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGEHQRLFSFTLL LTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESVTLSQLPVGIEYKLTEEDVTKDGYKTSATLK DGEQSSTYELGKDHKTDKSADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
An E box containing a conserved glutamic residue has been identified in SρyoM01000153. The E-box motif is underlined in SEQ ID NO: 246, below. The conserved glutamic acid (E), at amino acid residue 265, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of SpyoMO 1000153. Preferred fragments of SpyoMO 1000153 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. si^ibMWOBy'≡'?≡39
MKKNKLLLATAiLATALGMASMSQNIKAETAGVIDGSTLVVKKTFPSYTDDNVLMPKΆDYSFKVEADDNA
KGKTKDGLDIKPGVIDGLENTKTIRYSNSDKITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITY
DSQQWTVDVYVVNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGEHQRLFSFTLL LTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESVTLSQLPVGIEYKL TEEDVTKDGYKTSATLK
DGEQSSTYELGKDHKTDKSADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
SpyoM01000152 is a SrtC2 type sortase. An example of SpyoMO 1000152 is shown in SEQ ID NO: 247 SEQ ID NO: 247
MMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVI GWLNIPGTHIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIP KFLKKNFFNKHNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYISKRSKQFKPV KLKHHTKFVAFSTCENFSTDNRVIVVGTIQE
SpyoMO 1000151 is referred to as a hypothetical protein. An example of SpyoMO 1000151 is shown in SEQ ID NO: 248.
SEQ ED NO: 248
MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASF SPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRL VKPIPPRQPDIPKTPLPLΛGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
SpyoM01000151 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID NO: 248, above). In some recombinant host cell systems, it maybe preferable to remove this motif to facilitate secretion of a recombinant SpyoMO 1000151 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyoMO 1000151. The pilin motif sequence is underlined in SEQ ID NO: 248, below. Conserved lysine (K) residues are also marked in bold, at amino acid residue 138. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of SρyoM01000151 include the conserved lysine residue. Preferably, fragments include the pilin sequence.
SEQ ID NO: 248
MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASF SPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRL VKPIPPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
Two E boxes containing conserved glutamic residues have been identified in SρyoM01000151. The E-box motifs are underlined in SEQ ID NO: 248, below. The conserved glutamic acid (E) residues, at amino acid residues 58 arid 128, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of olIgόtaeϊc^πy4ϊkSllicώiIøϊSpySy:il000151. Preferred fragments of SpyoM01000151 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 248 MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIΆGSGRASF SPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRL VKPIPPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
SpyoM01000150 is referred to as aputative MsmRL. Anexample ofSpyoMO1000150 is set forthinSEQ IDNO: 249. SEQ ID NO: 249
MVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAIPQAAQDVIFYEGLFEESFM IFPLCHYIIAIGPFYPYSLNKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFD TQFETTCQQTIHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRI
HFSESLTNKSISDKRQMYNSVLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKI KEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI
SpyoMO 1000149 is a F2 like fibronectin-binding protein. An example of SpyoMO 1000149 is set forth in SEQ ID NO: 250.
SEQ ID NO: 250
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFEIKKVDQNNKPLSGATFSLTP KDGKGKPVQTFTSSEEGIIDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIIS KAGSKDVSSΞLQLENPKMSVVSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLD RRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTFTNYIAGLDKVQLSAELSLFLENKEVLENTN ISDFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDISRLNLRKDLEAKLPQGSTQGA NKRLRIDFGENLQGKAFVVKVTGKADQSGKELIVQSHLSSFNNWGSYKTLRPNSHVSFTNEIALSPSKGS GSGTSEFTKPAITVANLKR'VAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEIHFKDLTSG TYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKPHSFVKVEANKEVTIVNHKETLTFSGKKIWE NDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL GNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYAD GVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGP NLEIEETLPLESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATM ELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIV MVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKPSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTE IEDSKSSDVIIGGQGQVVETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFDNKEPESNSEIPKKDKPKSNT SIPΛTGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLLSC SpyoMO 1000149 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:
180 LPATG (shown in italics in SEQ ID NO: 250, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000149 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in SρyoM01000149. The pilin motif sequences are underlined in SEQ ID NO: 250, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 157 and 163, and 216 and 224. The pilin sequences, in particular the conserved lysine residues, are thought to be important fdFMθ^Ji^ailidyrrg^iQyibtriyϊi^'ϊiisαiM'Ifetructures. Preferred fragments of SpyoMO 1000149 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.
SEQ ID NO: 250 MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFEIKKVDQNNKPLSGATFSLTP KDGKGKPVQTFTSSEEGIIDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIIS KAGSKDVSSSLQLENPKMSVVSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLD RRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTFTNYIAGLDKVQLSAELSLFLENKEVLENTN ISDFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDISRLNLRKDLEAKLPQGSTQGA NKRLRIDFGENLQGKAFVVKVTGKADQSGKELIVQSHLSSFNNWGSYKTLRPNSHVSFTNEIALSPSKGS GSGTSEFTKPAITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEIHFKDLTSG TYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKPHSFVKVEANKEVTIVNHKETLTFSGKKIWE NDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL GNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYAD GVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGP NLEIEETLPLESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATM ELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIV MVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKPSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTE IEDSKSSDVIIGGQGQVVETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFDNKEPESNSEIPKKDKPKSNT SLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLLSC
Two E boxes containing conserved glutamic residues have been identified in SpyoMO 1000149. The E-box motifs are underlined in SEQ ID NO: 250, below. The conserved glutamic acid (E) residues, at amino acid residues 329 and 668, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of SpyoM01000149. Preferred fragments of SpyoM01000149 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.
SEQ ID NO: 250 MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFEIKKVDQNNKPLSGATFSLTP KDGKGKPVQTFTSSEEGIIDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIIS KAGSKDVSSSLQLENPKMSVVSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLD RRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTFTNYIAGLDKVQLSAELSLFLENKEVLENTN ISDFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDISRLNLRKDLEAKLPQGSTQGA NKRLRIDFGENLQGKAFVVKVTGKADQSGKELIVQSHLSSFNNWGSYKTLRPNSHVSFTNEIALSPSKGS GSGTSEFTKPAITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEIHFKDLTSG TYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKPHSFVKVEANKEVTIVNHKETLTFSGKKIWE NDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL GNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYAD GVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGP NLEIEETLPLESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATM ELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIV MVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKPSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTE IEDSKSSDVIIGGQGQVVETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFDNKEPESNSEIPKKDKPKSNT SLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLLSC
As discussed above, applicants have also determined the nucleotide and encoded amino acid sequence of fimbrial structural subunits in several other GAS AI-3 strains of bacteria. Examples of sequences of these fimbrial structural subunits are set forth below.
M3 strain isolate ISS 3040 is a GAS AI-3 strain of bacteria. ISS3040_fimbrial is thought to be a fimbrial structural subunit of M3 strain isolate ISS 3040. An example of a nucleotide sequence eiiE'oIini'thέΑfeiiϋliiUSriitHb'rfiprXritslQIDNO: 263)andanISS3040Jimbrialproteinamino acid sequence (SEQ ID NO: 264) are set forth below. SEQ ID NO: 263 gagacggcaggagtgtccgaaaatgcaaaattaatagtaaaaaagacatttgactcttat acagacaatgaagttttaatgccaaaagctgattatacttttaaagtagaggcagatagt acagctagtggcaaaacgaaagacggtttagagattaagccaggtattgttaatggttta acagaacagattatcagctatactaatactgataaaccagatagtaaagttaaaagtaca gagtttgatttttcaaaagtagtattccctggtattggtgtttaccgctatactgtttca gaaaaacaaggtgatgttgaaggaattacctacgatactaagaagtggacagtagatgtt tatgttggaaacaaagaaggtggtggttttgaacctaagtttattgtatctaaggaacaa ggaacagacgtcaaaaaaccagttaattttaacaactcgtttgcaactacttcgttaaaa gttaagaagaatgtatcggggaatactggagaattgcaaaaagaatttgactttacattg acgcttaatgaaagcacgaattttaaaaaagatcaaattgtttctttacaaaaaggaaac gagaaatttgaagttaagattggtactccctacaagtttaaactcaaaaatggggaatct attcaactagacaagttaccagttggtattacttataaagtcaatgaaatggaagctaat aaagatgggtataaaacaacagcatccttgaaagagggagatggtcaatctaaaatgtat caattggatatggaacaaaaaacagacgaatctgctgacgaaatcgttgtcacaaataag cgtgacactcaagttccaactggtgttgtaggcacccttgctccatttgcagttcttagc
SEQ ID NO: 264 ETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASG
KTKDGLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRYTVSEK QGDVEGITYDTKKWTVDVYVGNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLK VKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKNG ESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESADEIV VTNKRDTQVPTGVVGTLAPFAVLS
M44 strain isolate ISS 3776 is a GAS AI-3 strain of bacteria. ISS3776_fimbrial is thought to be a fimbrial structural subunit of M44 isolate ISS 3776. An example of a nucleotide sequence encoding the ISS3776_fimbrial protein (SEQ ID NO: 253) and an ISS3776_fimbrial protein amino acid sequence (SEQ ID NO: 254) are set forth below. SEQ ID NO: 253 ttggagagagaaaaaatgaaaaaaaacaaattattacttgctactgcaatcttagcaact gctttaggaacagcttctttaaatcaaaacgtaaaagctgagacggcaggggttgtaaca ggaaaatcactacaagttacaaagacaatgacttatgatgatgaagaggtgttaatgccc gaaaccgcctttacttttactatagagcctgatatgactgcaagtggaaaagaaggcagc ctagatattaaaaatggaattgtagaaggcttagacaaacaagtaacagtaaaatataag aatacagataaaacatctcaaaaaactaaaatagcacaatttgatttttctaaggttaaa tttccagctataggtgtttaccgctatatggtttcagagaaaaacgataaaaaagacgga attacgtacgatgataaaaagtggactgtagatgtttatgttgggaataaggccaataac gaagaaggtttcgaagttctatatattgtatcaaaagaaggtacttctagtactaaaaaa ccaattgaatttacaaactctattaaaactacttccttaaaaattgaaaaacaaataact ggcaatgcaggagatcgtaaaaaatcattcaacttcacattaacattacaaccaagtgaa tattataaaactggatcagttgtgaaaatcgaacaggatggaagtaaaaaagatgtgacg ataggaacgccttacaaatttactttgggacacggtaagagtgtcatgttatcgaaatta ccaattggtatcaattactatcttagtgaagacgaagcgaataaagacggctacactaca acggcaacattaaaagaacaaggcaaagaaaagagttccgatttcactttgagtactcaa aaccagaaaacagacgaatctgctgacgaaatcgttgtcacaaataagcgtgacactcaa gttccaactggtgttgtagggacccttgctccatttgcagttcttagcattgtggctatt ggtggagttatctatattacaaaacgtaaaaaagcttaa
SEQ ID NO: 254 MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVVTGKSL
QVTKTMTYDDEEVLMPETAFTFTIEPDMTASGKEGSLDIKNGIVEGLDKQVTVKYKNT DKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTVDVYVGNKΆNN EEGFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQITGNAGDRKKSFNFTLTLQP SEYYKTGSVVKIEQDGSKKDVTIGTPYKFTLGHGKSVMLSKLPIGINYYLSEDEANKD G^itiEkllLKiy^iilisiEFrLBoIdiirinlESlLDEiVVTNKRDTOVPTGVVGTLAPFAV
LSIVAIGGVI YITKRKKA
M77 strain isolate ISS4959 is a GAS AI-3 strain of bacteria. ISS4959_fimbrial is thought to be a fimbrial structural subunit of M77 strain ISS 4959. An example of a nucleotide sequence encoding the ISS4959_fimbrial protein (SEQ ID NO: 271) and an ISS4959_fimbrial protein amino acid sequence (SEQ ID NO: 272) are set forth below. SEQ ID NO: 271 gtaacagtaaaatataagaatacagataaaacatctcaaaaaactaaaatagcacaattt gatttttctaaggttaaatttccagctataggtgtttaccgctatatggtttcagagaaa aacgataaaaaagacggaattacgtacgatgataaaaagtggacngtagatgtttatgtt gggaataaggccaataacgaagaaggtttcgaagttctatatattgtatcaaaagaaggt acttctagtnctaaaaaaccaattgaatttacaaactctattaaaactacttccttaaaa attgaaaaacaaataactggcaatgcaggagatcgtaaaaaatcattcaacttcacattn acattacanccaagtgaatattataaaactggatcagttgtgaaaatcgaacaggatgga agtaaaaaagatgtgacgataggaacgccttacaaatttactttgggacacggtaagagt gtcatgttatcgaaattnccaattggtatcaattactatcttagtgaagacgaagcgaat aaagacggntacactacancggcaacattaaaagaacaaggcaaagaaaagagttccgat ttcactttgagtactcaaaaccagaaaacagacgaatctgctg
SEQ ID NO: 272 VTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKK
DGITYDDKKWTVDVYVGNKANNEEGFEVLYIVSKEGTSSXKKPIEFTNSIKTTSLKIE KQITGNAGDRKKSFNFTXTLXPSEYYKTGSVVKIEQDGSKKDVTIGTPYKFTLGHGKS VMLSKXPIGINYYLSEDEANKDGYTTXATLKEQGKEKSSDFTLSTQNQKTDESA
Examples of GAS AI-4 sequences from M 12 strain isolate A735 are set forth below. 19224133 is thought to be a RofA regulatory protein. An example of a nucleotide sequence encoding the RofA regulatory protein (SEQ ID NO: 104) and a RofA regulatory protein amino acid sequence (SEQ ID NO: 105) are set forth below. SEQ ID NO: 104
Figure imgf000186_0001
TTGAAAAAAAGTAGCCGTGATATTATCGAAACTTACTGCCAACTAAACTTTTCAGCAGGAGATTTGGACTACCTC
CAACTTTTTGAAGAAAATGATACTTTTCGCCTGCTTTTAAATCCTATCATCACTCTTTTACCTAACCTAAAAGAG CAAAAGGCTAGTTTAGTAAAAGCTCTTATGTTTTTTTCAAAATCATTCTTGTTTAATCTGCAACATTTTATTCCT
GAGTGGATGGCCAAACTTCCTGGTAAGCGTTACTTGAACCATAAGCATTTTCATCTTTTTTGCCACTATGTCGAG
Figure imgf000186_0002
AAAGAGGAAAAATTCCAAGCTGATTTAACCAAACAATTAACATAA
SEQ ID NO: 105
MTIQKRMISCQFTHPSKETYL YQL YAS SNVLQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALIPLLRNF ELKLSKNKIVGEEYRIRYLIALLYSKFGIKVYDLTQQDKNI IHSFLSHSSTHLKTSPWLSESFSFYDILLALSWK
RHQFSVTI PQTRI FQQLKKL FVYDSLKKSSRDI IETYCQLNFSAGDLDYLYLI YITANNS FASLQWTPEHIRQCC
QLFEENDTFRLLLNPIITLLPNLKEQKASLVKALMFFSKS FLFNLQHFIPETNLFVSPYYKGNQKL YTSLKLIVE YQI PDLKPDLVITHSQLIPFVHHELTKGIAVAEISFDESILSIQELMYQVKEEKFQADLTKQLT
19224134 is thought to be a protein F fibronectin binding protein. An example of a nucleotide sequence encoding the protein F fibronectin binding protein (SEQ ID NO: 106) and a protein F fibronectin binding protein amino acid sequence (SEQ ID NO: 107) are set forth below. SEQ ID NO: 106
ATGGTAAGCTCATATATGTTTGCGAGAGGAGAGAAAATGAATAACAΆAATGTTTTTGAACAAAGAAGCCGGTTTT GGTGCTATCGGTTTTGGTCAAGTAGCCTATGCTGCGGATGAGAAGACTGTGCCGAATTTTAAAAGCCCAGATCCA
Figure imgf000187_0001
SEQ ID NO: 107
MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACAGAIGFGQVAYAADEKTVPNFKSPDP DYPWYGYDSYRGIFARYHNLKVNLKGSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDE NLDKLEKNILNVIYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPLNDVNKMWEREVRNGEISESQVTLMR EALKKLIDPNLEATAANKIPSGYRLNIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKR
SLEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETE YITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEP EVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPG VLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPΛTGDIENVLAF LGILILSVLSIFSLLKNKQNNKV
19224134 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 181 LPATG (shown in italics in SEQ ID NO: 107, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224134 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of ώe'yφϊeye&pfbt&in'mϊys'bl1 cleaved during purification or the recombinant protein may¬ be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224134. The pilin motif sequence is underlined in SEQ ID NO: 107, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 275, 285, and 299. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of 19224134 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 107 MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACAGAIGFGQVAYAADEKTVPNFKSPDP DYPWYGYDSYRGIFARYHNLKVNLKGSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDE NLDKLEKNILNVIYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPLNDVNKMWEREVRNGEISESQVTLMR EALKKLIDPNLEATAANKIPSGYRLNIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKR SLEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETE YITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEP EVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPG VLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENVLAF LGILILSVLSIFSLLKNKQNNKV Two E boxes containing conserved glutamic residues have been identified in 19224134. The
, E-box motifs are underlined in SEQ ID NO: 107, below. The conserved glutamic acid (E) residues, at amino acid residues 487 and 524, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to-be important for the formation of oligomeric pilus-like structures of 19224134. Preferred fragments of 19224134 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ID NO: 107
MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACAGAIGFGQVAYAADEKTVPNFKSPDP
DYPWYGYDSYRGIFARYHNLKVNLKGSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDE
NLDKLEKNILNVIYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPLNDVNKMWEREVRNGEISESQVTLMR EALKKLIDPNLEATAANKIPSGYRLNIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKR
SLEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETE YITEVYGNQQNPV-DIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEP EVLMGGQSEΞVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPG VLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENVLAF LGILILSVLSIFSLLKNKQNNKV
19224135 is thought to be a capsular polysaccharide adhesin (Cpa) protein. An example of a nucleotide sequence encoding the Cpa protein (SEQ ID NO: 108) and a Cpa protein amino acid sequence (SEQ ID NO: 109) are set forth below. SEQ ID NO: 108
ACTTTAGTGGGAGTATTTTTAATGTTTTTGACCTTGGTAAGTTCCATGAGAGGTGCTCAAAGCATATTTGGAGAG
GAAAAGAGAATTGAAGAAGTCAGTGTTCCTAAAATAAAAAGTCCAGATGATGCCTACCCTTGGTATGGCTATGAT TCATATGACTCTAGTCATCCTTACTATGAACGTTTTAAAGTAGCACATGATTTAAGGGTTAATTTAAATGGAAGT
AAGAGCTACCAAGTATATTGCTTTAATATCAATTCTCATTATCCGAATAGAAAAAATGCTTTTTCTAAACAATGG TTTAAGAGAGTTGATGGGACAGGTGATGTGTTCACAAATTATGCTCAGACACCTAAGATTCGTGGAGAATCATTG AATAATAAACTTTTAAGTATTATGTACAACGCTTATCCTAAAAATGCTAATGGCTATATGGATΆAGATAGAACCA PMtffaliHIGWSVieTffiSSACMGClriSfTTGGTACTATTCTGACAGTTCTTATGGTAATATAAAAACGTTA TGGGCATCTGAGCTTAAAGACGGAAAAATAGATTTTGAACAAGTAAAATTAATGCGTGAAGCTTACTCAAAACTA ATTAGTGATGATTTAGAAGAAACATCTAAAAATAAGCTACCTCAAGGATCTAAACTGAATATTTTTGTTCCGCAA GATAAATCTGTTCAAAATTTATTAAGTGCAGAGTACGTGCCTGAATCCCCTCCGGCACCAGGTCAGTCTCCAGAA
GGAGCAACTTTGCGTTTAACAGGGGAAGATΆTCCTAGATTTTCAAGAAAAAGTCTTCCAAAGTAATGGAACAGGA
AAAGGACTAAAAAATGACTAA
SEQ ID NO: 109
MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMFLTLVSSMRGAQSIFGEEKRIEEVSVPKIKSPDDAYPWYGYD SYDSSHPYYERFKVAHDLRVNLNGSKSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESL NNKLLSIMYNAYPKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTLWASELKDGKIDFEQVKLMREAYSKL ISDDLEETSKNKLPQGSKLNIFVPQDKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLE GATLRLTGEDILDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKIAEPIKFRVVNKKVFIVQKDGSQVENPN KEVAEPYSVEAYSDMQDSNYINPETFTPYGKFYYAKNKDKSSQVVYCFNADLHSPPESEDGGGTIDPDISTMKEV KYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYNKKGDSYNGLTETQFRAATQLAIYYFTDSTDLKTLKTY NNGKGYHGFESMDEKTLAVTKELINYAQDNSAPQLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVI PVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYS YTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPΓGFITDGGTYLWLLLLVPFGLLVWFFGR
KGLKND
19224135 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID NO: 109, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224135 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224135. The pilin motif sequence is underlined in SEQ ID NO: 109, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 164 and 172. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of 19224135 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 109 SYDSSHPYYERFKVAHDLRVNLNGSKSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESL NNKLLSIMYNAYPKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTLWASELKDGKIDFEQVKLMREAYSKL ISDDLEETSKNKLPQGSKLNIFVPQDKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLE GATLRLTGEDILDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKIAEPIKFRVVNKKVFIVQKDGSQVENPN KEVAEPYSVEAYSDMQDSNYINPETFTPYGKFYYAKNKDKSSQVVYCFNADLHSPPESEDGGGTIDPDISTMKEV KYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYNKKGDSYNGLTETQFRAATQLAIYYFTDSTDLKTLKTY NNGKGYHGFESMDEKTLAVTKELINYAQDNSΆPQLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVI PVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYS YTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGFITDGGTYLWLLLLVPFGLLVWFFGR KGLKND
An E box containing a conserved glutamic residue has been identified in 19224135. The E- box motif is underlined in SEQ ID NO: 109, below. The conserved glutamic acid (E), at amino acid residue 339, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the foπnation of oligomeric pilus-like structures of 19224135. Preferred fragments of 19224135 include the conserved glutamic acid residue. Preferably, fragments include the E box motif. SEQ ED NO: 109 MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMFLTLVSSMRGAQSIFGEEKRIEEVSVPKIKSPDDAYPWYGYD SYDSSHPYYERFKVAHDLRVNLNGSKSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESL NNKLLSIMYNAYPKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTLWASELKDGKIDFEQVKLMREAYSKL ISDDLEETSKNKLPQGSKLNIFVPQDKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLE GATLRLTGEDILDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKIAEPIKFRVVNKKVFIVQKDGSQVENPN KEVAEPYSVEAYSDMQDSNYINPETFTPYGKFYYAKNKDKSSQVVYCFNADLHSPPESEDGGGTIDPDISTMKEV KYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYNKKGDSYNGLTETQFRAATQLAIYYFTDSTDLKTLKTY NNGKGYHGFESMDEKTLAVTKELINYAQDNSAPQLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVI PVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYS YTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGFITDGGTYLWLLLLVPFGLLVWFFGR KGLKND
19224136 is thought to be a LepA protein. An example of a nucleotide sequence encoding the LepA protein (SEQ ID NO: 110) and a LepA protein amino acid sequence (SEQ ID NO: 111) are set forth below. SEQ ID NO: 110
Figure imgf000190_0001
SEQ ID NO: 111 MTNYLNRLNENPLFKAFIRLVLKISIIGFLGYILFQYVFGVMIVNTNQMSPAVSAGDGVLYYRLTDRYHINDVVV YEVDNTLKVGRIAAQAGDEVSFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR YYGALPINQIKGKISTLLRVRGI
19224137 is thought to be a fimbrial protein. An example of a nucleotide sequence encoding the fimbrial protein (SEQ ID NO: 112) and a fimbrial protein amino acid sequence (SEQ ID NO: 113) are set forth below. SEQ ID NO: 112 Al GTAAAAGCTGAGACGGCAGGGGTTGTTAGCAGTGGTCAATTAACAATAAAAAAATCAATTACAAATTTTAATGAT
Figure imgf000191_0001
AGCATTGTGGCTATTGGTGGAGTTATCTATATTACAAAACGTAAAAAAGCTTAA
SEQ ID NO: 113
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTES NLPIKPGIAVNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHKKAFT^TLTLQPNEYYEASSVVKIEE NGQTKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE IVVTNNRDTQVPΓGVVGTLAPFAVLSIVΆIGGVIYITKRKKA
19224137 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO: 113, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224137 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224137. The pilin motif sequence is underlined in SEQ ID NO: 113, below. A conserved lysine (K) residue is also marked in bold, at amino acid residue 160. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of 19224137 include the conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ BD NO: 113
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTES NLPIKPGIAVNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPΞVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHKKAFTFTLTLQPNEYYEASSVVKIEE NGQTKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE IVVTNNRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
An E box containing a conserved glutamic residue has been identified in 19224137. The E- box motif is underlined in SEQ ID NO: 113, below. The conserved glutamic acid (E), at amino acid residue 263, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of 19224137. Preferred fragments of 19224137 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.
SEQ ID NO: 113 NLPlkpGIAVNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHKKAFTFTLTLQPNEYYEASSVVKIEE NGQTKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE IVVTNNRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
19224138 is thought to be a SrtC2-type sortase. An example of a nucleotide sequence encoding the SrtC2 sortase (SEQ ID NO: 114) and a SrtC2 sortase amino acid sequence (SEQ ID NO: 115) are set forth below. SEQ m NO: 114
Figure imgf000192_0001
AATTTTTCTACTGACAATCGTGTTATCGTTGTCGGTACTATTCAAGAATAA SEQ ID NO: 115
MMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNI PGTHIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFNK HNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTCE NFSTDNRVIVVGTIQE
19224139 is an open reading frame that encodes a sortase substrate motif LPXAG shown in italics in SEQ ID NO: 117. An example of a nucleotide sequence of the open reading frame (SEQ ID NO: 116) and the amino acid sequence encoded by the open reading frame (SEQ ID NO: 117) are set forth below. SEQ ID NO: 116
Figure imgf000192_0002
TTACTAGTTCTTCTTTATGTTAAAAAACTGAAGAG
SEQ ID NO: 117
MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKAΞFSPLTF TTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPN IPKTPiPIAGEVKSLLGILSIVLLGLLVLLYVKKLKSKL
19224139 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID NO: 117, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224139 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular
Figure imgf000193_0001
during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.
A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224139. The pilin motif sequence is underlined in SEQ ID NO: 117, below. A conserved lysine (K) residue is also marked in bold, at amino acid residue 138. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of 19224139 include the conserved lysine residue. Preferably, fragments include the pilin sequence. SEQ ID NO: 117 MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERΆGDSTPFSIALESIDAMKTIEEITIAGSGKΆSFSPLTF TTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYPEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPN IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSKL
Two E boxes containing conserved glutamic residues have been identified in 19224139. The E-box motifs are underlined in SEQ ID NO: 117, below. The conserved glutamic acid (E) residues, at amino acid residues 58 and 128, are marked in bold. The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of 19224139. Preferred fragments of 19224139 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif. SEQ ED NO: 117 MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTF TTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPN IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSKL
19224140 is thought to be a MsmRL protein. An example of a nucleotide sequence encoding the MsmRL protein (SEQ ID NO: 118) and a MsmRL protein amino acid sequence (SEQ ID NO: 119) are set forth below. SEQ ID NO: 118
Figure imgf000193_0002
CAATTGCTCAAGCAAGAAATCAATCGCATCCCCTTATCAAGTATCACCTCATCTTCTATTTCTGCTCTAAGGGCG
Figure imgf000193_0003
ATTTAA
SEQ ID NO: 119
MVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAIPQAAQDVIFYEGLFEESFMIFPLC HYIIAIGPFYPYSLNKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT ppvLEHLNHiMDLVKLGNPQLLKQEiNRi PLSSITSSSISΆLRA
EKNLTVIYLTRLLEFSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS VLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDT THFHKIFKKYTGISSKDYLAKYRDNI
19224141 is thought to be a protein F2 fibronectin binding protein. An example of a nucleotide sequence encoding the protein F2 fibronectin binding protein (SEQ ID NO: 120) and a protein F2 fibronectin binding protein amino acid sequence (SEQ ID NO: 121) are set forth below. SEQ ID NO: 120
Figure imgf000194_0001
GGAGAGGTTCACTTTAAGGACCTGACCTCGGGCACATATGACCTGTATGAAACAAAAGCGCCAAAAGGTTATCAG
Figure imgf000194_0002
GGCGGTCAAGGTGAAGTTGTTGACACAACAGAAGACACACAAAGTGGTATGACGGGCCATTCTGGCTCAACTACT GAAATAGAAGATAGCAAGTCTTCAGACGTTATCATTGGTGGTCAGGGGCAGGTTGTCGAGACAACAGAGGATACC
Figure imgf000194_0003
TCACTAAAATCCAAAAAACGCCTATCATCATGTTAA SEQ ID NO: 121
Figure imgf000195_0001
TSVQTFTSNDKGIVDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS LQLENPKMSVVSKYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLDRRLNPKGISQDIPKI IYDSANSPLAIGKYHAENHQLIYTFTDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVN
SΆELGEIQVYEVPEGEKLPSSYGVDVTKLTLRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGK PLVVQSNLASFRGASEYAAFTPVGGNVYFQNEIALSPSKGSGSGKSEFTKPSITVANLKRVAQLRFKKMSTDNVP LPEAAFELRSSNGNSQKLEASSNTQGEVHFKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGS PHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPK YDAKNQEYKYSVEEVNVPDGYKVSYLGNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKI IDEDTLTSFKGKK IWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDI IYSVKEVTVPTGYDVTYSA NDIINTKREVITQQGPKLEIEETLPLESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRD IDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATK GDTHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDLIIGGQGEVVDTTEDTQSGMTGHSGSTT EIEDSKSSDVIIGGQGQVVETTEDTQTGMYGDSGCKTEVEDTKLVQSFHFDNKEPESNSEI PKKDKPKSNTSIPA TGEKQHNMFFWMVTSCSLISSVFVISLKSKKRLSSC
19224141 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO: 181 LPATG (shown in italics in SEQ ID NO: 121, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224141 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition. Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in 19224141. The pilin motif sequences are underlined in SEQ ID NO: 121, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 157 and 163 and at amino acid residues 216, 224, and 238. The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of 19224141 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence. SEQ ED NO: 121
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFEIKKVDQNNKPLPGATFSLTSKDGKG
TSVQTFTSNDKGIVDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS LQLENPKMSVVSKYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLDRRLNPKGISQDIPKI
IYDSANSPLAIGKYHAENHQLIYTFTDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKΞTIGGQEITYKGTVN
SAELGEIQVYEVPEGEKLPSSYGVDVTKLTLRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGK PLVVQSNLASFRGASEYAAFTPVGGNVYFQNEIALSPSKGSGSGKSEFTKPSITVANLKRVAQLRFKKMSTDNVP LPEAAFELRSSNGNSQKLEASSNTQGEVHFKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGS PHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPK YDAKNQEYKYSVEEVNVPDGYKVSYLGNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKK IWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSA NDIINTKREVITQQGPKLEIEETLPLESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRD IDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATK GDTHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDLIIGGQGEVVDTTEDTQSGMTGHSGSTT EIEDSKSSDVIIGGQGQVVETTEDTQTGMYGDSGCKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKPKSNTSLPA TGEKQHNMFFWMVTSCSLISSVFVISLKSKKRLSSC
Two E boxes containing conserved glutamic residues have been identified in 19224141. The E-box motifs are underlined in SEQ ID NO: 121, below. The conserved glutamic acid (E) residues, at aflitilαaiϊd'reyiiSβ®ari'diSKC5e!≤a3:edinbold. The Eboxmotifs, inparticular theconserved glutamicacidresidues, are thought tobe important forthe formationofoligomericpilus-like structures of19224141. Preferredfragments of19224141 includeatleast one conservedglutamic acid residue. Preferably, fragments include at least one E boxmotif. SEQ π>NO: 121
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFEIKΪCVDQNNKPLPGATFSLTSKDGKG TSVQTFTSNDKGIVDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS LQLENPKMSVVSKYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLDRRLNPKGISQDIPKI IYDSANSPLAIGKYHAENHQLIYTFTDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVN
SAELGEIQVYEVPEGEKLPSSYGVDVTKLTLRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGK PLVVQSNLASFRGASEYAAFTPVGGNVYFQNEIALSPSKGSGSGKSEFTKPSITVANLKRVAQLRFKKMSTDNVP LPEAAFELRSSNGNSQKLEASSNTQGEVHFKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGS PHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPK YDAKNQEYKYSVEEVNVPDGYKVSYLGNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKK IWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSΆ NDIINTKREVITQQGPKLEIEETLPLEΞGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRD IDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATK GDTHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDLIIGGQGEVVDTTEDTQSGMTGHSGSTT EIEDSKSSDVIIGGQGQVVETTEDTQTGMYGDSGCKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKPKSN TSLPATGEKQHNMFFWMVTSCSLISSVFVISLKSKKRLSSC
As discussed above, applicants have also determined the nucleotide and encoded amino acid sequence of fimbrial structural subunits in several other GAS AI-4 strains of bacteria. Examples of sequences of these fimbrial structural subunits are set forth below. M12 strain isolate 20010296 is a GAS AI-4 strain of bacteria. 20010296_fimbrial is thought to be a fimbrial structural subunit of M12 strain isolate 20010296. An example of a nucleotide sequence encoding the 20010296_fimbrial protein (SEQ ID NO: 257) and a 20010296_fimbrial protein amino acid sequence (SEQ ID NO: 258) are set forth below. SEQ ID NO: 257 agcagtggtcaattaacaataaaaaaatcaattacaaattttaatgatgatacacttttg atgcctaagacagactatacttttagcgttaatccggatagtgcggctacaggtactgaa agtaatttaccaattaaaccaggtattgctgttaacaatcaagatattaaggtttcttat tctaatactgataagacatcaggtaaagaaaaacaagttgttgttgactttatgaaagtt acttttcctagcgttggtatttaccgttatgttgttaccgagaataaagggacagcagaa ggagttacatatgatgatacaaaatggttagttgacgtctatgttggtaataatgaaaag ggaggtcttgaaccaaagtatattgtatctaaaaaaggagattctgctactaaagaacca atccagtttaataattcattcgaaacaacgtcattaaaaattgaaaaggaagttactggt aatacaggagatcataaaaaagcatttaactttacattaacattgcaaccaaatgaatac tatgaggcaagttcggttgtgaaaattgaagagaacggacaaacgaaagatgtgaaaatt ggggaggcatataagtttactttgaacgatagtcagagtgtgatattgtctaaattacca gttggtattaattataaagttgaagaagcagaagctaatcaaggtggatatactacaaca gcaactttaaaagatggagaaaagttatctacttataacttaggtcaggaacataaaaca gacaagactgctgatgaaatcgt
SEQ ID NO: 258 SSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLP
IKPGIAVNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGV
TYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTG NTGDHKKAFNFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLNDSQSVILSK LPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIV P C '"UήUSiBόM&iMlioBBHbAS AI-4 strain ofbacteria.20020069_fϊmbrial is thought to beafimbrial structural subunit ofM12 strainisolate 20020069. Anexample ofanucleotide sequenceencoding the 20020069_fimbrialprotein (SEQ IDNO: 259) and a 20020069_fimbrial protein amino acidsequence (SEQ IDNO: 260) are set forthbelow. SEQ ID NO: 259 agcagtggtcaattaacaataaaaaaatcaattacaaattttaatgatgatacacttttg atgcctaagacagactatacttttagcgttaatccggatagtgcggctacaggtactgaa agtaatttaccaattaaaccaggtattgctgttaacaatcaagatattaaggtttcttat tctaatactgataagacatcaggtaaagaaaaacaagttgttgttgactttatgaaagtt acttttcctagcgttggtatttaccgttatgttgttaccgagaataaagggacagcagaa ggagttacatatgatgatacaaaatggttagttgacgtctatgttggtaataatgaaaag ggaggtcttgaaccaaagtatattgtatctaaaaaaggagattctgctactaaagaacca atccagtttaataattcattcgaaacaacgtcattaaaaattgaaaaggaagttactggt aatacaggagatcataaaaaagcatttaactttacattaacattgcaaccaaatgaatac tatgaggcaagttcggttgtgaaaattgaagagaacggacaaacgaaagatgtgaaaatt ggggaggcatataagtttactttgaacgatagtcagagtgtgatattgtctaaattacca gttggtattaattataaagttgaagaagcagaagctaatcaaggtggatatactacaaca gcaactttaaaagatggagaaaagttatctacttataacttaggtcaggaacataaaaca gacaagactgctgatgaaatcgt SEQ ID NO: 260
SSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLP IKPGIAVNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGV TYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTG NTGDHKKAFNFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLNDSQSVILSK LPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIV
M12 strain isolate CDC SS 635 is a GAS AI-4 strain of bacteria. CDC SS 635_fimbrial is thought to be a fimbrial structural subunit of M12 strain isolate CDC SS 635. An example of a nucleotide sequence encoding the CDC SS 635_fimbrial protein (SEQ ID NO: 261) and a CDC SS 635_fimbrial protein amino acid sequence (SEQ ID NO: 262) are set forth below. SEQ ID NO: 261 gagacggcaggggttgttagcagtggtcaattaacaataaaaaaatcaattacaaatttt aatgatgatacacttttgatgcctaagacagactatacttttagcgttaatccggatagt gcggctacaggtactgaaagtaatttaccaattaaaccaggtattgctgttaacaatcaa gatattaaggtttcttattctaatactgataagacatcaggtaaagaaaaacaagttgtt gttgactttatgaaagttacttttcctagcgttggtatttaccgttatgttgttaccgag aataaagggacagcagaaggagttacatatgatgatacaaaatggttagttgacgtctat gttggtaataatgaaaagggaggtcttgaaccaaagtatattgtatctaaaaaaggagat tctgctactaaagaaccaatccagtttaataattcattcgaaacaacgtcattaaaaatt gaaaaggaagttactggtaatacaggagatcataaaaaagcatttaactttacattaaca ttgcaaccaaatgaatactatgaggcaagttcggttgtgaaaattgaagagaacggacaa acgaaagatgtgaaaattggggaggcatataagtttactttgaacgatagtcagagtgtg atattgtctaaattaccagttggtattaattataaagttgaagaagcagaagctaatcaa ggtggatatactacaacagcaactttaaaagatggagaaaagttatctacttataactta ggtcaggaacataaaacagacaagactgctgatgaaatcgttgtcacaaataaccgtgac act
SEQ ID NO: 262
ETAGVVSSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATG TESNLPIKPGIAVNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPSVGIYRYVVTENK GTAEGVTYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKI EKEVTGNTGDHKKAFNFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLNDSQ SVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIVVT
NNRDT i";i1 C
Figure imgf000198_0001
SUS1S AI-4 strain of bacteria. ISS4883_fϊmbrial is thought to be a fimbrial structural subunit of M5 strain isolate ISS 4883. An example of a nucleotide sequence encoding the ISS4883_fimbrial protein (SEQ ID NO: 265) and an ISS4883_fimbrial protein amino acid sequence (SEQ ID NO: 266) are set forth below. SEQ ID NO: 265 gagacggcaggggttgtaacaggaaaatcactacaagttacaaagacaatgacttatgat gatgaagaggtgttaatgcccgaaaccgcctttacttttactatagagcctgatatgact gcaagtggaaaagaaggcgacctagatattaaaaatggaattgtagaaggcttagacaaa caagtaacagtaaaatataagaatacagataaaacatctcaaaaaactaaaatagcacaa tttgatttttctaaggttaaatttccagctataggtgtttaccgctatatggtttcagag aaaaacgataaaaaagacggaattaggtacgatgataaaaagtggactgtagatgtttat gttgggaataaggccaataacgaagaaggtttcgaagttctatatattgtatcaaaagaa ggtacttctagtactaaaaaaccaattgaatttacaaactctattaaaactacttcctta aaaattgaaaaacaaataactggcaatgcaggagatcgtaaaaaatcattcaacttcaca ttaacattacaaccaagtgaatattataaaaccggatcagttgtgaaaatcgaacaggat ggaagtaaaaaagatgtgacgataggaacgccttacaaatttactttgggacacggtaag agtgtcatgttatcgaaattaccaattggtatcaattactatcttagtgaagacgaagcg aataaagacggttacactacaacggcaacattaaaagaacaaggcaaagaaaagagttcc gatttcactttgagtactcaaaaccagaaaacagacgaatctgctgacgaaatcgttgtc acaaataagcgtgacactctcgag
SEQ ID NO: 266
ETAGVVTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTASGK EGDLDIKNGIVEGLDKQVTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKN DKKDGIRYDDKKWTVDVYVGNKANNEEGFEVLYIVSKEGTSSTKKPIEFTNSIKTTSL KIEKQITGNAGDRKKSFNFTLTLQPSEYYKTGSVVKIEQDGSKKDVTIGTPYKFTLGH GKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQNQKTDESAD EIVVTNKRDTLE
M50 strain isolate ISS4538 is a GAS AI-4 strain of bacteria. ISS4538_fimbrial is thought to be a fimbrial structural subunit of M50 strain ISS 4538. An example of a nucleotide sequence encoding the ISS4538_fimbrial protein (SEQ ID NO: 255) and an ISS4538_fimbrial protein amino acid sequence (SEQ ID NO: 256) are set forth below. SEQ ID NO: 255 atgaaaaaaaataaattattacttgctactgcaatcttagcaactgctttaggaacagct tctttaaatcaaaacgtaaaagctgagacggcaggggttgttagcagtggtcaattaaca ataaaaaaatcaattacaaattttaatgatgatacacttttgatgcctaagacagactat acttttagcgttaatccggatagtgcggctacaggtactgaaagtaatttaccaattaaa ccaggtattgctgttaacaatcaagatattaaggtttcttattctaatactgataagaca tcaggtaaagaaaaacaagttgttgttgactttatgaaagttacttttcctagcgttggt atttaccgttatgttgttaccgagaataaagggacagcagaaggagttacatatgatgat acaaaatggttagttgacgtctatgttggtaataatgaaaagggaggtcttgaaccaaag tatattgtatctaaaaaaggagattctgctactaaagaaccaatccagtttaataattca ttcgaaacaacgtcattaaaaattgaaaagaaagttactggtaatacaggagatcataaa aaagcatttaactttacattaacattgcaaccaaatgaatactatgaggcaagttcggtt gtgaaaattgaagagaacggacaaacgaaagatgtgaaaattggggaggcatataagttt actttgaacgatagtcagagtgtgatattgtctaaattaccagttggtattaattataaa gttgaagaagcagaagctaatcaaggtggatatactacaacagcaactttaaaagatgga gaaaagttatctacttataacttaggtcaggaacataaaacagacaagactgctgatgaa atcgttgtcacaaataancgngacactcnagttccaacnggtgtngtaggcaccccncct ccattcncagttcttancattgnggctantggtggngtnatntatnttacaaaacgnaaa aaagnataa
SEQ IDNO: 256
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKS ITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKTSG KfStWVfDfMϊwf&S^VSl.ΫR^VVΪgfϊKiSTfiΕGVTYDDTKWLVDVYVGNNEKGGLEPK
YIVSKKGDSATKEPIQFNNSFETTSLKIEKKVTGNTGDHKKAFNFTLTLQPNEYYEAS SVVKIEENGQTKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTAT LKDGEKLSTYNLGQEHKTDKTADEIVVTNXRDTXVPTGVVGTPPPFXVLXIXAXGGVX YXTKRKKX
There may be an upper limit to the number of GAS proteins which will be in the compositions of the invention. Preferably, the number of GAS proteins in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3. Still more preferably, the number of GAS proteins in a composition of the invention is less than 6, less than 5, or less than 4. Still more preferably, the number of GAS proteins in a composition of the invention is 3.
The GAS proteins and polynucleotides used in the invention are preferably isolated, i.e., separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose.
Examples Other Gram positive bacterial Adhesin Island Sequences
The Gram positive bacteria AI polypeptides of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from a gram positive bacteria, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form.
The Gram positive bacteria AI proteins of the invention may include polypeptide sequences having sequence identity to the identified Gram positive bacteria proteins. The degree of sequence identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more). Polypeptides having sequence identity include homologs, orthologs, allelic variants and mutants of the identified Gram positive bacteria proteins. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith- Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affinity gap search with parameters gap open penalty=12 and gap extension penalty=l.
The Gram positive bacteria adhesin island polynucleotide sequences may include polynucleotide sequences having sequence identity to the identified Gram positive bacteria adhesin island polynucleotide sequences. The degree of sequence identity may vary depending on the polynucleotide sequence in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more). K ' i! 1...» the ©raffl posittVe Sactέriaraiϊhesin island polynucleotide sequences of the invention may include polynucleotide fragments of the identified adhesin island sequences. The length of the fragment may vary depending on the polynucleotide sequence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
The Gram positive bacteria adhesin island amino acid sequences of the invention may include polypeptide fragments of the identified Gram positive bacteria proteins. The length of the fragment may vary depending on the amino acid sequence of the specific Gram positive bacteria antigen, but the fragment is preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferably the fragment comprises one or more epitopes from the sequence. The fragment may comprise at least one T-cell or, preferably, a B-cell epitope of the sequence. T- and B-cell epitopes can be identified empirically (e.g., using PEPSCAN [Geysen et al. (1984) PNAS USA 81:3998-4002; Carter (1994) Methods MoI. Biol. 36:207-223, or similar methods], or they can be predicted (e.g., using the Jameson- Wolf antigenic index [Jameson, BA et al. 1988, C4fi/O1Sr 4(l): 1818-186], matrix-based approaches [Raddrizzani and Hammer (2000) Brief Bioinform. 1(2): 179-189], TEPITOPE [De Lalla ef α/. (199) J. Immunol. 163:1725-1729], neural networks [Brusic et al. (1998) Bioinformatics 14(2): 121-130], OptiMer & EpiMer [Meister et al. (1995) Vaccine 13(6):581-591; Roberts et al. (1996) AIDS Res. Hum. Retroviruses 12(7):593-610], ADEPT [Maksyutov & Zagrebelnaya (1993) Comput. Appl. Biosci. 9(3):291-297], Tsites [Feller & de Ia Cruz (1991) Nature 349(6311):720-721], hydrophilicity [Hopp (1993) Peptide Research 6:183- 190], antigenic index [Welling et al. (198S)FEBS Lett. 188:215-218] or the methods disclosed in Davenport et al. (1995) Immunogenetics 42:392-297, etc. Other preferred fragments include (1) the N-terminal signal peptides of each identified Gram positive bacteria protein, (2) the identified Gram positive bacteria protein without their N-terminal signal peptides, (3) each identified Gram positive bacteria protein wherein up to 10 amino acid residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the N-terminus and/or the C-terminus e.g. the N-terminal amino acid residue may be deleted. Other fragments omit one or more domains of the protein (e.g. omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain), and (4) the polypeptides, but without their N-terminal amino acid residue. As indicated in the above text, nucleic acids and polypeptides of the invention may include sequences that:
(a) are identical (i.e., 100% identical) to the sequences disclosed in the sequence listing;
(b) share sequence identity with the sequences disclosed in the sequence listing;
(c) have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single nucleotide or amino acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b);
(d) when aligned with a particular sequence from the sequence listing using a pairwise alignment algorithm, a moving window of x monomers (amino acids or nucleotides) !>""" !i " •■''' lUvϊngΥrofe'klrt (N-ϊerfninius or 5') to end (C-terminus or 3'), such that for an alignment that extends top monomers (where p>x) there zxep-x+l such windows, each window has at least xy identical aligned monomers, where: x is sleeted from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and \ϊχ-y is not an integer then it is rounded up to the nearest integer. The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [Needlman &Wunsch (1970) /. MoI. Biol. 48, 443-453], using default parameters (e.g., with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [Rice et al. (2000) Trends Genet. 16:276-277].
The nucleic acids and polypeptides of the inention may additionally have further sequences to theN-terminus/5' and/or C-terminus/3' of these sequences (a) to (d).
All of the Gram positive bacterial sequences referenced herein are publicly available through PubMed on GenBank.
Streptococcus pneumoniae Adhesin Island Sequences
As discussed above, a S. pneumoniae AI sequence is present in the TIGR4 S. pneumoniae genome. Examples of S. pneumoniae AI sequences are set forth below. SrtD (SρO468) is a sortase. An example of an amino acid sequence of SrtD is set forth in
SEQ ID NO: 80. SEQ ID NO: 80
MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
SrtC (SpO467) is a sortase. An example of an amino acid sequence of SrtC is set forth in SEQ ID NO: 81. SEQ ID NO: 81
MSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIG YVEIPAI DQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGDIFYLHVLD QVLAYQVDQIVTVEPNDFEPVLIQHGEDYATI1LTCTPYMINSHRLLVRGKRI PYTAPIAERNRAVRERGQFWLWL LLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
SrtB (SP0466) is a sortase. An example of an amino acid sequence of SrtB is set forth in
SEQ ID NO: 82.
SEQ ID NO: 82
MAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM LEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGD KFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ
SpO465 is a hypothetical protein. An example of an amino acid sequence of SρO465 is set forth in SEQ ID NO: 83. MFLPFLSASLYLQTHHFIAFPNRQSYLLRETRKSHFFLIHHPF
RrgC (SP0464) is a cell wall surface anchor family protein. RrgC contains a sortase substrate motif VPXTG (SEQ ID NO: 137), shown in italics in SEQ ID NO: 84.
SEQ ID NO: 84
MISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHS WDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTK VKLIKVDQDHNRLEGVGFKLVSVARDVSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEP LAGYAVTTLDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVLQNGKEVVV TSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDΓGEETLYILML
VAILLFGSGYYLTKKPNN
RrgB (SpO463) is a cell wall surface anchor protein. RrgB contains a sortase substrate motif IPXTG (SEQ ID NO: 133), shown in italics in SEQ ID NO: 85. SEQ ID NO: 85
IAGVMFVWTNTNNEIIDENGQTLGVNIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKIYEIHSLSTY VGEDGATLTGSKAVPIEIELPLNDVVDAHVYPKNTEAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDVVEYEIV TKIPALANYATANWSDRMTEGLAFNKGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIPAGAEATFDLVNAQTG KVVQTVTLTTDKNTVTVNGLDKNTEYKFVERSIKGYSADYQEITTAGEIAVKNWKDENPKPLDPTEPKVVTYGKK FVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVSQEEKQLVVTTKDALDRAVAAYNALTAQQQTQQEKEKVDK AQAAYNAAVIAANNAFEWVADKDNENWKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY SATGQGIEYTAGSGKDDATKVVNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
RrgA (SpO462) is a cell wall surface anchor protein. RrgA contains a sortase substrate motif YPXTG (SEQ ID NO: 186), indicated in italics in SEQ ID NO: 86.
SEQ IDNO: 86 MLNRETHMKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDG TTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGT YPDVQTPYQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYEQKDKSVPL
DSLFWNYDQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQAR QNSQKVIFHITDGVPTMSYPINFNHATFAPSYQNQLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQM
NIAPDGYDVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITD PMGELIDLQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVLYDTTEKRIRVTGLYLGTDEKVT LTYNVRLNDEFVSNKFYDTNGRTTLHPKEVEQNTVRDFPIPKIRDVRKYPEITISKEKKLGDIEFIKVNKNDKKP LRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVN GEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKRERPI?RGGIGMLPFYLIGCMMMGGVLLYTRKHP
RIrA (SρO461) is a transcriptional regulator. An example of an amino acid sequence for RIrA is set forth in SEQ ID NO: 87. SEQ ID NO: 87
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL
As discussed above, aS.pneumoniaeAI sequenceispresentin theS.pneumoniaestrain670 genome. Examples ofS.pneumoniaeAI sequences are set forthbelow. F" C
Figure imgf000203_0001
acid sequence of orfl_670 is set forth in SEQ ID NO: 171. SEQ H) NO: 171
MEHINHTTLLIGIKDKNITLNKAIQHDTHIEVFATLDYHPPKCKHCKGKQIKYDFQKPSKIPFIEIGGFPSLIHL KKRRFQCKSCRKVTVAETTLVQKNCQISEMVRQKIAQLLLNREALTHIASKLAISTSTSTVYRKLKQFHFQEDYT TLPEILSWDEFSYQKGKLAFIAQDFNTKKIMTILDNRRQTTIRNHFFKYSKEARKKVKVVTVDMSGSYIPLIKKL FPNAKIVLDRFHIVQHMSRALNQTRINIMKQFDDKSLEYRALKYYWKFILKDSRKLSLKPFYARTFRETLTPREC LKKIFTLVPELKDYYDLYQLLLFHLQEKNTDQFWGLIQDTLPHLNRTFKTTLSTFICYKNYITNAIELPYSNAKL EATNKLIKDIKRNAFGFRNFENFKKRIFIALNIKKERTKFVLSRA
Orf2_670 is a transcriptional regulator. An example of an amino acid sequence of Orf2_670 is set forth in SEQ ID NO: 172. SEQ ID NO: 172
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK
LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR
LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL
Orf3_670 is a cell wall surface anchor family proten. An example of an amino acid sequence of Orf3_670 is set forth in SEQ ID NO: 173.
SEQ ID NO: 173 MLNRETHMKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDG TTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGT YPDVQTPYQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPL
DSALWTFDRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQAR PNSKKVIFHITDGVPTMSYPINFKYTGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQM
FTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTDWITNHGDPTTWYYNGNMA
QDGYDVFTVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMG
ELIDFQLGADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTY
NVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRD AVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEV
RDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
Orf4_670 is a cell wall surface anchor family protein. An example of an amino acid sequence of orf4_670 is set forth in SEQ ID NO: 174. SEQ ID NO: 174
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLK DLTGVVAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSΪGTKIPYVVNTTIPSN ATFATSFWSDEMTEGLTYNEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSE NNWTYTWSGLDNSIEYKVEEEYNGYSAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGY VEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
Orf5_670 is a cell wall surface anchor family protein. An example of an amino acid sequence of orf5_670 is set forth in SEQ ID NO: 175.
SEQ ID NO: 175
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDDRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK TΪKVKWΪ^&QDlN'Rlii'G^G'fkLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGN YRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL QNGKEWVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKPNN
Orf6_670 is a sortase. An example of an amino acid sequence of orf6_670 is set forth in SEQ
ID NO: 176. SEQ ED NO: 176
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT
HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAΆRKEVKVE
DGQQ Orf7_670 is a sortase. An example of an amino acid sequence of orf7_670 is set forth in SEQ
ID NO: 177. SEQ ID NO: 177
VSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIG YVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLD QVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWL LLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
Orf8_670 is a sortase. An example of an amino acid sequence of orf8_670 is set forth in SEQ ID NO: 178. SEQ ID NO: 178
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 19A Hungary 6 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 19A Hungary 6 are set forth below.
ORF2_19AH is a transcriptional regulator. An example of an amino acid sequence of ORF2J9AH is set forth in SEQ ID NO: 187.
SEQ ID NO: 187
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL
ORF3_19AH is acell wall surfaceprotein. Anexample ofanamino acid sequenceof ORF3_19AH is set forthin SEQ IDNO: 188.
SEQ ID NO: 188
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDGTTVSQRT EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDVVILLD NSNSMSNIRHNHAHRAEKAGEATRALVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILNDSALWTF DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVI FHITDGVPTMSYPINFKYTGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT TVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRLNDQ FVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIV PQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKNP
ORF4_19AH is a cell wall surfaceprotein. An exampleofanamino acid sequenceof ORF4J9AH is set forthin SEQ ID NO: 189. SEQ IDNO: 189
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLK DLTGVVAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSIGTKIPYVVNTTIPSN ATFATSFWSDEMTEGLTYNEDVTITLNNVAMDQADYEVTKGXNGFNLKLTEAGLAKINGKDADQKIQITYSATLN SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSE NNWTYTWSGLDNSIEYKVEEEYNGYSAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGY VEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
ORF5_19AH is a cell wall surface protein. An example of an amino acid sequence of
ORF5J9AH is set forth in SEQ ID NO: 190.
SEQ ID NO: 190
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDDRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGN YRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKPNN
ORF6_19AH is aputativesortase. Anexample ofanamino acidsequence ofORF6_19AH is set forthin SEQ IDNO: 191. SEQ ID NO: 191
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ ORF7_19AH is aputativesortase. An example ofanamino acidsequence ofORF7_19AH is set forthin SEQ IDNO: 192.
SEQ D) NO: 192
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
ORF8_19AH is a putative sortase. An example of an amino acid sequence of ORF8_19AH is set forth in SEQ ID NO: 193. SEQ ID NO: 193
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFMGILFVLWKLARLLRGK If"" C
Figure imgf000206_0001
AI sequence is present in the 6B Finland 12 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 6B Finland 12 are set forth below.
ORF2_6BF is a transcriptional regulator. An example of an amino acid sequence of ORF2_6BF is set forth in SEQ ID NO: 194.
SEQ ID NO: 194
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL ORF3_6BF is acell wall surfaceprotein. Anexample ofan amino acidsequence of ORF3_6BF is set forthin SEQIDNO: 195.
SEQ ID NO: 195
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDGTTVSQRT
EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDVVILLD
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVI FHITDGVPTMSYPINFKYTGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVF TVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRLNDQ FVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIV PQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
ORF4_6BF is a cell wall surface protein. An example of an amino acid sequence of
ORF4_6BF is set forth in SEQ ID NO: 196.
SEQ ID NO: 196
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLK DLTGVVAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
, NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPWDKRIADTLNYNDQNGLSIGTKIPYVVNTTIPSN
ATFATSFWSDEMTEGLTYNEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSE
NNWTYTWSGLDNSIEYKVEEEYNGYSAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGY
VEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
ORF5_6BF is acell wallsurfaceprotein. Anexampleofanamino acidsequence of ORF5_6BFis set forthin SEQIDNO: 197.
SEQ IDNO: 197
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDDRV QIVRDLHSWDENKLSSFKKTΞFEMTFLENQIEVSHIPNGLYYVRΞIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGN YRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL QNGKEWVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKPNN .S'i riut&tiVe Jorfiϊse?1'; An example of an amino acid sequence of ORF6_6BF is set forth in SEQ ID NO: 198. SEQ ID NO: 198
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHBYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ ORF7_6BF is a putative sortase. An example of an amino acid sequence of ORP7_6BF is set forth in SEQ ID NO: 199. SEQ ID NO: 199
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
ORF8 6BF is a putative sortase. An example of an amino acid sequence of ORF8_6BF is set forth in SEQ ID NO: 200. SEQ ID NO: 200
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 6B Spain 2 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 6B Spain 2 are set forth below.
ORF2_6BSP is a transcriptional regulator. An example of an amino acid sequence of ORF2_6BSP is set forth in SEQ ID NO: 201. SEQ ID NO: 201
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL
ORF3_6BSP is acellwall surfaceprotein. Anexample ofan amino acidsequenceof ORF3_6BSP is set forthin SEQ IDNO: 202.
SEQ ID NO: 202
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDGTTVSQRT EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDVVILLD
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVI FHITDGVPTMSYPINFKYTGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVF TVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRLNDQ FVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIV PQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP '■' ' ' O'F^Ψ_6B SP is a' cell'wali's'ύr'Face protein. An example of an amino acid sequence of
ORF4_6BSP is set forth in SEQ ID NO: 203.
SEQ ID NO: 203
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLK DLTGVVAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSIGTKIPYVVNTTIPSN
ATFATSFWSDEMTEGLTYNEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSE
NNWTYTWSGLDNSIEYKVEEEYNGYSAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGY
VEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
ORF5_6BSP is a cellwall surfaceprotein. An example ofanamino acid sequence of ORF5_6BSP is set forthin SEQ IDNO: 204.
SEQ ED NO: 204
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDDRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGN YRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQΆPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKPNN
ORF6_6BSP is aputativesortase. An example ofan amino acidsequence ofORF6_6BSP is setforthinSEQ IDNO: 205.
SEQ ID NO: 205
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
ORF7_6BSP is aputativesortase. An example ofan amino acid sequence ofORF7_6BSP is set forthin SEQ IDNO: 206. SEQ ID NO: 206
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
ORF8_6BSP is a putative sortase. An example of an amino acid sequence of ORF8_6BSP is set forth in SEQ ID NO: 207.
SEQ ID NO: 207
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 9V Spain 3 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 9V Spain 3 are set forth below.
ORF2_9VSP is a transcriptional regulator. An example of an amino acid sequence of ORF2_9VSP is set forth in SEQ ID NO: 208. MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVΆLTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL ORF3_9VSP is a cell wall surfaceprotein. Anexample ofan amino acid sequenceof ORF3_9VSP is set forth in SEQ IDNO: 209.
SEQ ID NO: 209
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTNGTTVSQRT
EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQRTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYERKDKSVPLDVVILLD
DQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVI FHITDGVPTMSYPINFNHATFAPSYQNQLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVY DVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELID LQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRL NDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPAITIAKEKKLGEIEFIKINKNDKKPLRDAVFS LQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVT SIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLLFYLIGCMMMGGVLLYTRKHP
ORF4_9VSP is a cellwall surfaceprotein. Anexample ofan amino acidsequence of
ORF4_9VSP is set forthin SEQIDNO: 210. SEQ ID NO: 210 IAGVMFVWTNTNNEIIDENGQTLGVNIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKIYEIHSLSTY VGEDGATLTGSKAVPIEIELPLNDVVDAHVYPKNTEAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDVVEYEIV TKIPALANYATANWSDRMTEGLAFNKGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIPAGAEATFDLVNAQTG KVVQTVTLTTDKNTVTVNGLDKNTEYKFVERSIKGYSADYQEITTAGEIAVKNWKDENPKPLDPTEPKVVTYGKK FVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVSQEEKQLVVTTKDALDRAVAAYNALTAQQQTQQEKEKVDK AQAAYNAAVIAANNAFEWVADKDNENVVKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY SATGQGIEYTAGSGKDDATKVVNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDEDQLA
ORF5_9VSP is acellwallsurfaceprotein. Anexampleofanamino acidsequenceof OPsP5_9VSP is set forthin SEQ IDNO: 211.
SEQ ID NO: 211
MTMQKMQKMQKMQKMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVW KLDDSYSYDNRVQIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMT DQTVEPLVIVAKKADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKN GEIVVTNLPLGTYRFKEVEPLAGYTVTTMDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKV MKEENGHYTPVLQNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNN KRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKTNN
ORF6_9VSP is a putative sortase. An example of an amino acid sequence of ORF6_9VSP is set forth in SEQ ID NO: 212.
SEQ ID NO: 212
MLIKMAKTKKQKRNNLLLGVVFFIGIAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVE DE ORF7_9VSP is a putative sortase. An example of an amino acid sequence of ORF7_9VSP is set forth in SEQ ID NO: 213. SEQ ID NO: 213 MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD ORF8_9VSP is a putative sortase. An example of an amino acid sequence of ORF8_9VSP is set forth in SEQ ID NO: 214. SEQ ID NO: 214
MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 14 CSR 105. pneumoniae genome. Examples of S. pneumoniae AI sequences from 14 CSR 10 are set forth below. ORF2_14CSR is a transcriptional regulator. An example of an amino acid sequence of
ORF2J4CSR is set forth in SEQ ID NO: 215.
SEQ ID NO: 215
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL
ORF3_14CSR is a cell wall surface protein. An example of an amino acid sequence of ORF3_14CSR is set forth in SEQ ID NO: 216.
SEQ ED NO: 216
MKKVRKIFQKAVAGLCCΪSQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDGTTVSQRT EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDVVILLD
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVI FHITDGVPTMSYPINFKYTGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVF TVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRLNDQ FVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIV PQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
ORF4_14CSRis acellwall surfaceprotein. Anexampleofanamino acid sequenceof ORF4J4CSRis set forthinSEQ IDNO: 217.
SEQIDNO: 217 MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLK DLTGVVAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSIGTKIPYVVNTTIPSN ATFATSFWSDEMTEGLTYNEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSE
Figure imgf000211_0001
NAQFWKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGY VEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV VNKKITIPQTGGIGTII FAVAGAAIMGIΆVYAYVKNNKDEDQLA
ORF5_14CSR is a cell wall surface protein. An example of an amino acid sequence of
ORF5J4CSR is set forth in SEQ ID NO: 218. SEQ ID NO: 218
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDDRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGN YRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKPNN
ORF6 14CSR is a putative sortase. An example of an amino acid sequence of ORF6_14CSR is set forth in SEQ ID NO: 219. SEQ ID NO: 219
MLIKMVKTKKQKRNNLLLGVVFFTGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ ORF7_14CSR is a putative sortase. An example of an amino acid sequence of ORF7_14CSR is set forth in SEQ ID NO: 220.
SEQ ID NO: 220
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
ORF8_14CSR is a putative sortase. An example of an amino acid sequence of ORF8_14CSR is set forth in SEQ ID NO: 221. SEQ ID NO: 221
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 19F Taiwan 145. pneumoniae genome. Examples of S. pneumoniae AI sequences from 19F Taiwan 14 are set forth below.
ORF2_19FTW is a transcriptional regulator. An example of an amino acid sequence of ORF2J 9FTW is set forth in SEQ ID NO: 222.
SEQ ID NO: 222
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL P C. TV U S O Eii ./ E!! 7 E 3 '9
ORF3_19FTW is a cell wall surface protein. An example of an amino acid sequence of ORF3_19FTW is set forth in SEQ ID NO: 223.
SEQ ID NO: 223 MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDGTTVSQRT EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYERKDKSVPLDVVILLD
DQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVI FHITDGVPTMSYPINFNHATFAPSYQNQLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVY
DVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELID LQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRL NDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPAITIAKEKKLGEIEFIKINKNDKKPLRDAVFS LQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVT SIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
ORF4_19FTW is acell wall surfaceprotein. An example ofanamino acid sequence of ORF4J9FTWis set forth in SEQID NO: 224. SEQ ID NO: 224
IAGVMFVWTNTNNEIIDENGQTLGVNIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKIYEIHSLSTY VGEDGATLTGSKAVPIEIELPLNDVVDAHVYPKNTEAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDVVEYEIV TKIPALANYATANWSDRMTEGLAFNKGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIPAGAEATFDLVNAQTG KVVQTVTLTTDKNTVTVNGLDKNTEYKFVERSIKGYSADYQEITTAGEIAVKNWKDENPKPLDPTEPKWTYGKK FVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVSQEEKQLVVTTKDALDRAVAAYNALTAQQQTQQEKEKVDK AQAAYNAAVIAANNAFEWVADKDNENVVKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY SATGQGIEYTAGSGKDDATKVVNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDEDQLA
ORF5_19FTW is a cell wall surface protein. An example of an amino acid sequence of
ORF5J9FTW is set forth in SEQ ID NO: 225.
SEQ ID NO: 225
MTMQKMQKMISRIFFVMALCFΞLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDNRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIVVTNLPLGT YRFKEVEPLAGYTVTTMDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVL QNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKTNN
OKF6_19FTW is aputative sortase. An example ofanamino acid sequence of
ORF6J9FTWis set forthin SEQ IDNO: 226.
SEQ ID NO: 226
MLIKMAKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVE DE ORF7_19FTW is a putative sortase. An example of an amino acid sequence of
ORF7J9FTW is set forth in SEQ ID NO: 227.
SEQ ID NO: 227
MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTDQEKKQGVSEYANMLKVHERIGYVEIPAIEQEIPMYVGTSEDILQKGAGLLEGASLPVGGE INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
ORF8_19FTW is a putative sortase. An example of an amino acid sequence of ORF8J9FTW is set forth in SEQ ID NO: 228. SEQ ID NO: 228
MSRTKLRALLGYLLML VACLIPI YCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSH VFFRHLDQLKVGDAL YYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 23F Taiwan 15 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 23F Taiwan 15 are set forth below. ORF2_23FTW is a transcriptional regulator. An example of an amino acid sequence of
ORF2_23FTW is set forth in SEQ ID NO: 229. SEQ ID NO: 229
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL
ORF3_23FTW is a cell wall surface protein. An example of an amino acid sequence of
ORP3_23FTW is set forth in SEQ ID NO: 230.
SEQ ID NO: 230
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGEGGALLGDAVFELKNNTDGTTVSQRT EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYEQKDKSVPLDVVILLD
DQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVI FHITDGVPTMSYPINFNHATFAPSYQNQLNAFFSKSPNKDGILLΞDFITQATSGEHTIVRGDGQSYQMFTDKTVY
DVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELID LQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVLYDTTEKRIRVTGLYLGTDEKVTLTYNVRL NDEFVSNKFYDTNGRTTLHPKEVEQNTVRDFPIPKIRDVRKYPEITISKEKKLGDIEFIKVNKNDKKPLRDAVFS LQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVT SIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
ORF4_23FTW is a cell wall surfaceprotein. An example ofan amino acidsequence of ORF4_23FTWis set forth in SEQ IDNO: 231.
SEQ ID NO: 231 MKSINKFLTILAALLLTVSSLFSAATVFAAEQKTKTLTVHKLLMTDQELDAWNSDAITTAGYDGSQNFEQFKQLQ GVPQGVTEISGVAFELQSYTGPQGKEQENLTNDAVWTAVNKGVTTETGVKFDTEVLQGTYRLVEVRKESTYVGPN GKVLTGMKAVPALITLPLVNQNGVVENAHVYPKNSEDKPTATKTFDTAΆGFVDPGEKGLAIGTKVPYIVTTTIPK NSTLATAFWSDEMTEGLDYNGDVVVNYNGQPLDNSHYTLEΆGHNGFILKLNEKGLEAINGKDAEATITLKYTATL NALAVADVPEANDVTFHYGNNPGHGNTPKPNKPKNGELTITKTWADAKDAPIAGVEVTFDLVNAQTGEVVKVPGH ETGIVLNQTNNWTFTATGLDNNTEYKFVERTIKGYSADYQTITETGKIAVKNWKDENPEPINPEEPRVKTYGKKF VKVDQKDERLKEAQFVVKNEQGKYLALKSAAQQAVNEKAAAEAKQALDAAIAΆYTNAADKNAAQAVVDAAQKTYN DNYRAARFGYVEVERKEDALVLTSNTDGQFQISGLAAGSYTLEETKAPEGFAKLGDVKFEVGAGSWNQGDFNYLK DVQKNDATKVVNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDEDQLA I" '" Il ORF!&£ 23 Ww'ϊs'a feell'wa'ϊl βulrrabe protein. An example of an amino acid sequence of
ORF5_23FTW is set forth in SEQ ID NO: 232.
SEQ ID NO: 232
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDNRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIVVTNLPLGT YRFKEVEPLAGYTVTTMDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVL QNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKTNN
ORF6_23FTW is aputative sortase. An example ofanamino acidsequence of
ORF6_23FTWis setforthin SEQ IDNO: 233.
SEQ ID NO: 233
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGQLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ ORF7_23FTW is a putative sortase. An example of an amino acid sequence of
ORF7_23FTW is set forth in SEQ ID NO: 234.
SEQ ID NO: 234
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGKDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
ORF8_23FTW is a putative sortase. An example of an amino acid sequence of ORF8_23FTW is set forth in SEQ ID NO: 235. SEQ ID NO: 235
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIP1TFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
As discussed above, a S. pneumoniae AI sequence is present in the 23F Poland 16 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 23F Poland 16 are set forth below.
ORF2_23FP is a transcriptional regulator. An example of an amino acid sequence of ORF2_23FP is set forth in SEQ ID NO: 236.
SEQ BD NO: 236
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQ TNYYFHQLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEK LKNLFMYPILMEHCQTYLEPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHR LYLFSLYLTETIFSSLPAIPIFIILNNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAMIAKKAHYLL ORF3_23FP is acellwall surfaceprotein. An example ofan amino acidsequence of ORF3_23FP is set forthin SEQ IDNO: 237.
SEQ BD NO: 237 M^MSKΪEQykil/SϊlSsdlii^ϊ'SffiϊwilϊilETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQRT EAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDVVILLD NSNSMSNIRHNHAHRAEKAGEATRALVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILNDSALWTF DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVI FHITDGVPTMSYPINFKYTGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVF TVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKRIRVTGLYLGTGEKVTLTYNVRLNDQ FVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIV PQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKNP
ORF4_23FP is a cell wall surface protein. An example of an amino acid sequence of ORF4 23FP is set forth in SEQ ID NO: 238. SEQ ID NO: 238
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLK DLTGVVAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSIGTKIPYVVNTTIPSN ATFATSFWSDEMTEGLTYNEDVTITLNNVAMDQADYEVTKGINGFNLKLTEAGLAKINGKDADQKIQITYSATLN SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSE NNWTYTWSGLDNSIEYKVEEEYNGYSAEYTVESKGKLGVKNWKDNNPAPINLEEPRVKTYGKKFVKVDQKDTRLE NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGY VEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV VNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDEDQLA
ORF5_23FP is a cell wall surface protein. An example of an amino acid sequence of
ORF5_23FP is set forth in SEQ ID NO: 239.
SEQ ID NO: 239
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDNRV QIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK KADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIVVTNLPLGT YRFKEVEPLAGYAVTTMDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVL QNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGE ETLYILMLVAILLFGSGYYLTKKTNN
ORF6_23FP is aputative sortase. Anexampleofan amino acid sequence ofORF6_23FP is set forthin SEQ IDNO: 240.
SEQ ID NO: 240
MLIKMAKTKKQKRNNLLLGVVFFIGIAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVE DE ORF7_23FP is aputative sortase. Anexampleofan amino acidsequenceofORF7_23FP is setforthin SEQ IDNO: 241.
SEQ ID NO: 241
MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAF NATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGΆSLPVGGE NTHTVVTAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYM INSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
ORF8_23FP is aputativesortase. Anexampleofan amino acid sequence ofORF8_23FP is setforthin SEQ IDNO: 242. sfφlΪD ivd:!bi$S Ci 5 / B 7 ≡ 39
MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDP FLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSH VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAV YQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
Immunogenic compositions of the invention comprising AI antigens may further comprise one or more antigenic agents. Preferred antigens include those listed below. Additionally, the compositions of the present invention may be used to treat or prevent infections caused by any of the below-listed microbes. Antigens for use in the immunogenic compositions include, but are not limited to, one or more of the following set forth below, or antigens derived from one or more of the following set forth below: Bacterial Antigens
N. meningitides: a protein antigen from N. meningitides serogroup A, C, W135, Y, and/or B
(1-7); an outer-membrane vesicle (OMV) preparation from N. meningitides serogroup B. (8, 9, 10,
11); a saccharide antigen, including LPS, from N. meningitides serogroup A, B, C W135 and/or Y, such as the oligosaccharide from serogroup C (see PCT/US99/09346; PCT IB98/01665; and PCT
IB99/00103);
Streptococcus pneumoniae: a saccharide or protein antigen, particularly a saccharide from Streptooccus pneumoniae;
Streptococcus agalactiae: particularly, Group B streptococcus antigens; Streptococcus pyogenes: particularly, Group A streptococcus antigens;
Enterococcus faecalis or Enterococcus faecium: Particularly a trisaccharide repeat or other Enterococcus derived antigens provided in US Patent No. 6,756,361 ;
Helicobacter pylori: including: Cag, Vac, Nap, HopX, HopY and/or urease antigen;
Bordetella pertussis: such as petussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also combination with pertactin and/or agglutinogens 2 and 3 antigen;
Staphylococcus aureus: including S. aureus type 5 and 8 capsular polysaccharides optionally conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, or antigens derived from surface proteins, invasins (leukocidin, kinases, hyaluronidase), surface factors that inhibit phagocytic engulfment (capsule, Protein A), carotenoids, catalase production, Protein A, coagulase, clotting factor, and/or membrane-damaging toxins (optionally detoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin);
Staphylococcus epidermis: particularly, S. epidermidis slime-associated antigen (SAA);
Staphylococcus saprophyticus: (causing urinary tract infections) particularly the 160 kDa hemagglutinin of S. saprophyticus antigen; Pseudomonas aeruginosa: particularly, endotoxin A, Wzz protein, P. aeruginosa LPS, more particularly LPS isolated from PAOl (05 serotype), and/or Outer Membrane Proteins, including Outer Membrane Proteins F (OprF) (Infect Immun. 2001 May; 69(5): 3510-3515); F" C "WacϊM^SMIιShis'"ζaMUrS):3ύiik as B. anthracis antigens (optionally detoxified) from A- components (lethal factor (LF) and edema factor (EF)), both of which can share a common B- component known as protective antigen (PA);
Moraxella catarrhalis: (respiratory) including outer membrane protein antigens (HMW- OMP), C-antigen, and/or LPS;
Yersinia pestis (plague): such as Fl capsular antigen (Infect Immun. 2003 Jan; 71(1)): 374- 383, LPS (Infect Immun. 1999 Oct; 67(10): 5395), Yersinia pestis V antigen (Infect Immun. 1997 Nov; 65(11): 4476-4482);
Yersinia enterocolitica (gastrointestinal pathogen): particularly LPS (Infect Immun. 2002 August; 70(8): 4414);
Yersinia pseudotuberculosis: gastrointestinal pathogen antigens;
Mycobacterium tuberculosis: such as lipoproteins, LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6 optionally formulated in cationic lipid vesicles (Infect Immun.
2004 October; 72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associated antigens (Proc Natl Acad Sci U S A. 2004 Aug 24; 101(34): 12652), and/or MPT51 antigens
(Infect Immun. 2004 July; 72(7): 3829);
Legionella pneumophila (Legionnairs' Disease): L. pneumophila antigens — optionally derived from cell lines with disrupted asd genes (Infect Immun. 1998 May; 66(5): 1898);
Rickettsia: including outer membrane proteins, including the outer membrane protein A and/or B (OmpB) (Biochim Biophys Acta. 2004 Nov 1 ; 1702(2): 145), LPS, and surface protein antigen (SPA) (JAutoimmun. 1989 Jun;2 Suppl:81);
E. coli: including antigens from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E. coli (EHEC); Vibrio cholerae: including proteinase antigens, LPS, particularly lipopolysaccharides of
Vibrio cholerae II, Ol Inaba O-specific polysaccharides, V. cholera 0139, antigens of IEM108 vaccine (Infect Immun. 2003 Oct;71(10):5498-504), and/or Zonula occludens toxin (Zot);
Salmonella typhi (typhoid fever): including capsular polysaccharides preferably conjugates (Vi, i.e. vax-TyVi); Salmonella typhimurium (gastroenteritis): antigens derived therefrom are contemplated for microbial and cancer therapies, including angiogenesis inhibition and modulation of flk;
Listeria monocytogenes (sytemic infections in immunocompromised or elderly people, infections of fetus): antigens derived from L. monocytogenes are preferably used as carriers/vectors for intracytoplasmic delivery of conjugates/associated compositions of the present invention; Porphyromonas gingivalis: particularly, P. gingivalis outer membrane protein (OMP);
Tetanus: such as tetanus toxoid (TT) antigens, preferably used as a carrier protein in conjunction/conjugated with the compositions of the present invention; i!';:" C Εiph'MemEϊM&kάsSά$Ε&eϊi&ioxoid, preferably CRMi97, additionally antigens capable of modulating, inhibiting or associated with ADP ribosylation are contemplated for combination/co- administration/conjugation with the compositions of the present invention, the diphtheria toxoids are preferably used as carrier proteins; Borrelia burgdorferi (Lyme disease): such as antigens associated with P39 and P13 (an integral membrane protein, Infect Immun. 2001 May; 69(5): 3323-3334), VIsE Antigenic Variation Protein (J CHn Microbiol. 1999 Dec; 37(12): 3997);
Haemophilus influenzae B: such as a saccharide antigen therefrom;
Klebsiella: such as an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid;
Neiserria gonorrhoeae: including, a Por (or porin) protein, such as PorB {see Zhu et al, Vaccine (2004) 22:660 — 669), a transferring binding protein, such as TbpA and TbpB (See Price et al., Infection and Immunity (2004) 71(1):277 - 283), a opacity protein (such as Opa), a reduction- modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations {see Plante et al., J Infectious Disease (2000) 182:848 - 855), also see e.g. WO99/24578, WO99/36544, WO99/57280, WO02/079243);
Chlamydia pneumoniae: particularly C. pneumoniae protein antigens;
Chlamydia trachomatis: including antigens derived from serotypes A, B, Ba and C are (agents of trachoma, a cause of blindness), serotypes L1, L2 & L3 (associated with Lymphogranuloma venereum), and serotypes, D-K;
Treponema pallidum (Syphilis): particularly a TinpA antigen; and Haemophilus ducreyi (causing chancroid): including outer membrane protein (DsrA). Where not specifically referenced, further bacterial antigens of the invention may be capsular antigens, polysaccharide antigens or protein antigens of any of the above. Further bacterial antigens may also include an outer membrane vesicle (OMV) preparation. Additionally, antigens include live, attenuated, split, and/or purified versions of any of the aforementioned bacteria. The bacterial or microbial derived antigens of the present invention may be gram-negative or gram-positive and aerobic or anaerobic.
Additionally, any of the above bacterial-derived saccharides (polysaccharides, LPS, LOS or oligosaccharides) can be conjugated to another agent or antigen, such as a carrier protein (for example
CRMi97). Such conjugation may be direct conjugation effected by reductive amination of carbonyl moieties on the saccharide to amino groups on the protein, as provided in US Patent No. 5,360,897 and Can J Biochem Cell Biol. 1984 May;62(5):270-5. Alternatively, the saccharides can be conjugated through a linker, such as, with succinamide or other linkages provided in Bioconjugate Techniques, 1996 and CRC, Chemistry of Protein Conjugation and Cross-Linking, 1993. PiIrWAmMhB ≡ / ≡ 7 Ξ 3 li:!
Influenza: including whole viral particles (attenuated), split, or subunit comprising hemagglutinin (HA) and/or neuraminidase (NA) surface proteins, the influenza antigens may be derived from chicken embryos or propogated on cell culture, and/or the influenza antigens are derived from influenza type A, B, and/or C, among others;
Respirator)' syncytial virus (RSV): including the F protein of the A2 strain of RSV (J Gen Virol. 2004 Nov; 85(Pt 11):3229) and/or G glycoprotein;
Parainfluenza virus (PIV): including PIV type 1, 2, and 3, preferably containing hemagglutinin, neuraminidase and/or fusion glycoproteins; Poliovirus: including antigens from a family of picornaviridae, preferably poliovirus antigens such as OPV or, preferably IPV;
Measles: including split measles virus (MV) antigen optionally combined with the Protollin and or antigens present in MMR vaccine;
Mumps: including antigens present in MMR vaccine; Rubella: including antigens present in MMR vaccine as well as other antigens from
Togaviridae, including dengue virus;
Rabies: such as lyophilized inactivated virus (RabAvert™);
Flaviviridae viruses: such as (and antigens derived therefrom) yelow fever virus, Japanese encephalitis virus, dengue virus (types 1, 2, 3, or 4), tick borne encephalitis virus, and West Nile virus;
Caliciviridae; antigens therefrom;
HIV: including HIV-I or HIV-2 strain antigens, such as gag (p24gag and p55gag), env (gpl60 and gp41), pol, tat, nef, rev vpu, miniproteins, (preferably p55 gag and gpl40v delete) and antigens from the isolates HIVmb, HIVSF2, HIVLAV, HIVLAi, HIVMN, HIV-1CM235, HIV-1US4) HIV-2; simian immunodeficiency virus (SIV) among others;
Rotavirus: including VP4, VP5, VP6, VP7, VP8 proteins (Protein Expr Purif. 2004 Dec;38(2):205) and/or NSP4;
Pestivirus: such as antigens from classical porcine fever virus, bovine viral diarrhoea virus, and/or border disease virus; Parvovirus: such as parvovirus B19;
Coronavirus: including SARS virus antigens, particularly spike protein or proteases therefrom, as well as antigens included in WO 04/92360;
Hepatitis A virus: such as inactivated virus;
Hepatitis B virus: such as the surface and/or core antigens (sAg), as well as the presurface sequences, pre-Sl and ρre-S2 (formerly called pre-S), as well as combinations of the above, such as sAg/pre-Sl, sAg/pre-S2, sAg/ρre-Sl/pre-S2, and pre-S l/pre-S2, (see, e.g., AHBV Vaccines - Human
Vaccines and Vaccination, pp. 159-176; and U.S. Patent Nos. 4,722,840, 5,098,704, 5,324,513;
Figure imgf000220_0001
Bimbaum et al., J. Virol. (1990) 64:3319-3330; and Zhou et al., /. Virol. (1991) 65:5457-5464);
Hepatitis C virus: such as El, E2, E1/E2 (see, Houghton et al., Hepatology (1991) 14:381), NS345 polyprotein, NS 345-core polyprotein, core, and/or peptides from the nonstructural regions (International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436);
Delta hepatitis virus (HDV): antigens derived therefrom, particularly δ-antigen from HDV (see, e.g., U.S. Patent No. 5,378,814);
Hepatitis E virus (HEV); antigens derived therefrom; Hepatitis G virus (HGV); antigens derived therefrom; Varcicella zoster virus: antigens derived from varicella zoster virus (VZV) (/. Gen. Virol.
(1986) 67:1759);
Epstein-Barr virus: antigens derived from EBV (Baer et al., Nature (1984) 310:207); Cytomegalovirus: CMV antigens, including gB and gH {Cytomegaloviruses (J.K. McDougall, ed., Springer-Verlag 1990) pp. 125-169); Herpes simplex virus: including antigens from HSV-I or HSV-2 strains and glycoproteins gB, gD and gH (McGeoch et al., J. Gen. Virol. (1988) 69: 1531 and U.S. Patent No. 5,171,568);
Human Herpes Virus: antigens derived from other human herpesviruses such as HHV6 and HHV7; and
HPV: including antigens associated with or derived from human papillomavirus (HPV), for example, one or more of El — E7, Ll , L2, and fusions thereof, particularly the compositions of the invention may include a virus-like particle (VLP) comprising the Ll major capsid protein, more particular still, the HPV antigens are protective against one or more of HPV serotypes 6, 11, 16 and/or 18.
Further provided are antigens, compostions, methods, and microbes included in Vaccines, 4lh Edition (Plotkin and Orenstein ed. 2004); Medical Microbiology 4th Edition (Murray et al. ed. 2002);
Virology, 3rd Edition (W.K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and
D.M. Knipe, eds. 1991), which are contemplated in conjunction with the compositions of the present invention.
Additionally, antigens include live, attenuated, split, and/or purified versions of any of the aforementioned viruses. Fungal Antigens
Fungal antigens for use herein, associated with vaccines include those described in: U.S. Pat. Nos. 4,229,434 and 4,368,191 for prophylaxis and treatment of trichopytosis caused by Trichophyton mentagrophytes; U.S. Pat. Nos. 5,277,904 and 5,284,652 for a broad spectrum dermatophyte vaccine for the prophylaxis of dermatophyte infection in animals, such as guinea pigs, cats, rabbits, horses and lambs, these antigens comprises a suspension of killed T. equinum, T. mentagrophytes (var. granulare), M. canis and/or M. gypseum in an effective amount optionally combined with an adjuvant; iφS.ffiaϊ N6IJfe]ii5l]iZ7i..ώSl633S".73feli:fl)r a ringworm vaccine comprising an effective amount of a homogenized, formaldehyde-killed fungi, i.e., Microsporum canis culture in a carrier; U.S. Pat. No. 5,948,413 involving extracellular and intracellular proteins for pythiosis. Additional antigens identified within antifungal vaccines include Ringvac bovis LTF-130 and Bioveta. Further, fungal antigens for use herein may be derived from Dermatophytres, including:
Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme.
Fungal pathogens for use as antigens or in derivation of antigens in conjunction with the compositions of the present invention comprise Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Ciγptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, and Saksenaea spp. Other fungi from which antigens are derived include Alternaria spp, Curvularia spp,
Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.
Processes for producing a fungal antigens are well known in the art (see US Patent No. " 6,333,164). In a preferred method a solubilized fraction extracted and separated from an insoluble fraction obtainable from fungal cells of which cell wall has been substantially removed or at least partially removed, characterized in that the process comprises the steps of: obtaining living fungal cells; obtaining fungal cells of which cell wall has been substantially removed or at least partially removed; bursting the fungal cells of which cell wall has been substantially removed or at least partially removed; obtaining an insoluble fraction; and extracting and separating a solubilized fraction from the insoluble fraction. vyS-TMAnti'fffms n κ» ,- --a -y ZΆ ";;:'.' iris
In particular embodiments, microbes (bacteria, viruses and/or fungi) against which the present compositions and methods can be implement include those that cause sexually transmitted diseases (STDs) and/or those that display on their surface an antigen that can be the target or antigen composition of the invention. In a preferred embodiment of the invention, compositions are combined with antigens derived from a viral or bacterial STD. Antigens derived from bacteria or viruses can be administered in conjunction with the compositions of the present invention to provide protection against at least one of the following STDs, among others: chlamydia, genital herpes, hepatitis (particularly HCV), genital warts, gonorrhoea, syphilis and/or chancroid (See, WO00/15255). In another embodiment the compositions of the present invention are co-administered with an antigen for the prevention or treatment of an STD.
Antigens derived from the following viruses associated with STDs, which are described in greater detail above, are preferred for co-administration with the compositions of the present invention: hepatitis (particularly HCV), HPV, HIV, or HSV. Additionally, antigens derived from the following bacteria associated with STDs, which are described in greater detail above, are preferred for co-administration with the compositions of the present invention: Neiserria gonorrhoeae, Chlamydia pneumoniae, Chlamydia trachomatis, Treponema pallidum, or Haemophilus ducreyi. Respiratory Antigens The antigen may be a respiratory antigen and could further be used in an immunogenic composition for methods of preventing and/or treating infection by a respiratory pathogen, including a virus, bacteria, or fungi such as respiratory syncytial virus (RSV), PIV, SARS virus, influenza, Bacillus anthracis, particularly by reducing or preventing infection and/or one or more symptoms of respiratory virus infection. A composition comprising an antigen described herein, such as one derived from a respiratory virus, bacteria or fungus is administered in conjunction with the compositions of the present invention to an individual which is at risk of being exposed to that particular respiratory microbe, has been exposed to a respiratory microbe or is infected with a respiratory virus, bacteria or fungus. The composition(s) of the present invention is/are preferably coadministered at the same time or in the same formulation with an antigen of the respiratory pathogen. Administration of the composition results in reduced incidence and/or severity of one or more symptoms of respiratory infection. Pediatric/Geriatric Antigens
In one embodiment the compositions of the present invention are used in conjunction with an antigen for treatment of a pediatric population, as in a pediatric antigen. In a more particular embodiment the pediatric population is less than about 3 years old, or less than about 2 years, or less than about 1 years old. In another embodiment the pediatric antigen (in conjunction with the composition of the present invention) is administered multiple times over at least 1, 2, or 3 years. ,rii .1 of the present invention are used in conjunction with
Figure imgf000223_0001
an antigen for treatment of a geriatric population, as in a geriatric antigen. Other Antigens
Other antigens for use in conjunction with the compositions of the present include hospital acquired (nosocomial) associated antigens.
In another embodiment, parasitic antigens are contemplated in conjunction with the compositions of the present invention. Examples of parasitic antigens include those derived from organisms causing malaria and/or Lyme disease.
In another embodiment, the antigens in conjunction with the compositions of the present invention are associated with or effective against a mosquito born illness. In another embodiment, the antigens in conjunction with the compositions of the present invention are associated with or effective against encephalitis. In another embodiment the antigens in conjunction with the compositions of the present invention are associated with or effective against an infection of the nervous system.
In another embodiment, the antigens in conjunction with the compositions of the present invention are antigens transmissible through blood or body fluids. Antigen Formulations
In other aspects of the invention, methods of producing microparticles having adsorbed antigens are provided. The methods comprise: (a) providing an emulsion by dispersing a mixture comprising (i) water, (ii) a detergent, (iii) an organic solvent, and (iv) a biodegradable polymer selected from the group consisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate. The polymer is typically present in the mixture at a concentration of about 1% to about 30% relative to the organic solvent, while the detergent is typically present in the mixture at a weight-to-weight detergent-to-polymer ratio of from about 0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about 0.1: 1, about 0.001: 1 to about 0.1:1, or about 0.005: 1 to about 0.1: 1); (b) removing the organic solvent from the emulsion; and (c) adsorbing an antigen on the surface of the microparticles. In certain embodiments, the biodegradable polymer is present at a concentration of about 3% to about 10% relative to the organic solvent.
Microparticles for use herein will be formed from materials that are sterilizable, non-toxic and biodegradable. Such materials include, without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride, PACA, and polycyanoacrylate. Preferably, microparticles for use with the present invention are derived from a ρoly(α-hydroxy acid), in particular, from a poly(lactide) ("PLA") or a copolymer of D,L-lactide and glycolide or glycolic acid, such as a ρoly(D,L-lactide-co-glycolide) ("PLG" or "PLGA"), or a copolymer of D,L-lactide and caprolactone. The microparticles may be derived from any of various polymeric starting materials which have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide: glycolide ratios, the selection of which will be largely a matter,, of. cliQic%".depf adiμg;; ι jn»ιpajj> an. the coadministered macromolecule. These parameters are discussed more fully below.
Further antigens may also include an outer membrane vesicle (OMV) preparation. Additional formulation methods and antigens (especially tumor antigens) are provided in U.S. Patent Serial No. 09/581,772. Antisen References
The following references include antigens useful in conjunction with the compositions of the present invention: ' 1 International patent application WO99/24578
2 International patent application WO99/36544.
3 International patent application WO99/57280.
4 International patent application WO00/22430.
5 Tettelin et al. (2000) Science 287:1809-1815. 6 International patent application WO96/29412.
7 Pizza et al. (2000) Science 287:1816-1820.
8 PCT WO 01/52885.
9 Bjune et al. (1991) Lancet 338(8775).
10 Fuskasawa et al. (1999) Vaccine 17:2951-2958. 11 Rosenqist et al. (1998) Dev. Biol. Strand 92:323-333.
12 Constantino et al. (1992) Vaccine 10:691-698.
13 Constantino et al. (1999) Vaccine 17: 1251-1263.
14 Watson (2000) Pediatr Infect Dis J 19:331-332.
15 Rubin (20000) Pediatr Clin North Am 47:269-285,v. 16 Jedrzejas (2001) Microbiol MoI Biol Rev 65:187-207.
17 International patent application filed on 3rd July 2001 claiming priority from GB- 0016363.4; WO 02/02606; PCT IB/01/00166.
18 Kalman et al. (1999) Nature Genetics 21:385-389.
19 Read et al. (2000) Nucleic Acids Res 28: 1397-406. 20 Shirai et al. (2000) J. Infect. Dis 181(Suppl 3):S524-S527.
21 International patent application WO99/27105.
22 International patent application WO00/27994.
23 International patent application WO00/37494.
24 International patent application WO99/28475. 25 Bell (2000) Pediatr Infect Dis J 19:1187-1188.
26 Iwarson (1995) APMIS 103:321-326.
27 Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.
28 Hsu et al. (1999) Clin Liver Dis 3:901-915.
29 Gastofsson et al. (1996) N. Engl. J. Med. 334-:349-355. 30 Rappuoli et al. (1991) TIBTECH 9:232-238.
31 Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.
32 Del Guidice et al. (1998) Molecular Aspects of Medicine 19: 1-70.
33 International patent application WO93/018150.
34 International patent application WO99/53310. 35 International patent application WO98/04702.
36 Ross et al. (2001) Vaccine 19:135-142.
37 Sutter et al. (2000) Pediatr Clin North Am 47:287-308.
38 Zimmerman & Spann (1999) Am Fan Physician 59:113-118, 125-126.
39 Dreensen (1997) Vaccine 15 Suppl"S2-6. 40 MMWR Morb Mortal Wklyrep 1998 Jan 16:47(1):12, 9.
41 McMichael (2000) Vaccinel9 Suppl l:S101-107. l!' 4? GB' patent appEcations'00Z6-ft^5, 0028727.6 & 0105640.7.
44 Dale (1999) Infect Disclin North Am 13:227-43, viii.
45 Ferretti et al. (2001) PNAS USA 98: 4658-4663. 46 Kuroda et al. (2001) Lancet 357(9264): 1225-1240; see also pages 1218-1219.
47 Ramsay et al. (2001) Lancet 357(9251):195-196.
48 Lindberg (1999) Vaccine 17 Suppl 2:S28-36.
49 Buttery & Moxon (2000) JR Coil Physicians Long 34:163-168.
50 Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii. 51 Goldblatt (1998) J. Med. Microbiol. 47:663-567.
52 European patent 0 477 508.
53 U.S. Patent No. 5,306,492.
54 International patent application WO98/42721.
55 Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114. 56 Hermanson (1996) Bioconjugate Techniques ISBN: 012323368 & 012342335X.
57 European patent application 0372501.
58 European patent application 0378881.
59 European patent application 0427347.
60 International patent application WO93/17712. 61 International patent application WO98/58668.
62 European patent application 0471177.
63 International patent application WO00/56360.
64 International patent application WO00/67161. The contents of all of the above cited patents, patent applications and journal articles are incorporated by reference as if set forth fully herein.
There may be an upper limit to the number of Gram positive bacterial proteins which will be in the compositions of the invention. Preferably, the number of Gram positive bacterial proteins in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3. Still more preferably, the number of Gram positive bacterial proteins in a composition of the invention is less than 6, less than 5, or less than 4. Still more preferably, the number of Gram positive bacterial proteins in a composition of the invention is 3. The Gram positive bacterial proteins and polynucleotides used in the invention are preferably isolated, i.e., separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose. Fusion Proteins: GBS AI sequences
The GBS AI proteins used in the invention may be present in the composition as individual separate polypeptides, but it is preferred that at least two {i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) of the antigens are expressed as a single polypeptide chain (a "hybrid" or "fusion" polypeptide). Such fusion polypeptides offer two principal advantages: first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable fusion partner that oyψcoxraes the prρhkq$3"Sepoi^y-;PθiprøPϊQJal manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful.
The fusion polypeptide may comprise one or more AI polypeptide sequences. Preferably, the fusion comprises an AI surface protein sequence. Preferably, the fusion polypeptide includes one or more of GBS 80, GBS 104, and GBS 67. Most preferably, the fusion peptide includes a polypeptide sequence from GBS 80. Accordingly, the invention includes a fusion peptide comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a GBS AI surface protein or a fragment thereof. Preferably, the first and second amino acid sequences in the fusion polypeptide comprise different epitopes.
Hybrids (or fusions) consisting of amino acid sequences from two, three, four, five, six, seven, eight, nine, or ten GBS antigens are preferred. In particular, hybrids consisting of amino acid sequences from two, three, four, or five GBS antigens are preferred.
Different hybrid polypeptides may be mixed together in a single formulation. Within such combinations, a GBS antigen may be present in more than one hybrid polypeptide and/or as a non-hybrid polypeptide. It is preferred, however, that an antigen is present either as a hybrid or as a non-hybrid, but not as both.
Hybrid polypeptides can be represented by the formula NH2-A-{-X-L-},,-B-COOH, wherein: X is an amino acid sequence of a GBS AI protein or a fragment thereof; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
If a -X- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein. In some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-terminus of the hybrid protein i. e. the leader peptide OfX1 will be retained, but the leader peptides of X2 ... Xn will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of Xi as moiety -A-.
For each n instances of {-X-L-}, linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH2-X1-Li-X2-L2-COOH, NH2-Xi-X2-COOH, NH2-XrLi-X2-COOH, NH2-Xi-X2-L2-COOH, etc. Linker amino acid sequence(s) -L- will typically be short {e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptide sequences which facilitate cloning, poly-glycine linkers (i.e. comprising GIyn where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. Hisπ where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG, with the GIy-S er dipeptide being formed from a BamΗΪ restriction site, thus aiding cloning and manipulation, and the (Gly)4 tetrapeptide being a typical poly-glycine linker.
-A- is an optional N-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 1,8.,. R4j6vl fjj,pljj|j| ||,i!l|2,.l |:;,!il'(Ji' 5, 4, 3, 2, 1). Examples include leader sequences to direct
Figure imgf000227_0001
protein trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. IfXi lacks its own N-terminus methionine, -A- is preferably an oligopeptide {e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine.
-B- is an optional C-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e.g. comprising histidine tags i.e. His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.
Most preferably, n is 2 or 3.
Fusion Proteins: Gram positive bacteria AI sequences The Gram positive bacteria AI proteins used in the invention may be present in the composition as individual separate polypeptides, but it is preferred that at least two (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) of the antigens are expressed as a single polypeptide chain (a "hybrid" or "fusion" polypeptide). Such fusion polypeptides offer two principal advantages: first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable fusion partner that overcomes the problem; second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful.
The fusion polypeptide may comprise one or more AI polypeptide sequences. Preferably, the fusion comprises an AI surface protein sequence. Accordingly, the invention includes a fusion peptide comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a Gram positive bacteria AI protein or a fragment thereof. Preferably, the first and second amino acid sequences in the fusion polypeptide comprise different epitopes.
Hybrids (or fusions) consisting of amino acid sequences from two, three, four, five, six, seven, eight, nine, or ten Gram positive bacteria antigens are preferred. In particular, hybrids consisting of amino acid sequences from two, three, four, or five Gram positive bacteria antigens are preferred.
Different hybrid polypeptides may be mixed together in a single formulation. Within such combinations, a Gram positive bacteria AI sequence may be present in more than one hybrid polypeptide and/or as a non-hybrid polypeptide. It is preferred, however, that an antigen is present either as a hybrid or as a non-hybrid, but not as both.
Hybrid polypeptides can be represented by the formula NH2-A-{-X-L-}n-B-COOH, wherein:
X is an amino acid sequence of a Gram positive bacteria AI sequence or a fragment thereof; L is an
Figure imgf000228_0001
If a -X- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein. In some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of Xi will be retained, but the leader peptides of X2 ■ - • Xn will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of Xi as moiety -A-.
For each n instances of {-X-L-}, linker amino acid sequence -L- may be present or absent. For instance, when «=2 the hybrid may be NH2-Xi-Li-X2-L2-COOH, NH2-X1-X2-COOH, NH2-Xi-Li-X2-COOH, NH2-Xi-X2-L2-COOH, etc. Linker amino acid sequence(s) -L- will typically be short {e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptide sequences which facilitate cloning, poly-glycine linkers {i.e. comprising GIyn where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG, with the Gly-Ser dipeptide being formed from a Bamϋl restriction site, thus aiding cloning and manipulation, and the (Gly)4 tetrapeptide being a typical poly-glycine linker.
-A- is an optional N-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. IfXi lacks its own N-terminus methionine, -A- is preferably an oligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine.
-B- is an optional C-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e.g. comprising histidine tags i.e. His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.
Most preferably, n is 2 or 3. Antibodies: GBS AI sequences
The GBS AI proteins of the invention may also be used to prepare antibodies specific to the GBS AI proteins. The antibodies are preferably specific to the an oligomeric or hyper-oligomeric form of an AI protein. The invention also includes combinations of antibodies specific to GBS AI proteins selected to provide protection against an increased range of GBS serotypes and strain isolates. For example, a combination may comprise a first and second antibody, wherein said first antippdyis §.pe(pi§G|Q §;;fir,stGp3'p AJ grafein and said second antibody is specific to a second GBS AI protein. Preferably, the nucleic acid sequence encoding said first GBS AI protein is not present in a GBS genome comprising a polynucleotide sequence encoding for said second GBS AI protein. Preferably, the nucleic acid sequence encoding said first and second GBS AI proteins are present in the genomes of multiple GBS serotypes and strain isolates.
The GBS specific antibodies of the invention include one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a GBS polypeptide. The antibodies of the invention include antibodies which specifically bind to a GBS AI protein. The invention includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature ■ 349: 293-299; and US Patent No. 4,816,567; F(ab')2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sd USA 69:2659-2662; and Ehrlich et al. (1980) Biochem jj):4091-4096); single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5897-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31.: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, for example, Riechmann et al. (1988) Nature yΩ/3τh-l>τi; Verhoeyan et al (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 September 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule. The invention further includes antibodies obtained through non- conventional processes, such as phage display.
Preferably, the GBS specific antibodies of the invention are monoclonal antibodies. Monoclonal antibodies of the invention include an antibody composition having a homogeneous antibody population. Monoclonal antibodies of the invention may be obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. See, e.g., Cote, et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p 77.
The antibodies of the invention may be used in diagnostic applications, for example, to detect the presence or absence of GBS in a biological sample. The antibodies of the invention may also be used in the prophylactic or therapeutic treatment of GBS infection. Antibodies: Gram positive bacteria AI sequences
The Gram positive bacteria AI proteins of the invention may also be used to prepare antibodies specific to the Gram positive bacteria AI proteins. The antibodies are preferably specific to the an oligomeric or hyper-oligomeric form of an AI protein. The invention also includes combinations of antibodies specific to Gram positive bacteria AI proteins selected to provide protection against an increased range of Gram positive bacteria genus, species, serotypes and strain isolates. „„ ,,Epr .example axonibin,a.tiQ,n nra ^comprise a first and second antibody, wherein said first F" IL 1 / ti1 & U 3.••'" e; ./ id: ..::* sa» antibody is specific to a first Gram positive bacteria AI protein and said second antibody is specific to a second Gram positive bacteria AI protein. Preferably, the nucleic acid sequence encoding said first Gram positive bacteria AI protein is not present in a Gram positive bacterial genome comprising a polynucleotide sequence encoding for said second Gram positive bacteria AI protein. Preferably, the nucleic acid sequence encoding said first and second Gram positive bacteria AI proteins are present in the genomes of multiple Gram positive bacteria genus, species, serotypes or strain isolates.
As an example of an instance where the combination of antibodies provides protection against an increased range of bacteria serotypes, the first antibody may be specific to a first GAS AI protein and the second antibody may be specific to a second GAS AI protein. The first GAS AI protein may comprise a GAS AI-I surface protein, while the second GAS AI protein may comprise a GAS AI-2 or AI-3 surface protein.
As an example of an instance where the combination of antibodies provides protection against an increased range of bacterial species, the first antibody may be specific to a GBS AI protein and the second antibody may be specific to a GAS AI protein. Alternatively, the first antibody may be specific to a GAS AI protein and the second antibody may be specific to a S. pneumoniae AI protein.
The Gram positive specific antibodies of the invention include one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a Gram positive bacteria AI polypeptide. The antibodies of the invention include antibodies which specifically bind to a Gram positive bacteria AI protein. The invention includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349: 293-299; and US Patent No. 4,816,567; F(ab')2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sd USA £5:5897-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 3_1 :1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 September 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule. The invention further includes antibodies obtained through non-conventional processes, such as phage display.
Preferably, the Gram positive specific antibodies of the invention are monoclonal antibodies. Monoclonal antibodies of the invention include an antibody composition having a homogeneous antibody population. Monoclonal antibodies of the invention may be obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas,, Se§v,e,gr,.,£otβ,,,%:tMl,,MQnoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p
P' if,! Ii ■••' U '.:;::iι Liι .b / ιι:::" / »';::: ..:::!! ''J' 77.
The antibodies of the invention may be used in diagnostic applications, for example, to detect the presence or absence of Gram positive bacteria in a biological sample. The antibodies of the invention may also be used in the prophylactic or therapeutic treatment of Gram positive bacteria infection. Nucleic Acids
The invention provides nucleic acids encoding the Gram positive bacteria sequences and/or the hybrid fusion polypeptides of the invention. The invention also provides nucleic acid encoding the GBS antigens and/or the hybrid fusion polypeptides of the invention. Furthermore, the invention provides nucleic acid which can hybridise to these nucleic acids, preferably under "high stringency" conditions (eg. 650C in a O.lxSSC, 0.5% SDS solution).
Polypeptides of the invention can be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, non-glycosylated, lipidated, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other GAS or host cell proteins).
Nucleic acid according to the invention can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other GBS or host cell nucleic acids).
The term "nucleic acid" includes DNA and RNA, and also their analogues, such as those containing modified backbones (e.g. phosphorothioates, etc.), and also peptide nucleic acids (PNA), etc. The invention includes nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing purposes). The invention also provides a process for producing a polypeptide of the invention, comprising the step of culturing a host cell transformed with nucleic acid of the invention under conditions which induce polypeptide expression.
The invention provides a process for producing a polypeptide of the invention, comprising the step of synthesising at least part of the polypeptide by chemical means. The invention provides a process for producing nucleic acid of the invention, comprising the step of amplifying nucleic acid using a primer-based amplification method (e.g. PCR).
The invention provides a process for producing nucleic acid of the invention, comprising the step of synthesising at least part of the nucleic acid by chemical means. Purification and Recombinant Expression The Gram positive bacteria AI proteins of the invention may be isolated from the native Gram positive bacteria, or they may be recombinantly produced, for instance in a heterologous host. For example, the GAS, GBS, and S. pneumoniae antigens of the invention may be isolated from Strg^φcpccμq qgala§tyae,β^gyogβnβ$®&p}ieumoniae, or they may be recombinantly produced, for instance, in a heterologous host. Preferably, the GBS antigens are prepared using a heterologous host.
The heterologous host may be prokaryotic (e.g. a bacterium) or eukaryotic. It is preferably E.coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g. M. tuberculosis), S. gordonii, L. lactis, yeasts, etc.
Recombinant production of polypeptides is facilitated by adding a tag protein to the Gram positive bacteria AI sequence to be expressed as a fusion protein comprising the tag protein and the Gram positive bacteria antigen. For example, recombinant production of polypeptides is facilitated by adding a tag protein to the GBS antigen to be expressed as a fusion protein comprising the tag protein and the GBS antigen. Such tag proteins can facilitate purification, detection and stability of the expressed protein. Tag proteins suitable for use in the invention include a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag,, chitin-binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination anti-terminiantion factor (NusA), E. coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred tag proteins include His-tag and GST. A full discussion on the use of tag proteins can be found at Terpe et al., "Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems", Appl Microbiol Biotechnol (2003) 60:523 - 533. After purification, the tag proteins may optionally be removed from the expressed fusion protein, i.e., by specifically tailored enzymatic treatments known in the art. Commonly used proteases include enterokinase, tobacco etch virus (TEV), thrombin, and factor X3. GBS polysaccharides
The compositions of the invention may be further improved by including GBS polysaccharides. Preferably, the GBS antigen and the saccharide each contribute to the immunological response in a recipient. The combination is particularly advantageous where the saccharide and polypeptide provide protection from different GBS serotypes.
The combined antigens may be present as a simple combination where separate saccharide and polypeptide antigens are administered together, or they may be present as a conjugated combination, where the saccharide and polypeptide antigens are covalently linked to each other.
Thus the invention provides an immunogenic composition comprising (i) one or more GBS AI proteins and (ii) one or more GBS saccharide antigens. The polypeptide and the polysaccharide may advantageously be covalently linked to each other to form a conjugate.
Between them, the combined polypeptide and saccharide antigens preferably cover (or provide protection from) two or more GBS serotypes (e.g. 2, 3, 4, 5, 6, 7, 8 or more serotypes). The serotypes of the polypeptide and saccharide antigens may or may not overlap. For example, the polypeptide might protect against serogroup II or V, while the saccharide protects against either serogroups Ia, Ib, or III. Preferred combinations protect against the following groups of serotypes: (|)(!Spio,to^||I|,|»||^:;P}'S^ot^^4| jξ≠ π> (3) serotypes Ia and III, (4) serotypes Ia and IV, (5) serotypes Ia and V, (6) serotypes Ia and VI, (7) serotypes Ia and VII, (8) serotypes Ia and VIII, (9) serotypes Ib and II, (10) serotypes Ib and III, (11) serotypes Ib and IV, (12) serotypes Ib and V, (13) serotypes Ib and VI, (14) serotypes Ib and VII, (15) serotypes Ib and VIII, 16) serotypes II and III, (17) serotypes II and IV, (18) serotypes II and V, (19) serotypes II and VI1 (20) serotypes II and VII, (21) serotypes II and VII, (22) serotypes III and IV, (23) serotypes III and V, (24) serotypes III and VI, (25) serotypes III and VII, (26) serotypes III and VIII, (27) serotypes IV and V, (28) serotypes IV and VI, (29) serotypes IV and VII, (30) serotypes IV and VIII, (31) serotypes V and VI, (32) serotypes V and VII, (33) serotypes V and VIII, (34) serotypes VI and VII, (35) serotypes VI and VIII, and (36) serotypes VII and VIII.
Still more preferably, the combinations protect against the following groups of serotypes: (1) serotypes Ia and II, (2) serotypes Ia and V, (3) serotypes Ib and II, (4) serotypes Ib and V, (5) serotypes III and II, and (6) serotypes III and V. Most preferably, the combinations protect against serotypes III and V. Protection against serotypes II and V is preferably provided by polypeptide antigens.
Protection against serotypes Ia, Ib and/or III may be polypeptide or saccharide antigens. Immunogenic compositions and medicaments
Compositions of the invention are preferably immunogenic compositions, and are more preferably vaccine compositions. The pH of the composition is preferably between 6 and 8, preferably about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen-free. The composition may be isotonic with respect to humans.
Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Accordingly, the invention includes a method for the therapeutic or prophylactic treatment of a Gram positive bacteria infection in an animal susceptible to such gram positive bacterial infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic composition of the invention. For example, the invention includes a method for the therapeutic or prophylactic treatment of a Streptococcus agalactiae, S. pyogenes, or S. pneumoniae infection in an animal susceptible to streptococcal infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention.
The invention also provides a composition of the invention for use of the compositions described herein as a medicament. The medicament is preferably able to raise an immune response in a mammal (Le. it is an immunogenic composition) and is more preferably a vaccine.
The invention also provides the use of the compositions of the invention in the manufacture of a medicament for raising an immune response in a mammal. The medicament is preferably a vaccine.
The invention also provides kits comprising one or more containers of compositions of the invention. Compositions can be in liquid form or can be lyophilized, as can individual antigens.
Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Couplers cap bg;;foiped from.a.iVarietjVήGf materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The composition may comprise a first component comprising one or more Gram positive bacteria AI proteins. Preferably, the AI proteins are surface AI proteins. Preferably, the AI surface proteins are in an oligomeric or hyperoligomeric form. For example, the first component comprises a combination of GBS antigens or GAS antigens, or S. pneumoniae antigens. Preferably said combination includes GBS 80. Preferably GBS 80 is present in an oligomeric or hyperoligomeric form.
The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including 'other buffers, diluents, filters, needles, and syringes.
The kit can also comprise a second or third container with another active agent, for example an antibiotic.
The kit can also comprise a package insert containing written instructions for methods of inducing immunity against S agalactiae andor S. pyogenes and/or S pneumoniae or for treating S agalactiae andor S. pyogenes and/or S pneumoniae infections. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug
Administration (FDA) or other regulatory body.
The invention also provides a delivery device pre- filled with the immunogenic compositions of the invention.
The invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. This immune response will preferably induce long lasting (e.g., neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to one or more GBS and/or GAS and/or S. pneumoniae antigens. The method may raise a booster response.
The invention provides a method of neutralizing GBS, GAS, or S. pneumoniae infection in a mammal comprising the step of administering to the mammal an effective amount of the immunogenic compositions of the invention, a vaccine of the invention, or antibodies which recognize an immunogenic composition of the invention.
The mammal is preferably a human. Where the vaccine is for prophylactic use, the human is preferably a female (either of child bearing age or a teenager). Alternatively, the human may be elderly (e.g., over the age of 50, 55, 60, 65, 70 or 75) and may have an underlying disease such as diabetes or cancer. Where the vaccine is for therapeutic use, the human is preferably a pregnant female or an elderly adult.
These uses and methods are preferably for the prevention and/or treatment of a disease caused by Streptococcus agalactiae, or S. pyogenes, or S. pneumoniae. The compositions may also be effectiy.eragains>o,therμstreptococ£ Abacteria. The compositions may also be effective against other
B"1'' Ii,- Il ■• 1I"" Sl' U -3ι / IC ..>''' C ..3 "'3I' Gram positive bacteria.
One way of checking efficacy of therapeutic treatment involves monitoring Gram positive bacterial infection after administration of the composition of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses against the Gram positive bacterial antigens in the compositions of the invention after administration of the composition.
One way of checking efficacy of therapeutic treatment involves monitoring GBS infection after administration of the composition of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses against the GBS antigens in the compositions of the invention after administration of the composition.
A way of assessing the immunogenicity of the component proteins of the immunogenic compositions of the present invention is to express the proteins recombinantly and to screen patient sera or mucosal secretions by immunoblot. A positive reaction between the protein and the patient serum indicates that the patient has previously mounted an immune response to the protein in question- that is, the protein is an immunogen. This method may also be used to identify immunodominant proteins and/or epitopes.
Another way of checking efficacy of therapeutic treatment involves monitoring GBS or GAS or S pneumoniae infection after administration of the compositions of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses both systemically (such as monitoring the level of IgGl and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the GBS and/or ~GAS and/or S pneumoniae antigens in the compositions of the invention after administration of the composition. Typically, GBS and/or GAS and/or S pneumoniae serum specific antibody responses are determined post-immunization but pre- challenge whereas mucosal GBS and/or GAS and/or S pneumoniae specific antibody body responses are determined post-immunization and post-challenge.
The vaccine compositions of the present invention can be evaluated in in vitro and in vivo animal models prior to host, e.g., human, administration.
The efficacy of immunogenic compositions of the invention can also be determined in vivo by challenging animal models of GBS and/or GAS and/or S pneumoniae infection^ e.g., guinea pigs or mice, with the immunogenic compositions. The immunogenic compositions may or may not be derived from the same serotypes as the challenge serotypes. Preferably the immunnogenic compositions are derivable from the same serotypes as the challenge serotypes. More preferably, the immunogenic composition and/or the challenge serotypes are derivable from the group of GBS and/or GAS and/or S pneumoniae serotypes. In vivo efficacy models include but are not limited to: (i) A murine infection model using human GBS and/or GAS and/or S pneumoniae serotypes; (ii) a murine disease model which is a murine model using a mouse-adapted GBS and/or GAS and/or S pneumoniae strain, such as those strains outh'ned.abpyt.whickis.paijjcularly virulent in mice and (iii) a primate model using human p' Li' il .■■"' '!■■■" '""i" Ui !bι / ic» >>'' H::::; ™:i> " J> GBS or GAS or S pneumoniae isolates.
The immune response may be one or both of a THl immune response and a TH2 response. The immune response may be an improved or an enhanced or an altered immune response. The immune response may be one or both of a systemic and a mucosal immune response.
Preferably the immune response is an enhanced system and/or mucosal response. An enhanced systemic and/or mucosal immunity is reflected in an enhanced THl and/or TH2 immune response. Preferably, the enhanced immune response includes an increase in the production of IgGl and/or IgG2a and/or IgA Preferably the mucosal immune response is a TH2 immune response. Preferably, the mucosal immune response includes an increase in the production of IgA.
Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections. Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immune response may result in the production of IgGl, IgE, IgA and memory B cells for future protection.
A TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgGl, IgE, IgA and memory B cells. Preferably, the enhanced TH2 immune resonse will include an increase in IgGl production. A THl immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a THl immune response (such as IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. Preferably, the enhanced THl immune response will include an increase in IgG2a production.
Immunogenic compositions of the invention, in particular, immunogenic composition comprising one or more GAS antigens of the present invention may be used either alone or in combination with other GAS antigens optionally with an immunoregulatory agent capable of eliciting a ThI and/or Th2 response.
Compositions of the invention will generally be administered directly to a patient. Certain routes may be favored for certain compositons, as resulting in the generation of a more effective immune response, preferably a CMI response, or as being less likely to induce side effects, or as being easier for administration. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intradermally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g. see WO 99/27961) or transcutaneous (e.g. see WO 02/074244 and WO 02/064162), intranasal (e.g. see WO03/028760), ocular, aural, pulmonary or other mucosal administration.
The invention may be used to elicit systemic and/or mucosal immunity. ,„. .,In one paytipu|jaj ly,,pr;^ep-ec| ernhQdiment, the immunogenic composition comprises one or more GBS or GAS or S pneumoniae antigen(s) which elicits a neutralising antibody response and one or more GBS or GAS or S pneumoniae antigen(s) which elicit a cell mediated immune response. In this way, the neutralising antibody response prevents or inhibits an initial GBS or GAS or S pneumoniae infection while the cell-mediated immune response capable of eliciting an enhanced ThI cellular response prevents further spreading of the GBS or GAS or S pneumoniae infection. Preferably, the immunogenic composition comprises one or more GBS or GAS or S pneumoniae surface antigens and one or more GBS or GAS or S pneumoniae cytoplasmic antigens. Preferably the immunogenic composition comprises one or more GBS or GAS or S pneumoniae surface antigens or the like and one or other antigens, such as a cytoplasmic antigen capable of eliciting a ThI cellular response.
Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
The compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, such as antibiotics, as needed. By 'immunologically effective amount', it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention, or increases a measurable immune response or prevents or reduces a clinical symptom. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Further,|,Comppnente, of., the Composition..,, iP' L. Il ..■ o .:;;;iι ii.,.ii 31 ..■• ic ,i|1 n:;;:; .,;;;!> "1SiI
The composition of the invention will typically, in addition to the components mentioned above, comprise one or more 'pharmaceutically acceptable carriers', which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, , glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. A thorough discussion of pharmaceutically acceptable excipients is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th ed., ISBN: 0683306472.
Adjuvants
Vaccines of the invention may be administered in conjunction with other immunoregulatory agents. In particular, compositions will usually include an adjuvant. Adjuvants for use with the invention include, but are not limited to, one or more of the following set forth below: A. Mineral Containing Compositions
Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminum salts and calcium salts. The invention includes mineral salts such as hydroxides {e.g. oxyhydroxides), phosphates {e.g. hydroxyphosphates, orthophosphates), sulfates, etc. {e.g. see chapters 8 & 9 of Vaccine Design... (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.), or mixtures of different mineral compounds {e.g. a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form {e.g. gel, crystalline, amorphous, etc.), and with adsorption to the salt(s) being preferred. The mineral containing compositions may also be formulated as a particle of metal salt (WO 00/23105).
Aluminum salts may be included in vaccines of the invention such that the dose OfAl3+ is between 0.2 and 1.0 mg per dose. B. Oil-Emulsions
Oil-emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). See WO90/14837. See also, Podda, "The adjuvanted influenza vaccines with novel adjuvants: experience with the MF59-adjuvanted vaccine", Vaccine (2001) 19: 2673-2680; Frey et al., "Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults", Vaccine (2003) 21:4234-4237. MF59 is used as the adjuvant in the FLUAD™ influenza virus trivalent subunit vaccine. ..„., „, , Earticylar,ly, nrefeiTedcadiUiVaiitSifor^use in the compositions are submicron oil-in- water e
Figure imgf000239_0001
mulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80 ™ (polyoxyelthylenesorbitan tnonooleate), and/or 0.25-1.0% Span 85™ (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D- isogluatminyl-L-alanine-2-(r-2'-dipalmitoyl-jw-glycero-3-huydroxyphosphophoiyloxy)-ethylamine (MTP-PE), for example, the submicron oil-in-water emulsion known as "MF59" (International Publication No. WO 90/14837; US Patent Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties; and Ott et al., "MF59 — Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines" in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M.F. and Newman, MJ. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene (e.g. 4.3%), 0.25-0.5% w/v Tween 80™, and 0.5% w/v Span 85™ and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA). For example, MTP-PE may be present in an amount of about 0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100 μg/dose. As used herein, the term "MF59-0" refers to the above submicron oil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes a formulation that contains MTP-PE. For instance, "MF59-100" contains 100 μg MTP-PE per dose, and so on. MF69, another submicron oil-in- water emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v Tween 80™, and 0.75% w/v Span 85™ and optionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 0.4% Tween 80™, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion. MF75-MTP denotes an MF75 foπnulation that includes MTP, such as from 100-400 μg MTP-PE per dose.
Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO 90/14837 and US Patent Nos. 6,299,884 and 6,45 1,325, incorporated herein by reference in their entireties.
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention. C. Saponin Formulations
Saponin formulations, may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs. μ Saμo^η,compjθsitiGii§, h^vejbeen^urified using High Perfoπnance Thin
Figure imgf000240_0001
Chromatography (HP-LC) and Reversed Phase High Performance Liquid Chromatography HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in US Patent No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO96/33739).
Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulating Complexs (ISCOMs). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP0109942, WO 96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid of additional detergent. See WO 00/07621.
A review of the development of saponin based adjuvants can be found at Barr, et al., "ISCOMs and other saponin based adjuvants", Advanced Drug Delivery Reviews (1998) 32:247-271. See also Sjolander, et al., "Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines", Advanced Drug Delivery Reviews (1998) 32:321-338. D. Virosomes and Virus Like Particles (VLPs)
Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi). VLPs are discussed further in WO 03/024480, WO 03/024481, and Niikura et al., "Chimeric Recombinant Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle Presenting Foreign Epitopes", Virology (2002) 293:273-280; Lenz et al., "Papillomarivurs-Like Particles Induce Acute Activation of Dendritic Cells", Journal of Immunology (2001) 5246-5355; Pinto, et al., "Cellular Immune
Responses to Human Papillomavirus (HPV)-16 Ll Healthy Volunteers Immunized with Recombinant HPV-16 Ll Virus-Like Particles", Journal of Infectious Diseases (2003) 188:327-338; and Gerber et al., "Human Papillomavrisu Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Entertoxin Mutant R192G or CpG", Journal of Virology (2001) 75(10):4752-4760. Virosomes are discussed further in, for example, Gluck et al.,
"New Technology Platforms in the Development of Vaccines for the Future", Vaccine (2002) 20:B10 -B 16. Immunopotentiating reconstituted influenza virosomes (IRIV) are used as the subunit antigen delivery system in the intranasal Myal/mt JNFLEXAL™ product {Mischler & Metcalfe (2002)
P1J"1:: "l" „• "■ Uf1S SJ Ξ' .■■■■■ I= ■•' »;"" --ft ■' -i'1
Vactϊne 20 Suppl 5:B17-23} and the INFLUVAC PLUS™ product.
E. Bacterial or Microbial Derivatives
Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as: (1) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS)
Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. See Johnson et al. (1999) BioorgMed Chem Lett 9:2273-2278.
(2) Lipid A Derivatives
Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM- 174. OM-174 is described for example in Meraldi et al., "OM- 174, a New Adjuvant with a Potential for Human Use, Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242-310 from the circumsporozoite protein of Plasmodium berghei", Vaccine (2003) 21:2485-2491; and Pajak, et al., "The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo", Vaccine (2003) 21:836-842. (3) Immunostimulatoty oligonucleotides
Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine may be replaced with an analog such as 2'-deox-y-7-deazaguanosine. See Kandimalla, et al., "Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles", Nucleic Acids Research (2003) 31(9): 2393-2400; WO02/26757 and WO99/62923 for examples of possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, "CpG motifs: the active ingredient in bacterial extracts?", Nature Medicine (2003) 9(7): 831-835; McCluskie, et al., "Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA", FEMS Immunology and Medical Microbiology (2002) 32:179-185; WO98/40100; US Patent No. 6,207,646; US Patent No. 6,239,116 and US Patent No. 6,429,199.
The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. See
Kandimalla, et al., "Toll-like receptor 9: modulation of recognition and cytokine induction by novel ytithetiαCpG nNAs'^BipcheiKpal, Soaef Transactions (2003) 31 (part 3): 654-658. The CpG
Figure imgf000242_0001
sequence may be specific for inducing immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al., "CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha", J. Immunol. (2003)
170(8):4061-4068; Krieg, "From A to Z on CpG", TRENDS in Immunology (2002) 23(2): 64-65 and WO01/95935. Preferably, the CpG is a CpG-A ODN.
Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3 ' ends to form "immunomers". See, for example, Kandimalla, et al., "Secondary structures in CpG oligonucleotides affect immunostimulatory activity", BBRC (2003) 306:948-953; Kandimalla, et al., "Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic GpG DNAs", Biochemical Society Transactions (2003) 3J,(part 3):664-658; Bhagat et al., "CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents" BBRC (2003) 300:853-861 and WO 03/035836.
(4) ADP-ribosylating toxins and detoxified derivatives thereof. Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin "LT), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211 and as parenteral adjuvants in WO98/42375.
Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTRl 92G. The use of ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al., "The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin", Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al., "Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants", Vaccine (2001) 19:2534-2541; Pizza, et al., "LTK63 and LTR72, two mucosal adjuvants ready for clinical trials" Int. J. Med. Microbiol (2000) 290(4-5) :455-461; Scharton-Kersten et al., "Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits and Unrelated Adjuvants", Infection and Immunity (2000) 68(9):5306-5313; Ryan et al., "Mutants of Escherichia coli Heat- Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on ThI and Th2 Cells" Infection and Immunity (1999) 67(12):6270-6280; Partidos et al., "Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides", Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al., "Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines", Vaccines (2003) 2(2):285-293; and Pine et al., (2002) "Intranasal φmunirøtipn^withinf ueijza: »ya|)omp'gn4;'p detoxified mutant of heat labile entero
Figure imgf000243_0001
amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al., MoI. Microbiol (1995) 15(6): 1165- 1167, specifically incorporated herein by reference in its entirety.
F. Bioadhesives and Mucoadhesives
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al. (2001) /. Cont. ReIe. 70:267-276) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention. E.g. "WO99/27960.
G. Microparticles
Microparticles may also be used as adjuvants in the invention. Microparticles {i.e. a particle of ~100nm to ~150μm in diameter, more preferably ~200nm to ~30μm in diameter, and most preferably ~500nm to ~10μm in diameter) formed from materials that are biodegradable and non-toxic {e.g. a ρoly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly^actide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB). H. Liposomes
Examples of liposome formulations suitable for use as adjuvants are described in US Patent No. 6,090,406, US Patent No. 5,916,588, and EP 0 626 169.
/. Polyoxyethylene ether and P olyoxy ethylene Ester Formulations
Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters. WO99/52549. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152).
Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9- lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, ρolyoxytheylene-8-steoryl ether, ρolyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23 -lauryl ether.
J. Polyphosphazene (PCPP)
PCPP formulations are described, for example, in Andrianov et al., "Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions", Biomaterials (1998) 19(1-
3): 109-115 and Payne et al., "Protein Release from Polyphosphazene Matrices", Adv. Drug. Delivery Review (1998) 31(3):185-196. PC f-ΛϊTPiϊPStϊ^B 7 ii:;;i: 3 «:$
Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-noπnurarayl-1-alanyl-d-isoglutamine (nor- MDP), and N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3- hydiOxyphosphoryloxy)-ethylamine MTP-PE).
L. Imidazoquinolone Compounds.
Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues, described further in Stanley, "Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential" Clin Exp Dermatol (2002) 27(7):571-577 and Jones, "Resiquimod 3M", Curr Opin Investig Drugs (2003) 4(2):214-218.
The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention:
(1) a saponin and an oil-in-water emulsion (WO 99/11241);
(2) a saponin (e.g.., QS21) + a non-toxic LPS derivative (e.g. 3dMPL) (see WO 94/00153); (3) a saponin (e.g.., QS21) + a non-toxic LPS derivative (e.g. 3dMPL) + a cholesterol;
(4) a saponin {e.g. QS21 ) + 3 dMPL + IL- 12 (optionally + a sterol) (WO 98/57659);
(5) combinations of 3 dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231);
(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr- MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.
(7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™);
(8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dPML).
(9) one or more mineral salts (such as an aluminum salt) + an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif). Combination No. (9) is a preferred adjuvant combination.
M. Human Immunomodulators
Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor. Aluminum salts and MF59 are preferred adjuvants for use with injectable influenza vaccines.
Bacterial toxins and bioadhesives are preferred adjuvants for use with mucosally-delivered vaccines, such as nasal vaccines. ,_„ ,r,,,,.ijFh& iqTOumQgBpip''(jj∞ipQgitiθ|[S;;:iρf the present invention may be administed in combination with an antibiotic treatment regime. In one embodiment, the antibiotic is administered prior to administration of the antigen of the invention or the composition comprising the one or more of the antigens of the invention. In another embodiment, the antibiotic is administered subsequent to the adminstration of the one or more antigens of the invention or the composition comprising the one or more antigens of the invention. Examples of antibiotics suitable for use in the treatment of the Steptococcal infections of the invention include but are not limited to penicillin or a derivative thereof or clindamycin or the like. Further antigens
The compositions of the invention may further comprise one or more additional Gram positive bacterial antigens which are not associated with an AI. Preferably, the Gram positive bacterial antigens that are not associated with an AI can provide protection across more than one serotype or strain isolate. For example, a first non-AI antigen, in which the first non-AI antigen is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) homologous to the amino acid sequence of a second non-AI antigen, wherein the first and the second non-AI antigen are derived from the genomes of different serotypes of a Gram positive bacteria, may be further included in the compositions. The first non-AI antigen may also be homologous to the amino acid sequence of a third non-AI antigen, such that the first non-AI antigen, the second non-AI antigen, and the third non- AI antigen are derived from the genomes of different serotypes of a Gram positive bacteria. The first non-AI antigen may also be homologous to the amino acid sequence of a fourth non-AI antigen, such that the first non-AI antigen, the second non-AI antigen, the third non-AI antigen, and the fourth non- AI antigen are derived from the genomes of different serotypes of a Gram positive bacteria.
The first non-AI antigen may be GBS 322. The amino acid sequence of GBS 322 across GBS strains from serotypes Ia, Ib, II, III, V, and VIII is greater than 90%. Alternatively, the first non-AI antigen may be GBS 276. The amino acid sequence of GBS 276 across GBS strain from serotypes Ia, Ib, II, III, V, and VIII is greater than 90%. Table 13 provides the percent amino acid sequence identity of GBS 322 and GBS 276 across different GBS strains and serotypes.
Table 13: Conservation of GBS 322 and GBS 276 amino acid sequences
Figure imgf000245_0001
Figure imgf000246_0002
As an example, inclusion of a non-AI protein, GBS 322, in combination with AI antigens GBS 67, GBS 80, and GBS 104 provided protection to newborn mice in an active maternal immunization assay.
Table 14: Active maternal immunization assay for a combination of fragments from GBS 322, GBS 80, GBS 104, and GBS 67
Figure imgf000246_0001
In fact, the non-AI GBS 322 antigen may itself provide protection to newborn mice in an active maternal immunization assay.
Figure imgf000247_0001
Thus, inclusion of a non-AI protein in an immunogenic composition of the invention may provide increased protection a mammal.
The immunogenic compositions comprising S. pneumonaie AI polypeptides may further secondary SP protein antigens which include (a) any of the SP protein antigens disclosed in WO
02/077021 or U.S. provisional application , filed April 20, 2005 (Attorney Docket
Number 002441.00154), (2) immunogenic portions of the antigens comprising at least 7 contiguous amino acids, (3) proteins comprising amino acid sequences which retain immunogenic! ty and which are at least 95% identical to these SP protein antigens (e.g., 95%, 96%, 97%, 98%, 99%, or 99.5% identical), and (4) fusion proteins, including hybrid SP protein antigens, comprising (l)-(3).
Alternatively, the invention may include an immunogenic composition comprising a first and a second Gram positive bacteria non-AI protein, wherein the polynucleotide sequence encoding the sequence of the first non-AI protein is less than 90% (i.e., less than 90, 88, 86, 84, 82, 81, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35, or 30 percent) homologous than the corresponding sequence in the genome of the second non-AI protein.
The compositions of the invention may further comprise one or more additional non-Gram positive bacterial antigens, including additional bacterial, viral or parasitic antigens. The compositions of the invention may further comprise one or more additional non-GBS antigens, including additional bacterial, viral or parasitic antigens.
In another embodiment, the GBS antigen combinations of the invention are combined with one or more additional, non-GBS antigens suitable for use in a vaccine designed to protect elderly or immunocomprised individuals. For example, the GBS antigen combinations may be combined with an antigen derived from the group consisting of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitides, influenza, and Parainfluenza virus ('PIV). ^heraa^saccharide^c^Aphy^ftte antigen is used, it is preferably conjugated to a earner pr
Figure imgf000248_0001
otein in order to enhance immunogenicity {e.g. Ramsay et al. (2001) £α«ce£ 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon (2000) JR Coll Physicians Lond 34:163-168; Ahmad & Chapnick (1999) InfectDis Clin North Am 13:113-133, viϊ.; Goldblatt (1998) J. Med. Microbiol. 47:563-567; European patent 0 477 508; US Patent No. 5,306,492; International patent application WO98/42721; Conjugate Vaccines (eds. Cruse et al) ISBN 3805549326, particularly vol. 10:48-114; and Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X}. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRMi97 diphtheria toxoid is particularly preferred {Research Disclosure, 453077 (Jan 2002)} . Other carrier polypeptides include the N meningitidis outer membrane protein (EP-A-
0372501), synthetic peptides (EP-A-0378881; EP-A-0427347), heat shock proteins (WO 93/17712; WO 94/03208), pertussis proteins (WO 98/58668; EP A 0471177), protein D from H.influenzae (WO 00/56360), cytokines (WO 91/01146), lymphokines, hormones, growth factors, toxin A or B from C.difficile (WO00/61761), iron-uptake proteins (WO01/72337), etc. Where a mixture comprises capsular saccharides from both serogroups A and C, it may be preferred that the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g. 2:1, 3: 1, 4:1, 5:1, 10:1 or higher). Different saccharides can be conjugated to the same or different type of carrier protein. Any suitable conjugation reaction can be used, with any suitable linker where necessary.
Toxic protein antigens may be detoxified where necessary e.g. detoxification of pertussis toxin by chemical and/or genetic means.
Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. Antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
As an alternative to using protein antigens in the composition of the invention, nucleic acid encoding the antigen may be used {e.g. refs. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9:471-480; Apostolopoulos & Plebanski (2000) Curr Opin MoI Titer 2:441-447; Ilan (1999) Curr Opin MoI Ther 1:116-120; Dubensky et al. (2000) MoI Med 6:723-732; Robinson & Pertmer (2000) Adv Virus Res 55: 1-74; Donnelly et al. (2000) Am JRespir Crit Care Med 162(4 Pt 2):S190-193; and Davis (1999) Mt. Sinai J. Med. 66:84-90}. Protein components of the compositions of the invention may thus be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid) that encodes the protein. Definitions , „ „,„, ,,„... „ .....,, ,..., . „.„ pll 'i ,.•■' llJlb iiJ b / id! / id: .J 11SiI
The term "comprising" means "including" as well as "consisting" e.g. a composition
"comprising" X may consist exclusively of X or may include something additional e.g. X + Y.
The term "about" in relation to a numerical value x means, for example, x+10%. References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) Supplement 30. A preferred alignment is determined by the Smith- Waterman homology search algorithm using an affϊne gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith- Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.
The invention is further illustrated, without limitation, by the following examples. EXAMPLE 1: Binding of an Adhesin Island surface protein, GBS 80, to Fibrinogen and Fibronectin.
This example demonstrates that an Adhesin Island surface protein, GBS 80 can bind to fibrinogen and fibronectin.
An enzyme-linked immunosorbent assay (ELISA) was used to analyse the in vitro binding ability of recombinant GBS 80 to immobilized extra-cellular matrix (ECM) proteins but not to bovine serum albumin (BSA). Microtiter plates were coated with ECM proteins (fibrinogen, fibronectin, laminin, collagen type IV) and binding assessed by adding varying concentrations of a recombinant form of GBS 80, over-expressed and purified from E. coli (FIGURE 5A). Plates were then incubated sequentially with a) mouse anti-GBS 80 primary antibody; b) rabbit anti-mouse AP-conjugated secondary antibody; c) pNPP colorimetric substrate. Relative binding was measured by monitoring absorbance at 405 nm, using 595 nm as a reference wavelength. Figure 5b shows binding of recombinant GBS 80 to immobilized ECM proteins (1 μg) as a function of concentration of GBS 80. BSA was used as a negative control. Data points represent the means of OD405 values ± standard deviation for 3 wells.
Binding of GBS 80 to the tested ECM proteins was found to be concentration dependent and exhibited saturation kinetics. As is also evident from FIGURE 5, binding of GBS 80 to fibronectin and fibrinogen was greater than binding to laminin and collagen type IV at all the concentrations tested. EXAMPLE 2: GBS 80 is required for surface localization of GBS 104.
This example demonstrates that co-expression of GBS 80 is required for surface localization ofGBS 104.
The polycistronic nature of the Adhesin Island I mRNA was investigated through reverse transcriptase-PCR (RT-PCR) analysis employing primers designed to detect transcripts arising from contiguous genes. Total RNA was isolated from GBS cultures grown to an optical density at 600 nm (OD600) of 0,3 in, ,JHR,(,To,ddrHew4t,,brQth) by the RNeasy Total RNA isolation method (Qiagen)
IP1 C T-'''' U ?.::;ι'i Ui ;b ..•■' »:~. ■''' el: ..r.'li ■' J accorSing to the manufacturer's instructions. The absence of contaminating chromosomal DNA was confirmed by failure of the gene amplification reactions to generate a product detectable by agarose gel electrophoresis, in the absence of reverse transcriptase. RT-PCR analysis was performed with the Access RT-PCR system (Promega) according to the manufacturer's instructions, employing PCR cycling temperatures of 60°C for annealing and 700C for extension. Amplification products were visualized alongside 100-bρ DNA markers in 2% agarose gels after ethidium bromide staining.
FIGURE 5 shows that all the genes are co-transcribed as an operon. A schematic of the AI-I operon is shown above the agarose gel analysis of the RT-PCR products. Large rectangular arrows indicate the predicted transcript direction. Primer pairs were selected such as "1-4" cross the 3'fϊnish- 5'start of successive genes and overlap each gene by at least 200 bp. Additionally, "1" crosses a putative rho-independent transcriptional terminator. "5" is an internal GBS 80 control and "6" is an unrelated control from a highly expressed gene. Lanes: "a": RNA plus RTase enzyme; "b" RNA without RTase; "c": genomic DNA control. In the effort to elucidate the functions of the AI- 1 proteins, in frame deletions of all of the genes within the operon have been constructed and the resulting mutants characterized with respect to surface exposure of the encoded antigens (see FIGURE 8).
Each in-frame deletion mutation was constructed by splice overlap extension PCR (SOE- PCR) essentially as decribed by Horton et al. [Horton R. M., Z. L. Cai, S. N. Ho, L. R. Pease (1990) Biotechniques 8:528-35] using suitable primers and cloned into the temperature sensitive shuttle vector pJRS233 to replace the wild type copy by allelic exchange [Perez-Casal, J., J. A. Price, et al. (1993) MoI Microbiol 8(5): 809-19.]. AU plasmid constructions utilized standard molecular biology techniques, and the identities of DNA fragments generated by PCR were verified by sequencing. Following SOE-PCR, the resulting mutant DNA fragments were digested with Xhol and EcoRJ, and ligated into a similarly digested pJRS233. The resuting vectors were introduced by electroporation into the chromosome of 2603 and COHl GBS strains in a three-step process, essentially as described in Framson et al. [Framson, P. E., A. Nittayajarn, J. Merry, P. Youngman, and C. E. Rubens. (1997) Appl. Environ. Microbiol. 63(9):3539-47]. Briefly, the vector ρJRS233 contains an erm gene encoding erythromycin resistance and a temperature-sensitive gram-positive replicon that is active at 300C but not at 37°C. Initially, the constructs are electroporated into GBS electro-competent cells prepared as described by Frameson et al., and transformants containing free plasmid are selected by their ability to grow at 300C on Todd-Hewitt Broth (THB) agar plates containing 1 μg/ml erythromycin. The second step includes a selection step for strains in which the plasmid has integrated into the chromosome via a single recombination event over the homologous plasmid insert and chromosome sequence by their ability to grow at 37°C on THB agar medium containing 1 mg/ml erythromycin. In the third step, GBS cells containing the plasmid integrated within the chromosome (integrants) are serially passed in broth culture in the absence of antibiotics at 300C. Plasmid excision from the chromosome^yia^^pcctpidirec^nibinatioii event over the duplicated target gene sequence eflhet'cόrnpleteoi the allelic exchange or reconstituted the wild- type genotype. Subsequent loss of the plasmid in the absence of antibiotic selection pressure resulted in an erythromycin-sensitive phenotype. In order to assess gene replacement a screening of erythromycin-sensitive colonies was performed by analysis of the target gene PCR amplicons.
FIGURE 7 reports a schematic of the IS-I operon for each knock-out strain generated, along with the deletion position within the amino acidic sequence. Most data presented here concern the COHl deletion strains, in which the expression of each of the antigens is higher by DNA microarray analysis (data not shown) as well as detectable by FACS analysis (see FIGURE 8). The double mutant in 2603 Δ80, Δ104 double mutant was constructed by sequential allelic exchanges of the shown alleles.
Immunization protocol
Immune sera for FACS experiments were obtained as follows.
Groups of 4 CD-I outbred female mice 6-7 weeks old (Charles River Laboratories, Calco Italy) were immunized with the selected GBS antigens, (20 μg of each recombinant GBS antigen), suspended in 100 μl of PBS. Each group received 3 doses at days 0, 21 and 35. Immunization was performed through intra-peritoneal injection of the protein with an equal volume of Complete
Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses. In each immunization scheme negative and positive control groups are used. Immune response was monitored by using serum samples taken on day 0 and 49.
FACS analysis
Preparation of paraformaldehyde treated GBS cells and their FACS analysis were carried out as follows.
GBS serotype COHl strain cells were grown in Todd Hewitt Broth (THB; Difco Laboratories, Detroit, Mich.) to OD600nm = 0.5. The culture was centrifuged for 20 minutes at 5000 rpm and bacteria were washed once with PBS, resuspended in PBS containing 0.05% paraformaldehyde, and incubated for 1 hours at 37 0C and then overnight at 4°C. 50μl of fixed bacteria (OD600 0.1) were washed once with PBS, resuspended in 20μl of Newborn Calf Serum, (Sigma) and incubated for 20 min. at room temperature. The cells were then incubated for 1 hour at 4°C in lOOμl of preimmune or immune sera, diluted 1:200 in dilution buffer (PBS, 20% Newborn Calf Serum, 0.1% BSA). After centrifugation and washing with 200μl of washing buffer (0.1% BSA in PBS), samples were incubated for 1 hour at 4°C with 50μl of R-Phicoerytrin conjugated F(ab)2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; Inc.), diluted 1 : 100 in dilution buffer. Cells were washed with 200μl of washing buffer and resuspended in 200μl of PBS. Samples were analysed using a FACS Calibur apparatus (Becton Dickinson, Mountain View, Calf.) and data were analyzed using the Cell Quest Software (Becton Dickinson). A shift in mean fluorescence intensity of > 75 channels compared to preimmune sera from the same mice was considered positive. This cutoff ♦ „ „ ., • • e v» u* ■ Λ was αetermineα irom me mean.βlujj.tjvo Standard deviations of shirts obtained with control sera raised ifii1 f": "!"./'" 1,1 H LiI :!:;;n „■■ id! / n::::: ,d> 11J against mock purified recombinant proteins from cultures of E. coli carrying the empty expression vector and included in every experiment. Artifacts due to bacterial lysis were excluded using antisera raised against 6 different known cytoplasmic proteins all of which were negative FACS data on COHl single KO mutants for GBS 104 and GBS 80 indicated that GBS 80 is required for surface localization of GBS 104.
As shown in FIGURE 8, GBS 104 is not surface exposed in the Δ80 strain (second column, bottom), but is present in the whole protein extracts (see FIGURE 10). Mean shift values suggest that GBS 104 is partially responsible for GBS 80 surface exposure (Mean shift of GBS 80 is reduced to ~60% wild-type levels in Δ104), and that GBS 80 is over-expressed in the complemented strain (mean shift value -200% wild-type level). The Δ80/pGBS 80 strain contains the GBS 80 orf cloned in the shuttle-vector p AM401 (Wirth, R., F. Y. An, et al. (1986). J Bacteriol 165(3): 831-6). The vector alone does not alter the secretion pattern of GBS 104 (right column). FACS was performed on mid- log fixed bacteria with mouse polyclonal antibodies as indicated at left. Black peak is pre-immune sera, colored peaks are sera from immunized animals.
EXAMPLE 3: Deletion of GBS 80 causes attenuation in vivo.
This example demonstrates that deletion of GBS 80 causes attenuation in vivo, suggesting that this protein contributes to bacterial virulence. By using a mouse animal model, we studied the role of GBS 80 and GBS 104 in the virulence of S. agalactiae.
Groups of ten outbred female mice 5-6 week weeks old (Charles River Laboratories, Calco Italy) were inoculated intraperitoneally with different dilutions of the mutant strains and LD50 (lethal dose 50) were calculated according to the method of Reed and Muench [Reed, L. J. and H. Muench (1938).The American Journal of Hygiene 27(3): 493-7]. As presented in the table below the number of colony forming units (cfu) counted for both the Δ80 and the Δ80, Δ104 double mutants is about 10 fold higher when compared to the wild type strain suggesting that inactivation of GBS 80 but not GBS 104 is responsible for an attenuation in virulence. This finding indicates that GBS 80 gene in the AI-I might contribute to virulence. Table Lethal dose 50% analysis of AI-I mutants in the 2603 strain background. LD50s were performed by IP injection of female CDl mice at an age of 5-6 weeks. LD50s were calculated by the method of Reed and Muench (8).
Figure imgf000252_0001
EXAMPLE 4: Effect of Adhesin Island Sortase Deletions on Surface Antigen Presentation n ,,T,his example of adhesin island sortase deletions on surface antigen
Figure imgf000253_0001
presentation.
FACS analysis results set forth in FIGURE 9 show that a deletion in sortase SAG0648 prevented GBS 104 from reaching the surface and slightly reduced the surface exposure of GBS 80 (fourth panel; mean shift value -60% wild-type COHl). In the double sortase knock-out strain, neither antigen was surface exposed (far right panel). Either sortase alone was sufficient for GBS 80 to arrive at the bacterial surface (third and fourth columns, top). No effect was seen on surface exposure of antigens GBS 80 or GBS 104 in the ΔGBS 52 strain. Antibodies derived from purified GBS 52 were either non-specific or were FACS negative for GBS 52 (data not shown). FACS analysis was performed as described above (see EXAMPLE 2).
As shown in FIGURE 10, inactivation of GBS 80 has no effect on GBS 104 expression as much as GBS 104 knock out doesn't change the total amount GBS 80 expressed. The Western blot of whole protein extracts (strains noted above lanes) probed with anti-GBS 80 antisera is shown in panel A. Arrow indicates expected size of GBS 80 (60 kDa). GBS 80 antibodies recognize a doublet, the lower band is not present in ΔGBS 80 strains. Panel B shows a Western blot of whole protein extracts probed with anti-GBS 104 antisera. Arrow indicates expected size of GBS 104 (99.4 kDa). Protein extracts were prepared from the same bacterial cultures used for FACS (FIGURES 8 and 9). In conclusion, although GBS 104 does not arrive at the surface in the Δ80 strain by FACS (FIGURE 8, second column), it is present at approximately wild-type levels in the whole protein preps (B, second lane). Approximately 20 μg of each protein extract was loaded per lane. Western-blot analysis
Aliquots of total protein extract mixed with SDS loading buffer (Ix: 60 mM TRIS-HCl pH 6.8, 5% w/v SDS, 10% v/v glycerin, 0.1% Bromophenol Blue, 100 mM DTT) and boiled 5 minutes at 95° C, were loaded on a 12.5% SDS-PAGE precast gel (Biorad). The gel is run using a SDS-PAGE running buffer containing 250 mM TRIS, 2.5 mM Glycine and 0.1 %SDS. The gel is electroblotted onto nitrocellulose membrane at 200 mA for 60 minutes. The membrane is blocked for 60 minutes with PBS/0.05 % Tween-20 (Sigma), 10% skimmed milk powder and incubated O/N at 4° C with PBS/0.05 % Tween 20, 1% skimmed milk powder, with the appropriate dilution of the sera. After washing twice with PBS/0.05 % Tween, the membrane is incubated for 2 hours with peroxidase- conjugated secondary anti-mouse antibody (Amersham) diluted 1:4000. The nitrocellulose is washed three times for 10 minutes with PBS/0.05 % Tween and once with PBS and thereafter developed by Opti-4CN Substrate Kit (Biorad).
Example 5: Binding of Adhesin Island proteins to epithelial cells and effect of Adhesin Island proteins on capacity of GBS to adhere to epithelial cells. This example illustrates the binding of AI proteins to epithelial cells and the effect of AI proteins on the capacity of GBS to adhere to epithelial cells. .
Applicants analysed whether recombinant AI surface proteins GBS 80 or GBS 104 would demonstrate binding to various epithelial cells in a FACS analysis. Applicants also analysed whether deletion of AI surface .proteins CTP1S11S1O11OI; GBS 104 would i e < ffect the capacity of GBS to adhere to and pr TV U b U & ■■'■ »;;;" " »::;" -=* ;il! invade'ME180 cervical eeppiitthheelliiaall c i ells.
As shown in Figure 28, deletion of GBS 80 sequence from GBS strain isolate 2603 (serotype V) did not affect the capacity of the mutated GBS to adhere to and invade MEl 80 cervical epithelial cells. Here MEl 80 cervical carcinoma epithelial cells were infected with wild type GBS 2603 or
GBS 2603 Δ80 isogenic mutant. After two hours of infection, non-adherent bacteria were washed off and infection prolonged for a further two hours and four hours. In invasion experiments, after each time point, was followed by a two hour antibiotic treatment. Cells were then lysed with 1% saponin and lysates platedon TSA plates. As shown in Figure 28, there was little difference between the percent invasion or percent adhesion of wild type and mutant strains up to the four hour time point. Figure 30 repeats this experiment with both Δ104 and Δ 80 mutants from a different strain isolate. Here, ME180 cervical carcinoma epithelial cells were infected with GBS strain isolate COH (serotype III) wild type or COHl ΔGBS 104 or COHl Δ80 isogenic mutant. After one hour of infection, non-adherent bacteria were washed off and the cells were lysed with 1% saponin. The lysates were plated on TSA plates. As shown in Figure 30, while there was little difference in the percent invasion, there was a significant decrease in the percent association of the Δ104 mutant compared to both the wild type and Δ80 mutant.
The affect of AI surface proteins on the ability of GBS to translocate through an epithelial monolayer was also analysed. As shown in Figure 31, a GBS 80 knockout mutant strain partially loses the ability to translocate through an epithelial monolayer. Here epithelial monolayers were inoculated with wildtype or knockout mutant in the apical chamber of a transwell system for two hours and then non-adherent bacteria were washed off. Infection was prolonged for a further two and four hours. Samples were taken from the media of the basolateral side and the number of colony forming unties measured. Transepithelial electrical resistance measured prior to and after infection gave comparable values, indicating the maintenance of the integrity of the monolayer. By the six hour time point, the Δ80 mutants demonstrated a reduced percent transcytosis.
A similar experiment was conducted with GBS 104 knock out mutants. Here, as shown in Figure 22, the Δ104 mutants also demonstrated a reduced percent transcytosis, indicating that the mutant strains translocate through an epithelial monolayer less efficiently than their isogenic wild type counterparts.
Applicants also studied the effect of AI proteins on the capacity of a GBS strain to invade J774 macrophage-like cells. Here, J774 cells were infected with GBS COHl wild type or COHl ΔGBS104 or COHl ΔGBS80 isogenic mutants. After one hour of infection, non-adherent bacteria were washed off and intracellular bacteria were recovered at two, four and six hours post antibiotic treatment. At each time point, cells were lysed with 0.25% Triton X-100 and lysates plated on TSA plates. As shown in Figure 32, the Δ104 mutant demonstrated a significantly reduced percent invasion compared to both the wild type and Δ80 mutant. Example.6; nHupfiFoligomerjie'StraetoFeis comprising AI surface proteins GBS 80 and GBS 104.
P'r Iu "S ■■•' ' 'U" -! "" U .3 ..1 ic .>■' ii..: I1 si'
This example illustrates hyperoligomeric structures comprising AI surface proteins GBS 80 and GBS 104. A GBS isolate COHl (serotype III) was adapted to increase expression of GBS 80. Figure 34 presents a regular negative stain electron micrograph of this mutant; no pilus or hyperoligomeric structures are distinguishable on the surface of the bacteria. When the EM stain is based on anti-GBS 80 antibodies labelled with 10 or 20 run gold particles, the presence of GBS 80 throughout the hyperoligomeric structure is clearly indicated (Figures 36, 37 and 38). EM staining against GBS 104 (anti-GBS 104 antibodies labelled with 10 nm gold particles) also reveals the presence of GBS 104 primarily on or near the surface of the bacteria or potentially associated with bacterial peptidoglycans (Figure 39). Analysis of this same strain (over-expressing GBS 80) with a combination of both anti-GBS 80 (using 20 nm gold particles) and anti-GBS 104 (using 10 nm gold particles) reveals the presence of GBS 104 on the surface and within the hyperoligomeric structures (see Figures 40 and 41).
Example 7: GBS 80 is necessary for polymer formation and GBS 104 and sortase SAG0648 are necessary for efficient pili assembly
This example demonstrates that GBS 80 is necessary for formation of polymers and that GBS 104 and sortase SAG0648 are necessary for efficient pili assembly. GBS 80 and GBS 104 polymeric assembly was systematically analyzed in Cohl strain single knock out mutants of each of the relevant coding genes in AI-I (GBS 80, GBS 104, GBS 52, sagO647, and sagO648). Figure 41 provides
Western blots of total protein extracts (strains noted above lanes) probed with either anti-GBS 80 (left panel) sera or anti-GBS 104 sera (right panel) for each of these Cohl and Cohl knock out strains. (Cohl, wild type Cohl; Δ80, Cohl with GBS 80 knocked out; Δ104, Cohl with GBS 104 knocked out; Δ52, Cohl with GBS 52 knocked out; Δ647, Cohl with SAG0647 knocked out; Δ648, Cohl with SAG0648 knocked out, Δ647-8, Cohl with SAG0647 and SAG0648 knocked out; Δ8O/ρGBS8O,
Cohl with GBS 80 knocked out but complemented with a high copy number plasmid expressing GBS 80. Asterisks identify the monomer of GBS 80 and GBS 104.)
The smear of immunoreactive material observed in the wild type strain, along with its disappearance in Δ80 and Δ104 mutants, is consistent with the notion that such high molecular weight structures are composed of covalently linked (SDS-resistant) GBS 80 and GBS 104 subunits. The immunoblotting with both anti-GBS 80 (α-GBS 80) and anti-GBS 104 (α-GBS 104) revealed that deletion of sortase SAG0648 also interferes with the assembly of high molecular weight species, whereas the knock out mutant of the second sortase (SAG0647), even if somehow reduced, still maintains the ability to form polymeric structures. Total extracts form GBS were prepared as follows. Bacteria were grown in 50 ml of Todd-
Hewitt broth (Difco) to an OD600nIn of 0.5-0.6 and successively pelleted. After two washes in PBS the pellet was resuspended and incubated 3 hours at 37°C with mutanolisin. Cells were then lysed with at least three reez ng-t aw ng cyc es n ry ce an a 370C ath. e ysate was t en centrifuged to eli P lmmi Clnnaa Tttee t f/hhee U ccee Slllluupllaarr 5 ddeebb/rriiss 5 aann Tdd ttihheeI ss 3uuppee !9rrnn;atant was quantified. Approximately 40 μg of each protein extract was separated on SDS-PAGE. The gel was then subjected to immunoblotting with mice antisera and detected with chemiluminescence.
Example 8: GBS 80 is polymerized by an AI-2 sortase
This example illustrates that GBS 80 can be polymerized not only by AI-I sortases, but also by AI-2 sortases. Figure 42 shows total cell extract immunoblots of GBS 515 strain, which lacks AI- 1. The left panel, where an anti-GBS 67 sera was used, shows that GBS 67 from AI-2 is assembled into high-molecular weight-complexes, suggesting the formation of a second type of pilus. The same high molecular structure is observed when GBS 80 is highly expressed by reintroducing the gene within a plasmid (pGBS 80). By using anti-GBS 80 (right panel) sera on the same extracts, again it is observed that, with GBS 80 over expression (515/ρGBS 80), a high-molecular weight structure is assembled. This implies that, in the absence of AI-I sortases, AI-2 sortases (SAG1405 and SAG1406) can complement the lacking function, still being able to assemble GBS 80 in a pilus structure.
Example 9: Cohl produces a high molecular weight molecule, the GBS 80 pilin
This example illustrates that Cohl produces a high molecular weight molecule, greater than 1000 kDa, which is the GBS 80 pilin. Figure 43 provides silver-stained electrophoretic gels that show that Cohl produces two macromolecules. One of these macromolecules disappears in the Cohl GBS 80 knock out cells, but does not disappear in the Cohl GBS 52 knock out mutant cells. The last two lanes on the right were loaded with 15 times the amount loaded in the other lanes. This was done in order to be able to count the bands. By doing this, a conservative size estimate of the top bands was calculated by starting at 240 kDa and considering each of 14 higher bands as the result of consecutive additions of a GBS 80 monomer.
Cohl, wild type Cohl; Δ80, Cohl cells with GBS 80 knocked out; Δ52, Cohl cells with GBS 52 knocked out; Δ80/pGBS 80, Cohl cells with GBS 80 knocked out and complemented with a high copy number construct expressing GBS 80.
Example 10. GBS 52 is a minor component of the GBS pilus
This example illustrates that GBS 52 is present in the GBS pilus and is a minor component of the pilus. Figure 45 shows an immunoblot of total cell extracts from a GBS Cohl strain and a GBS Cohl strain knocked out for GBS 52 (Δ52). The total cell extracts were immunoblotted anti-GBS 80 antisera (left) and anti-GBS 52 antisera (right). Immunoblotting was performed using a 3-8% Tris- acetate polyacrylamide gel (Invitrogen) which provided excellent separation of large molecular weight proteins (see figure 41). When the gel was incubated with anti-GBS 80 sera, the bands from the Cohl wild-type strain appeared shifted when compared to the Δ52 mutant. This observation indicated a different size, of , the pjjtøs,,p,o4y,meric components in the two strains. When the same gel
F" C "i",/ ' U H t.» --'I .-•• IC ■>>' π:;,;; ,,,;:Ϊ« "•:.!' was stripped and incubated with anti-GBS 52 sera the high-molecular subunits in the Cohl wild-type strain showed similar molecular size of those in the correspondent lane in the left panel. These findings confirmed that GBS 52 is indeed associated with GBS 80 macro-molecular structures but represents a minor component of the GBS pilus.
Example 11: Pilus structures are present in the supernatant of GBS bacterial cultures
This example illustrates that the pilus structure assembled in Cohl GBS is present in the supernatant of a bacterial cell culture. Figure 46 shows an immunoblot where the protein extract of the supernatant from cultures of different GBS mutant strains (117 = Cohl GBS 80 knockout; 159= Cohl GBS 104 knockout; 202= Cohl GBS 52 knockout; 206= Cohl GBS sagO647 knockout; 208= Cohl GBS sagO648 knockout; 197= Cohl GBS sag0647/sag0648 knockout; 179= Cohl GBS 80 knockout complemented with a high copy plasmid expressing GBS 80). GBS 80 antisera detects the presence of pilus structures in the appropriate Cohl strains. The protein extract was prepared as follows. Bacteria were grown in THB to an ODgoonm of
0.5-0.6 and the supernatant was separated from the cells by centrifugation. The supernatant was then filtered (0 0.2 μm) and 1 ml was added with 60% TCA for protein precipitation. GBS pili were also extracted from the fraction of surface-exposed proteins in Cohl strain and its GBS 80 knock out mutant as described hereafter. Bacteria were grown to an ODόoonm of 0.6 in 50 ml of THB at 37°C. Cells were washed once with PBS and the pellet was then resuspended in 0.1 M KPO4 pH 6.2, 40% sucrose, 10 mM MgC12, 400U/ml mutanolysin and incubated 3 hours at 37°C. Protoplasts were separated by centrifugation and the supernatant was recovered and its protein content measured.
In order to study the dynamics of pilus production during different growth phases, 1 ml supernatant of a culture at different OD60Omn was TCA precipitated and loaded onto a 3-8% SDS-
PAGE as described before. Figure 47 shows the corresponding Western blot with GBS 80 anti-sera. The first group of lanes (left five sample lanes) refer to a Cohl strain growth (OD6oonm are noted above the lanes) whereas the second group of lanes (right five samples) are from a GBS 80 knock out strain over expressing GBS 80. The experiment shows that pilus macromolecular structures can be found in the supernatant in all of the growth phases tested.
Example 12: In GBS strain Cohl, only GBS 80 and a sortase (sagO647 or sagO648) is required for polymerization
This example describes requirements for pilus formation in Cohl. Figure 48 shows a Western blot of total protein extracts (prepared as described before) using anti-GBS 80 sera on Cohl clones. (Cohl, wild type Cohl; Δ104, Cohl knocked out for GBS 104, Δ647, Cohl knocked out for sagO647, Δ648, Cohl knocked for sagO648, Δ647-8, Cohl knocked out for sagO647 and sagO648; 515, wild tyJS'lf^tfϊaϊ itøJtøS OliϊiWfrϊfel WS a!!A|r 1 ; p80 a high copy number plasmid which expresses GBS 80.) The data show that only the double sortase mutant is unable to polymerize GBS 80 indicating that the 'conditio sine qua non ' for pilus polymerization is the co-existence of GBS 80 with at least one sortase. This result leads to a reasonable assumption that SAG 1405 and SAG 1406 are responsible for polymerization in this strain.
Example 13: GBS 80 can be expressed in L. lactis under its own promoter and terminator sequences
This example demonstrates that L. lactis, a non-pathogenic bacterium, can express GBS AI polypeptides such as GBS 80. L. lactis M1363 {J. Bacterial. 154 (1983): 1-9) was transformed with a construct encoding GBS 80. Briefly, the construct was prepared by cloning a DNA fragment containing the gene coding for GBS 80 under its own promoter and terminator sequences into plasmid pAM401 (a shuttle vector for E. coli and other Gram positive bacteria; J. Bacteriol. 163 (1986):831- 836). Total extracts of the transformed bacteria in log phase were separated on SDS-PAGE, transferred to membranes, and incubated with antiserum against GBS 80. A polypeptide corresponding to the molecular weight of GBS 80 was detected in the lanes containing total extracts of L. lactis transformed with the GBS 80 construct. See Figures 133 A and 133B, lanes 6 and 7. This same polypeptide was not detected in the lane containing total extracts of L. lactis not transformed with the GBS 80 construct, lane 9. This example shows that Z. lactis can express GBS 80 under its own promoter and terminator.
Example 14: L. lactis modified to express GBS AI-I under the GBS 80 promoter and terminator sequences expresses GBS 80 in polymeric structures
This example demonstrates the ability of L. lactis to express GBS AI-I polypeptides and to incorporate at least some of the polypeptides into oligomers. L. lactis was transformed with a construct containing the genes encoding GBS Al-I polypeptides. Briefly, the construct was prepared by cloning a DNA fragment containing the genes for GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences into construct pAM401. The construct was transformed into L. lactis M1363. Total extracts of log phase transformed bacteria were separated on reducing SDS-PAGE, transferred to membranes, and incubated with antiserum against GBS 80. A polypeptide with a molecular weight corresponding to the molecular weight of GBS 80 was detected in the lanes containing L. lactis transformed with the GBS AI-I encoding construct. See Figure 134, lane 2. In addition, the same lane also showed immunoreactivity of polypeptides having higher molecular weights than the polypeptide having the molecular weight of GBS 80. These higher molecular weight polypeptides are likely oligomers of GBS 80. Oligomers of similar molecular wfglfg
Figure imgf000259_0001
of the culture supernatant of the transformed L. lactis.
See lane 4 of Figure 135. Thus, this example shows that L. lactis transformed to express GBS AI-I can efficiently polymerize GBS 80 in the form of a pilus. This pilus structure can likely be purified from either the cell culture supernatant or cell extracts.
Example 15: Cloning and Expression of S. pneumoniae SpO462
This example describes the production of a clone encoding a SρO462 polypeptide and expression of the clone. To produce a clone encoding SpO462, the open reading frame encoding SpO462 was amplified using primers that annealed within the full-length SpO462 open reading frame sequence. Figure 150A provides a 893 amino acid sequence of SpO462. The primers used to produce a clone encoding the SρO462 polypeptide are shown in Figure 150B. These primers annealed to the nucleotide sequences encoding the amino acid residues indicated by underlining in Figure 150A. Amplification of the open reading frame encoding SpO462 using these primers produced the amplicon shown at lane 2 of the agarose gel provided in Figure 160. The SρO462 clone encodes amino acid residues 38-862 of the 893 amino acid residue SpO462 protein; the italicized residues in Figure 150A were eliminated. Figure 15 IA provides a schematic depiction of the recombinant SpO462 polypeptide. Figure 15 IB shows a schematic depiction of the fiill-length SρO462 polypeptide. Both the recombinant SpO462 encoded by the clone and the full-length SpO462 protein have two collagen binding protein type B (Cna B) domains and a von Hillebrand factor A (vWA) domain. The cloned recombinant SpO462 lacks the LPXTG motif present in the full-length SpO462 protein. Western blot analysis for expression of the SpO462 clone did not result in detection of polypeptides with serum obtained from S. pneumoniae-infected patients (Figure 152A) or GBS 80 antiserum (Figure 152B).
Example 16: Cloning and Expression of S. pneumoniae SpO463 This example describes the production of a clone encoding a SpO463 polypeptide and detection of recombinant SρO463 polypeptide expressed from the clone. To produce a clone encoding SpO463, the open reading frame encoding SρO463 was amplified using primers that annealed within the full-length SpO463 open reading frame sequence. Figure 153A provides a 665 amino acid sequence of SpO463. The primers used to produce the clone encoding SpO463 polypeptide are shown in Figure 153B. These primers annealed to the nucleotide sequences encoding the amino acid residues indicated by underlining in Figure 153 A. Amplification of the open reading frame encoding SρO463 using these primers produced the amplicon shown at lane 3 of the agarose gel provided in Figure 160. The SρO463 clone encodes amino acid residues 23-627 of the 665 amino acid residue SpO463 protein; the italicized residues in Figure 153A were eliminated. Figure 154A provides a schematic depiction of the recombinant SpO463 polypeptide. Figure 154B shows a schematic depiction of the full-length SpO463 polypeptide. Both the recombinant SpO463 encoded by the clone and the full-length SpO463 protein have a Cna B domain and an E box motif. The cloned recombinant
Figure imgf000260_0001
Spj^jg If cks f$3gEf§T§ m^ilf ' JMfSetfgiffiifhe full-length SρO463 protein. Expression of the SpO463 clone resulted in the detection of a 60 kD polypeptide, the expected molecular weight of the recombinant SpO463 polypeptide, by Western blot analysis. See Figure 155.
Example 17: Cloning and Expression of S. pneumoniae SpO464
This example describes the production of a clone encoding a SpO464 polypeptide and detection of recombinant SpO464 polypeptide expressed from the clone. To produce a clone encoding SpO464, the open reading frame encoding SρO464 was amplified using primers that annealed either within the full-length SρO464 open reading frame sequence. Figure 157A provides a 393 amino acid sequence of SpO464. The primers used to produce a clone encoding the SpO464 polypeptide are shown in Figure 157B. These primers annealed to the nucleotide sequences encoding the amino acid residues indicated by underlining in Figure 157A. Amplification of the open reading frame encoding SpO464 using these primers produced the amplicon shown at lane 4 of the agarose gel provided in Figure 160. The SpO464 clone encodes amino acid residues 19-356 of the 393 amino acid residue SpO464 protein; the italicized residues in Figure 157A were eliminated. Figure 158 A provides a schematic depiction of the recombinant SpO464 polypeptide. Figure 158B shows a schematic depiction of the full-length SpO464 polypeptide. Both the recombinant SpO464 encoded by the clone and the full-length SpO464 protein have two Cna B domains. The cloned recombinant SpO464 lacks the LPXTG motif present in the full-length SpO464 protein. Expression of the SpO464 clone resulted in the detection of a 38 kD polypeptide, the expected molecular weight of the recombinant SpO464 polypeptide, by Western blot analysis. See Figure 159.
Example 18: Intranasal Immunization of Mice with Recombinant L. lactis Expressing GBS 80 and Subsequent Challenge This example describes a method of intranasally immunizing mice using L. lactis that express
GBS 80. Intranasal immunization consisted of 3 doses at days 0, 14 and 28, each dose administered in three consecutive days. Each day, groups of 3 CD-I outbred female mice 6-7 weeks old (Charles River Laboratories, Calco Italy) were immunized intranasally with 109 or 1010 CFU of the recombinant Lactococcus lactis suspended in 20 μl of PBS. In each immunization scheme negative (wild-type L. lactis) and positive (recombinant GBS 80) control groups were used. The immune response of the dams was monitored by using serum samples taken on day 0 and 49. The female mice were bred 2-7 days after the last immunization (at approximately t= 36 - 37), and typically had a gestation period of 21 days. Within 48 hours of birth, the pups were challenged via LP. with GBS in a dose approximately equal to an amount which would be sufficient to kill 90 % of immunized pups (as determined by empirical data gathered from PBS control groups). The GBS challenge dose is preferably administered in 50ml of THB medium. Preferably, the pup challenge takes place at 56 to 61 days after the first immunization. The challenge inocula were prepared starting from frozen with THB prior to use. Survival of pups was
Figure imgf000261_0001
monitored for 5 days after challenge.
Example 19: Subcutaneous Immunization of Mice with Recombinant L. lactis Expressing GBS 80 and Subsequent Challenge
This example describes a method of subcutaneous immunization mice using L. lactis that express GBS 80. Subcutaneous immunization consists of 3 doses at days 0, 14 and 28. Groups of 3 CD-I outbred female mice 6-7 weeks old (Charles River Laboratories, Calco Italy) were injected subcutaneously with 109 or 1010 CFU of the recombinant Lactococcus lactis suspended in 100 μl of PBS. In each immunization scheme, negative (wild-type L. lactis) and positive (recombinant GBS80) control groups were used. The immune response of the dams was monitored by using serum samples taken on day 0 and 49. The female mice were bred 2-7 days after the last immunization (at approximately t= 36 - 37), and typically had a gestation period of 21 days. Within 48 hours of birth, the pups were challenged via LP. with GBS in a dose approximately equal to an amount which would be sufficient to kill 90 % of immunized pups (as determined by empirical data gathered from PBS control groups). The GBS challenge dose is preferably administered in 50ml of THB medium. Preferably, the pup challenge takes place at 56 to 61 days after the first immunization. The challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB prior to use. Survival of pups was monitored for 5 days after challenge.
Example 20: Immunization of Mice with GAS AI polypeptides and Subsequent Intranasal Challenge
This example describes a method of immunizing mice with GAS AI polypeptides and subsequently intranasally challenging the mice with GAS bacteria. Groups of 10 CDl female mice aged between 6 and 7 weeks are immunized with a combination of GAS antigens of the invention GAS 15, GAS 16, and GAS 18, (15 μg of each recombinant antigen, derived from Ml strain SF370) or Z. lactis expressing the Ml strain SF370 adhesin island, suspended in 100 μl of suitable solution. Each group receives 3 doses at days 0, 21 and 45. Immunization is performed through subcutaneous or intraperitoneal injection for the GAS 15, GAS 16, GAS 18 protein combination. The protein combination is administered with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses. Immunization is performed intranasally for thei. lactis expressing the Ml strain SF370 adhesin island. In each immunization scheme negative and positive control groups are used.
The negative control group for the mice immunized with the GAS 15, GAS 16, GAS 18 protein combination included mice immunized with PBS. The negative control group for the mice immunized with L. lactis expressing the Ml strain SF370 adhesin island, included mice immunized wiiffc gffifϊyilldpφlg; igcftϊjgS!' ($£?$; «r$nsformed with the pAM401 expression vector lacking any cloned adhesin island sequence.
The positive control groups included mice immunized with purified Ml strain SF370 M protein. Immunized mice are then anaesthetized with Zoletil and challenged intranasally with a 25 μL suspension containing 1.2 x 106 or 1.2 x 108 CFU of ISS 3348 in THB. Animals are observed daily and checked for survival.
Example 21: Active Maternal Immunization Assay As used herein, an Active Maternal Immunization assay refers to an in vivo protection assay where female mice are immunized with the test antigen composition. The female mice are then bred and their pups are challenged with a lethal dose of GBS. Serum titers of the female mice during the immunization schedule are measured as well as the survival time of the pups after challenge. Mouse immunization
Specifically, groups of 4 CD-I outbred female mice 6-8 weeks old (Charles River Laboratories, Calco Italy) are immunized with one or more GBS antigens, (20 μg of each recombinant GBS antigen), suspended in 100 μl of PBS. Each group receives 3 doses at days 0, 21 and 35. Immunization is performed through intra-peritoneal injection of the protein with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses. In each immunization scheme negative and positive control groups are used.
Immune response is monitored by using serum samples taken on day 0 and 49. The sera are analyzed as pools from each group of mice. Active maternal immunization
A maternal immunization/neonatal pup challenge model of GBS infection was used to verify the protective efficacy of the antigens in mice. The mouse protection study was adapted from Rodewald et al. (Rodewald et al. J. Infect. Diseases 166, 635 (1992)). In brief, CD-I female mice (6-8 weeks old) were immunized before breeding, as described above. The mice received 20 μg of protein per dose when immunized with a single antigen and 60 μg of protein per dose (15 μg of each antigen) when immunized with the combination of antigens. Mice were bred 2-7 days after the last immunization. Within 48 h of birth, pups were injected intraperitoneally with 50 μl of GBS culture. Challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB before use. In preliminary experiments (not shown), the challenge doses per pup for each strain tested were determined to cause 90% lethality. Survival of pups was monitored for 2 days after challenge. Protection was calculated as (percentage
Figure imgf000263_0001
divided by percentage deadControl multiplied by
100. Data were evaluated for statistical significance by Fisher's exact test.
The invention encompasses, but is not limited to, the embodiments enumerated below.
1. An immunogenic composition comprising a purified Group B Streptococcus (GBS) adhesin island (AI) polypeptide in oligomeric form. 2. The immunogenic composition of embodiment 1 wherein the GBS AI polypeptide is selected from a GBS AI-I.
3. The immunogenic composition of embodiment 1 wherein the GBS AI polypeptide is selected from a GBS AI-2.
1. An immunogenic composition comprising a purified Group B Streptococcus (GBS) adhesin island (AI) polypeptide in oligomeric form.
2. The immunogenic composition of embodiment 1 wherein the GBS AI polypeptide is selected from a GBS AI-I.
3. The immunogenic composition of embodiment 1 wherein the GBS AI polypeptide is selected from a GBS AI-2. 4. The immunogenic composition of any of embodiments 1-3 wherein the GBS AI polypeptide comprises a sortase substrate motif.
5. The immunogenic composition of embodiment 4 wherein the sortase substrate motif is an LPXTG motif.
6. The immunogenic composition of embodiment 5 wherein the LPXTG motif is represented by the amino acid sequence XPXTG, wherein the X at amino acid position 1 is an L, an I, or an F and the X at amino acid position 3 is any amino acid residue.
7. The immunogenic composition of any one of embodiments 1-3 wherein the GBS AI polypeptide affects the ability of GBS bacteria to adhere to epithelial cells.
8. The immunogenic composition of any one of embodiments 1-3 wherein the GBS AI polypeptide affects the ability of GBS bacteria to invade epithelial cells.
9. The immunogenic composition of any one of embodiments 1-3 wherein the GBS AI polypeptide affects the ability of GBS bacteria to translocate through an epithelial cell layer.
10. The immunogenic composition of any one of embodiments 1-3 wherein the GBS AI polypeptide is capable of associating with an epithelial cell surface. 11. The immunogenic composition of embodiment 10 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface.
12. The immunogenic composition of any of embodiments 1-3 wherein the GBS AI polypeptide is a full-length GBS AI protein.
13. The immunogenic composition of any of embodiments 1-3 wherein the GBS AI polypeptide is a fragment of a full-length GBS AI protein.
14. The immunogenic composition of embodiment 13 wherein the fragment comprises at least 7 contiguous amino acid residues of the GBS AI protein. ,,,,.,, ||...,,
Figure imgf000265_0001
of embodiment 2 wherein the GBS AI polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof.
16. The immunogenic composition of embodiment 3 wherein the GBS AI polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof.
17. The immunogenic composition of embodiment 15 wherein the GBS AI polypeptide is GBS 80.
18. The immunogenic composition of any of embodiments 1-3 or 15-17 wherein the oligomeric form is a hyperoligomer. 19. The immunogenic composition of any of embodiments 1-3, or 15-17 further comprising a
Gram positive bacterium antigen not associated with an AI.
20. The immunogenic composition of embodiment 19 wherein the antigen is selected from the group consisting of GBS 322 and GBS 276.
21. The immunogenic composition of embodiment 20 wherein the antigen is GBS 322. 22. An immunogenic composition comprising a purified Gram positive bacteria adhesin island (AI) polypeptide in an oligomeric form.
23. The immunogenic composition of embodiment 22 wherein the Gram positive bacteria is of a genus selected from the group consisting of Streptococcus, Enterococcus, Staphylococcus, or
Listeria. 24. The immunogenic composition of embodiment 23 wherein the Gram positive bacteria is of the genus Streptococcus.
25. The immunogenic composition of any of embodiments 22-24 wherein the Gram positive bacteria AI polypeptide comprises a sortase substrate motif.
26. The immunogenic composition of embodiment 25 wherein the sortase substrate motif is an LPXTG motif.
27. The immunogenic composition of any one of embodiments 22-24 wherein the Gram positive bacteria AI polypeptide affects the ability of Gram positive bacteria to adhere to epithelial cells.
28. The immunogenic composition of any one of embodiments 22-24 wherein the Gram positive bacteria AI polypeptide affects the ability of Gram positive bacteria to invade epithelial cells.
29. The immunogenic composition of any one of embodiments 22-24 wherein the Gram positive bacteria AI polypeptide affects the ability of Gram positive bacteria to translocate through an epithelial cell layer.
30. The immunogenic composition of any one of embodiments 22-24 wherein the Gram positive bacteria AI polypeptide is capable of associating with an epithelial cell surface.
31. The immunogenic composition of embodiment 30 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface. ,p, ip" 'cjømpgitfpn of any of embodiments 22-24 wherein the Gram positive
Figure imgf000266_0001
bacteria AI polypeptide is a full-length Gram positive bacteria AI protein.
33. The immunogenic composition of any of embodiments 22-24 wherein the Gram positive bacteria AI polypeptide is a fragment of a full-length Gram positive bacteria AI protein. 34. The immunogenic composition of embodiment 33 wherein the fragment comprises at least 7 contiguous amino acid residues of the Gram positive bacteria AI protein.
35. The immunogenic composition of embodiment 24 wherein the genus Streptococcus bacteria is Group A Streptococcus (GAS) bacteria and the Gram positive bacteria AI polypeptide is a GAS AI polypeptide. 36. The immunogenic composition of embodiment 35 wherein the GAS AI polypeptide is selected from a GAS AI-I.
37. The immunogenic composition of embodiment 35 wherein the GAS AI polypeptide is selected from a GAS AI-2.
38. The immunogenic composition of embodiment 35 wherein the GAS AI polypeptide is selected from a GAS AI-3.
39. The immunogenic composition of embodiment 35 wherein the GAS AI polypeptide is selected from a GAS AI-4.
40. The immunogenic composition of any of embodiments 35-39 wherein the GAS AI polypeptide comprises a sortase substrate motif. 41. The immunogenic composition of embodiment 40 wherein the sortase substrate motif is an LPXTG motif.
42. The immunogenic composition of embodiment 41 wherein the LPXTG motif is represented by XXXXG, wherein the X at the first amino acid position is an L, a V, an E, or a Q, wherein the X at the second amino acid position is P if the X at the first amino acid position is an L, the X at the second amino acid position is a V if the X at the first amino acid position is an E or a Q, or the X at the second amino acid position is a V or a P if the X at the first amino acid position is a V, wherein the X at the third amino acid position is any amino acid residue, and wherein the X at the fourth amino acid position is a T if the X at the first amino acid position is a V, an E, or a Q, or the X at the fourth amino acid position is a T, an S, or an A if the X at the first amino acid position is an L. 43. The immunogenic composition of any one of embodiments 35-39 wherein the GAS AI polypeptide affects the ability of GAS bacteria to adhere to epithelial cells.
44. The immunogenic composition of any one of embodiments 35-39 wherein the GAS AI polypeptide affects the ability of GAS bacteria to invade epithelial cells.
45. The immunogenic composition of any one of embodiments 35-39 wherein the GAS AI polypeptide affects the ability of GAS bacteria to translocate through an epithelial cell layer.
46. The immunogenic composition of any one of embodiments 35-39 wherein the GAS AI polypeptide is capable of associating with an epithelial cell surface. p Ij'"" 'ψ-/ ^Ψ: \}ψ^ΑWPBψ^ψ'(ψ^Ψ^}^011 °f embodiment 46 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface.
48. The immunogenic composition of any of embodiments 35-39 wherein the GAS AI polypeptide is a full-length GAS AI protein. 49. The immunogenic composition of any of embodiments 35-39 wherein the GAS AI polypeptide is a fragment of a full-length GAS AI protein.
50. The immunogenic composition of embodiment 49 wherein the fragment comprises at least 7 contiguous amino acid residues of the GAS AI protein.
51. The immunogenic composition of embodiment 36 wherein the GAS AI-I polypeptide is selected from the group consisting of M6_SpyO157, M6_SρyO159, M6_Spy0160, CDC SS
410_fimbrial, ISS3650_fimbrial, DSM2071_fϊmbrial, and fragments thereof.
52. The immunogenic composition of embodiment 37 wherein the GAS AI-2 polypeptide is selected from the group consisting of GAS 15, GAS 16, GAS 18, and fragments thereof.
53. The immunogenic composition of embodiment 38 wherein the GAS AI-3 polypeptide is selected from the group consisting of SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104,
SPsOlOO, SPs0102, SPs0104, SPs0106, orf78, orf80, orf82, orf84, spyM18_0126, spyM18_0128, spyM18_O13O, spyM18_0132, SpyoM01000156, SpyoMO 1000155, SpyoMO 1000154, SpyoM01000153, SpyoMO 1000152, SpyoM01000151, SpyoMO 1000150, SpyoMO 1000149, ISS3040_fimbrial, ISS3776_fimbrial, ISS4959_fimbrial, and fragments thereof. 53. The immunogenic composition of embodiment 39 wherein the GAS AI-4 polypeptide is selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, ISS4538_funbrial, and fragments thereof.
54. The immunogenic composition of embodiment 24 wherein the Streptococcus bacteria is Streptococcus pneumoniae and the Gram positive bacteria AI polypeptide is a S. pneumoniae AI polypeptide.
55. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide comprises a sortase substrate motif.
56. The immunogenic composition of embodiment 55 wherein the sortase substrate motif is an LPXTG motif.
57. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide affects the ability of S. pneumoniae to adhere to epithelial cells.
58. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide affects the ability of S. pneumoniae to invade epithelial cells. 59. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide affects the ability of S. pneumoniae to translocate through an epithelial cell layer.
60. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide is capable of associating with an epithelial cell surface. njji a δl./ ijTjIiffiijiij^njEjnog^mciii'^Qitnppgjfion of embodiment 60 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface.
62. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide is a full-length S. pneumoniae AI protein. 63. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide is a fragment of a full-length S. pneumoniae AI protein.
64. The immunogenic composition of embodiment 63 wherein the fragment comprises at least 7 contiguous amino acid residues of the S. pneumoniae AI protein.
65. The immunogenic composition of embodiment 54 wherein the S. pneumoniae AI polypeptide is selected from the group consisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670, ORF3_14CSR, ORF4_14CSR, ORF5J4CSR, ORF3_19AH, ORF4J9AH, ORF5_19AH, ORF3_19FTW, ORF4_19FTW, ORF5J9FTW, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, and fragments thereof. 66. The immunogenic composition of any one of embodiments 22-24, 35-39, 51-54, or 65 wherein the oligomeric form is a hyperoligomer.
67. The immunogenic composition of any one of embodiments 22-24, 35-39, 51-54, or 65 further comprising a Gram positive bacteria antigen not associated with an AI.
68. The immunogenic composition of embodiment 67 wherein the antigen is selected from the group consisting of GBS 322 and GBS 276.
69. An immunogenic composition comprising a first and a second Group B Streptococcus (GBS) adhesin island (AI) polypeptide.
70. The immunogenic composition of embodiment 69 wherein a full-length polynucleotide sequence encoding for the first GBS AI polypeptide is not present in a GBS bacteria genome comprising a polynucleotide sequence encoding for the second GBS AI polypeptide.
71. The immunogenic composition of embodiment 69 wherein polynucleotides encoding the first and the second GBS AI polypeptide are each present in genomes of more than one GBS serotype and strain isolate.
72. The immunogenic composition of embodiment 69 wherein the first GBS AI polypeptide is encoded by a GBS AI-I .
73. The immunogenic composition of embodiment 69 wherein the first GBS AI polypeptide is encoded by a GBS AI-2.
74. The immunogenic composition of embodiment 72 wherein the second GBS AI polypeptide is encoded by a GBS AI-2. , 75. The immunogenic composition of embodiment 73 wherein the second GBS AI polypeptide is encoded by a GBS AI-2.
76. The immunogenic composition of embodiment 72 wherein the second GBS AI polypeptide is encoded by a GBS AI-I. P Iu ψ'-/''' lFte IB1SS1J0^F?' ffSP'-f*'011 0^ embodiment 73 wherein the second GBS AI polypeptide is encoded by a GBS AI-I .
78. The immunogenic composition of embodiment 72 wherein the first GBS AI polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof. 79. The immunogenic composition of embodiment 73 wherein the first GBS AI polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof.
80. The immunogenic composition of embodiment 74 or 75 wherein the second GBS AI polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof, and wherein the first and the second GBS AI polypeptide are not the same polypeptide.
81. The immunogenic composition of embodiment 76 or 77 wherein the second GBS AI polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof, and wherein the first and the second GBS AI polypeptide are not the same polypeptide. 82. The immunogenic composition of any one of embodiments 69-77 wherein the first GBS
AI polypeptide comprises a sortase substrate motif.
83. The immunogenic composition of embodiment 82 wherein the sortase substrate motif is an LPXTG motif.
84. The immunogenic composition of embodiment 83 wherein the LPXTG motif is represented by the sequence XPXTG, wherein the X at amino acid position 1 is an L, an I, or an F and the X at amino acid position 3 is any amino acid residue.
85. The immunogenic composition of any one of embodiments 69-77 wherein the first GBS AI polypeptide affects the ability of GBS bacteria to adhere to epithelial cells.
86. The immunogenic composition of any one of embodiments 69-77 wherein the first GBS AI polypeptide affects the ability of GBS bacteria to invade epithelial cells.
87. The immunogenic composition of any one of embodiments 69-77 wherein the first GBS AI polypeptide affects the ability of GBS bacteria to translocate through an epithelial cell layer.
88. The immunogenic composition of any one of embodiments 69-77 wherein the first GBS AI polypeptide is capable of associating with an epithelial cell surface. 89. The immunogenic composition of embodiment 88 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface.
90. The immunogenic composition of any of embodiments 69-77 wherein the first GBS AI polypeptide is a full-length GBS AI protein.
91. The immunogenic composition of any of embodiments 69-77 wherein the first GBS AI polypeptide is a fragment of a full-length GBS AI protein.
92. The immunogenic, composition of embodiment 91 wherein the fragment comprises at least 7 contiguous amino acid residues of the first GBS AI protein. p..,. ,r»
Figure imgf000270_0001
cpi|pos|tjy of any one of embodiments 69-79 wherein the first GBS
AI polypeptide is in oligomeric form.
94. The immunogenic composition of any one of embodiments 69-77 wherein the second GBS AI polypeptide is in oligomeric form. 95. The immunogenic composition of any one of embodiments 69-79 wherein the first and the second GBS AI polypeptide are associated in a single oligomeric form.
96. The immunogenic composition of embodiment 95 wherein the first and the second GBS AI polypeptides are chemically associated.
97. The immunogenic composition of embodiment 95 wherein the first and the second GBS AI polypeptides are physically associated.
98. The immunogenic composition of embodiment 93 wherein the oligomeric form is a hyperoligomer.
99. The immunogenic composition of embodiment 94 wherein the oligomeric form is a hyperoligomer. 100. The immunogenic composition of embodiment 76 wherein the first GBS AI polypeptide is GBS 80 and the second GBS AI polypeptide is GBS 104.
101. The immunogenic composition of embodiment 74 wherein the first GBS AI polypeptide is GBS 80 and the second GBS AI polypeptide is GBS 67.
102. The immunogenic composition of any one of embodiments 69-79, 100, or 101 further comprising a GBS polypeptide not associated with an AI.
103. The immunogenic composition of embodiment 102 wherein the GBS polypeptide not associated with an AI is selected from the group consisting of GBS 322 and GBS 276.
104. The immunogenic composition of embodiment 103 wherein the GBS polypeptide not associated with an AI is GBS 322. 105. An immunogenic composition comprising a first and a second Gram positive bacteria adhesin island (AI) polypeptide.
106. The immunogenic composition of embodiment 105 wherein a full length polynucleotide sequence encoding for the first Gram positive bacteria AI polypeptide is not present in a genome of a Gram positive bacteria comprising a full length polynucleotide sequence encoding for the second Gram positive bacteria AI polypeptide.
107. The immunogenic composition of embodiment 105 wherein polynucleotides encoding the first and the second Gram positive bacteria AI polypeptide are each present in genomes of more than one Gram positive bacteria serotype and strain isolate.
108. The immunogenic composition of embodiment 105 wherein the first and the second Gram positive bacteria AI polypeptides are of different Gram positive bacteria species.
109. The immunogenic composition of embodiment 105 wherein the first and the second Gram positive bacteria AI polypeptides are of the same Gram positive bacteria species. the first and the second
Figure imgf000271_0001
111. The immunogenic composition of embodiment 105 wherein the first and the second Gram positive bacteria AI polypeptides are from the same AI subtype. 112. The immunogenic composition of embodiment 105 wherein the first Gram positive bacteria AI polypeptide has detectable surface exposure on a first Gram positive bacteria strain or serotype but not a second Gram positive bacteria strain or fsubtype and the second Gram positive bacteria AI polypeptide has detectable surface exposure on the second Gram positive bacteria strain or serotype but not the first Gram positive bacteria strain or serotype. 113. The immunogenic composition of embodiment 105 wherein the Gram positive bacteria is S. pneumonaie, S. mutans, E. faecalis, E. faecium, C. difficile, L. monocytogenes, or C. diphtheriae.
114. The immunogenic composition of any of embodiments 105-113 wherein the first and the second Gram positive bacteria AI polypeptides comprise a sortase substrate motif.
115. The immunogenic composition of embodiment 114 wherein the sortase substrate motif is an LPXTG motif.
116. The immunogenic composition of embodiment 115 wherein the LPXTG motif is represented by XXXXG, wherein the X at amino acid position 1 is an L, a V, an E, an I, an F, or a Q, wherein X at amino acid position 2 is a P if X at amino acid position 1 is an L, an I, or an F, wherein X at amino acid position 2 is a V if X at amino acid position 1 is a E or a Q, wherein X at amino acid position 2 is a V or a P if X at amino acid position 1 is a V, wherein X at amino acid position 3 is any amino acid residue, wherein X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, I, F, or Q, and wherein X at amino acid position 4 is a T, S, or A if X at amino acid position 1 is an L.
117. The immunogenic composition of embodiment 105 wherein the first Gram positive bacteria AI polypeptide is a first Group A Streptococcus (GAS) AI polypeptide. 118. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide comprises a sortase substrate motif.
119. The immunogenic composition of embodiment 118 wherein the sortase substrate motif is an LPXTG motif.
120. The immunogenic composition of embodiment 119 wherein the LPXTG motif is represented by XXXXG, wherein the X at the first amino acid position is an L, a V, an E, or a Q, wherein the X at the second amino acid position is P if the X at the first amino acid position is an L, the X at the second amino acid position is a V if the X at the first amino acid position is an E or a Q, or the X at the second amino acid position is a V or a P if the X at the first amino acid position is a V, wherein the X at the third amino acid position is any amino acid residue, and wherein the X at the fourth amino acid position is a T if the X at the first amino acid position is a V, an E, or a Q, or the X at the fourth amino acid position is a T, an S, or an A if the X at the first amino acid position is an L.
121. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide affects the ability of GAS bacteria to adhere to epithelial cells. IP !-.. 'I22.,- j| Jfl^p|[;|r^uil^|efflcj;:;:jpα|jpϊ),jpsition of embodiment 117 wherein the first GAS AI polypeptide affects the ability of GAS bacteria to invade epithelial cells.
123. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide affects the ability of GAS bacteria to translocate through an epithelial cell layer. 124. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is capable of associating with an epithelial cell surface.
125. The immunogenic composition of embodiment 117 wherein the associating with an epithelial. cell surface is binding to the epithelial cell surface.
126. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is a full-length GAS AI protein.
127. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is a fragment of a full-length GAS AI protein.
128. The immunogenic composition of embodiment 127 wherein the fragment comprises at least 7 contiguous amino acid residues of the GAS AI protein. 129. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is a first GAS AI-I polypeptide.
130. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is a first GAS AI-2 polypeptide.
131. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is a first GAS AI-3 polypeptide.
132. The immunogenic composition of embodiment 117 wherein the first GAS AI polypeptide is a first GAS AI-4 polypeptide.
133. The immunogenic composition of any one of embodiments 117 or 129-132 wherein the second Gram positive bacteria AI polypeptide is a second GAS AI polypeptide. 134. The immunogenic composition of embodiment 133 wherein the second GAS AI polypeptide is a second GAS AI-I polypeptide.
135. The immunogenic composition of embodiment 133 wherein the second GAS AI polypeptide is a second GAS AI-2 polypeptide.
136. The immunogenic composition of embodiment 133 wherein the second GAS AI polypeptide is a second GAS AI-3 polypeptide.
137. The immunogenic composition of embodiment 133 wherein the second GAS AI polypeptide is a second GAS AI-4 polypeptide.
138. The immunogenic composition of embodiment 129 wherein the first GAS AI-I polypeptide is selected from the group consisting of M6_Sρy0157, M6_Spy0159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, DSM2071_fimbrial, and fragments thereof.
139. The immunogenic composition of embodiment 130 wherein the first GAS AI-2 polypeptide is selected from the group consisting of GAS 15, GAS 16, GAS 18, and fragments thereof. in, U ij40/
Figure imgf000273_0001
of embodiment 131 wherein the first GAS AI-3 polypeptide is selected from the group consisting of SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPs0106, orf78, orfSO, orf82, orf84, spyM18_0126, spyM18_O128, spyM18_O13O, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoMO 1000152, SpyoMOl 000151, SpyoMO 1000150, SpyoMO 1000149, ISS3040_fimbrial, ISS3776_fimbrial, ISS4959Jϊmbrial, and fragments thereof.
141. The immunogenic composition of embodiment 132 wherein the first GAS Al-4 polypeptide is selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, ISS4538_fimbrial, and fragments thereof.
142. The immunogenic composition of embodiment 134 wherein the second GAS AI-I polypeptide is selected from the group consisting of M6_Spy0157, M6_SρyO159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fϊmbrial, DSM2071_fimbrial, and fragments thereof.
143. The immunogenic composition of embodiment 135 wherein the second GAS AI-2 polypeptide is selected from the group consisting of GAS 15 , GAS 16, GAS 18 , and fragments thereof.
144. The immunogenic composition of embodiment 136 wherein the second GAS AI-3 polypeptide is selected from the group consisting of SpyM3_OO98, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPsOlOo, orf78, orfSO, orfS2, orf84, spyM18_0126, spyM18_O128, spyM18_O13O, spyM18_0132, SρyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoMO 1000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, ISS4959_fimbrial, and fragments thereof.
145. The immunogenic composition of embodiment 137 wherein the second GAS AI-4 polypeptide is selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296Jϊmbrial, 20020069_fimbrial, CDC SS 635_fϊmbrial, ISS4883_Fimbrial, ISS4538_fϊmbrial, and fragments thereof.
146. The immunogenic composition of any one of embodiments 117-132 or 138-141 wherein the second Gram positive bacteria AI polypeptide is a Group B Streptococcus (GBS) AI polypeptide.
147. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide comprises a sortase substrate motif. 148. The immunogenic composition of embodiment 147 wherein the sortase substrate motif is an LPXTG motif.
149. The immunogenic composition of embodiment 148 wherein the LPXTG motif is represented by the amino acid sequence XPXTG, wherein the X at amino acid position 1 is an L, an I, or an F and the X at amino acid position 3 is any amino acid residue. 150. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide affects the ability of GBS bacteria to adhere to epithelial cells.
151. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide affects the ability of GBS bacteria to invade epithelial cells. Ip1 J,...., 41152, ' of embodiment 146 wherein the GBS AI polypeptide
Figure imgf000274_0001
affects the ability of GBS bacteria to translocate through an epithelial cell layer.
153. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide is capable of associating with an epithelial cell surface. 154. The immunogenic composition of embodiment 146 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface.
155. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide is a full-length GBS AI protein. »
156. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide is a fragment of a full-length GBS AI protein.
157. The immunogenic composition of embodiment 156 wherein the fragment comprises at least 7 contiguous amino acid residues of the GBS AI protein.
158. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide is a GBS AI-I polypeptide. 159. The immunogenic composition of embodiment 146 wherein the GBS AI polypeptide is a GBS AI-2 polypeptide.
160. The immunogenic composition of embodiment 158 wherein the GBS AI-I polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof.
161. The immunogenic composition of embodiment 159 wherein the GBS AI-2 polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof.
162. The immunogenic composition of any one of embodiments 117-132 or 138-141 wherein the second Gram positive bacteria AI polypeptide is a Streptococcus pneumoniae AI polypeptide.
163. The immunogenic composition of embodiment 162 wherein the S. pneumoniae AI polypeptide comprises a sortase substrate motif.
164. The immunogenic composition of embodiment 163 wherein the sortase substrate motif is an LPXTG motif.
165. The immunogenic composition of embodiment 162 wherein the S. pneumoniae AI polypeptide affects the ability of S. pneumoniae to adhere to epithelial cells. 166. The immunogenic composition of embodiment 162 S. pneumoniae AI polypeptide affects the ability of S. pneumoniae to invade epithelial cells.
167. The immunogenic composition of embodiment 162 wherein the S. pneumoniae AI polypeptide affects the ability of S. pneumoniae to translocate through an epithelial cell layer.
168. The immunogenic composition of embodiment 162 wherein the S. pneumoniae AI polypeptide is capable of associating with an epithelial cell surface.
169. The immunogenic composition of embodiment 168 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface. ».»ι, Il -WO," ||T{te;
Figure imgf000275_0001
ffiPmP^ftion of embodiment 162 wherein the S. pneumoniae AI polypeptide is a full-length S. pneumoniae AI protein.
171. The immunogenic composition of embodiment 162 wherein the S. pneumoniae AI polypeptide is a fragment of a full-length S. pneumoniae AI protein. 172. The immunogenic composition of embodiment 162 wherein the fragment comprises at least 7 contiguous amino acid residues of the S. pneumoniae AI protein.
173. The immunogenic composition of embodiment 162 wherein the S. pneumoniae AI polypeptide is selected from the group consisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670,
ORF3J4CSR, ORF4_14CSR, ORF5_14CSR, ORF3 19AH, ORF4_19AH, ORF5J9AH, ORF3J9FTW, ORF4_19FTW, ORF5_19FTW, ORF3_23FP, ORF4_23FP, ORF5_23FP,
ORF3_23FTW, ORF4_23FTW, ORF5J23FTW, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF3_6BSP,
ORF4_6BSP, ORF5_6BSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, and fragments thereof.
174. The immunogenic composition of any one of embodiments 105-117 wherein the first
Gram positive bacteria AI polypeptide is in oligomeric form. 175. The immunogenic composition of embodiment 174 wherein the oligomeric form is a hyperoligomer.
176. The immunogenic composition of embodiment 174 wherein the second Gram positive bacteria AI polypeptide is in oligomeric form.
177. The immunogenic composition of embodiment 176 wherein the oligomeric form is a hyperoligomer.
178. The immunogenic composition of embodiment 176 wherein the first and the second Gram positive bacteria AI polypeptide are associated in a single oligomeric form.
179. The immunogenic composition of embodiment 178 wherein the first and the second Gram positive bacteria AI polypeptide are chemically associated. 180. The immunogenic composition of embodiment 178 wherein the first and the second
Gram positive bacteria AI polypeptide are physically associated.
181. The immunogenic composition of any one of embodiments 105-117 further comprising a Gram positive bacteria polypeptide not associated with an AI.
182. The immunogenic composition of embodiment 181 wherein the Gram positive bacteria polypeptide not associated with an AI is selected from the group consisting of GBS 322 and GBS 276.
183. The immunogenic composition of embodiment 182 wherein the Gram positive bacteria polypeptide not associated with an AI is GBS 322.
184. A modified Gram positive bacterium adapted to produce increased levels of AI surface protein. 185. The modified Gram positive bacterium of embodiment 184 wherein the AI surface protein is in oligomeric form.
186. The modified Gram positive bacterium of embodiment 185 wherein the oligomeric form is a hyperoligomer. .p. ,,,,,, ,1,3.7. ,. positive, bacterium of any one of embodiments 184-186 which is a
Figure imgf000276_0001
Group B Streptococcus bacterium.
188. The modified Gram positive bacterium of any one of embodiments 184-186 which is a Group A Streptococcus bacterium. 189. The modified Gram positive bacterium of any one of embodiments 184-186 which is a non-pathogenic Gram positive bacterium.
190. The modified Gram positive bacterium of embodiment 189 wherein the non-pathogenic Gram positive bacterium is Streptococus gordonii.
191. The modified Gram positive bacterium of embodiment 189 wherein the non-pathogenic Gram positive bacterium is Lactococcus lactis.
192. The modified Gram positive bacterium of any one of embodiments 184-186 which has been inactivated and wherein the AI surface protein is exposed on the surface of the Gram positive bacterium.
193. The modified Gram positive bacterium of any one of embodiments 184-186 which has been attenuated- and wherein the AI surface protein is exposed on the surface of the Gram positive bacterium.
194. The modified GBS bacterium of embodiment 187 which has been inactivated and wherein the AI surface protein is exposed on the surface of the GBS bacterium.
195. The modified GBS bacterium of embodiment 187 which has been attenuated and wherein the AI surface protein is exposed on the surface of the GBS bacterium.
196. The modified GAS bacterium of embodiment 188 which has been inactivated and wherein the AI surface protein is exposed on the surface of the GAS bacterium.
197. The modified GAS bacterium of embodiment 188 which has been attenuated and wherein the AI surface protein is exposed on the surface of the GAS bacterium. 198. The modified non-pathogenic bacterium of embodiment 189 which has been inactivated and wherein the AI surface protein is exposed on the surface of the non-pathogenic Gram positive bacterium.
199. The modified non-pathogenic bacterium of embodiment 189 which has been attenuated and wherein the AI surface protein is exposed on the surface of the non-pathogenic Gram positive bacterium.
200. A method for manufacturing an oligomeric adhesin island (AI) surface antigen comprising: culturing a Gram positive bacterium that expresses an oligomeric AI surface antigen and isolating the expressed oligomeric AI surface antigen. 201. The method of embodiment 200 wherein the step of isolating is performed by collecting said oligomeric AI surface antigen from Gram positive bacterium secretions in the Gram positive bacterium culture.
202. The method of embodiment 200 further comprising a step of purifying. wherein the oligomeric AI surface antigen is purified
Figure imgf000277_0001
from the Gram positive bacterium cell
204. The method of embodiment 200 wherein the Gram positive bacterium is adapted for increased AI protein expression. 205. The method of any one of embodiments 200-204 wherein the Gram positive bacterium is a Group A Streptococcus bacterium.
206. The method of any one of embodiments 200-204 wherein the Gram positive bacterium is a Group B Streptococcus bacterium.
207. The method of any one of embodiments 200-204 wherein the oligomeric AI surface antigen is in hyperoligomeric form.
208. The method of embodiment 200 wherein the Gram positive bacterium expresses the oligomeric AI surface antigen recombinantly.
209. The method of embodiment 208 wherein the Gram positive bacterium further manipulated expresses at least 1 AI sortase. 210. The modified Gram positive bacterium of any one of embodiments 184-186 which is a
S. pneumoniae bacterium.
211. The method of any one of embodiments 200-204 wherein the Gram positive bacterium is S. pneumoniae.

Claims

WE.CLAIM:,
1. An immunogenic composition comprising a purified Group B Streptococcus (GBS) adhesin island (AI) polypeptide in oligomeric form.
2. The immunogenic composition of claim 1 wherein the GBS AI polypeptide is selected from a GBS AI-I .
3. The immunogenic composition of claim 1 wherein the GBS AI polypeptide is selected from a GBS AI-2.
4. The immunogenic composition of claim 2 wherein the GBS AI polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof. 5. The immunogenic composition of claim 3 wherein the GBS AI polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof.
6. The immunogenic composition of claim 4 wherein the GBS AI polypeptide is GBS 80.
7. The immunogenic composition of any of claims 1-6 wherein the oligomeric form is a hyperoligomer.
8 (22). An immunogenic composition comprising a purified Gram positive bacteria adhesin island (AI) polypeptide in an oligomeric form.
9 (23). The immunogenic composition of claim 8 wherein the Gram positive bacteria is of a genus selected from the group consisting of Streptococcus, Enterococcus, Staphylococcus, Clostridium, Corynebacteriuni, or Listeria. 10 (24). The immunogenic composition of claim 9 wherein the Gram positive bacteria is of the genus Streptococcus.
11 (35). The immunogenic composition of claim 10 wherein the genus Streptococcus bacteria is Group A Streptococcus (GAS) bacteria and the Gram positive bacteria AI polypeptide is a GAS AI polypeptide.
12 (36). The immunogenic composition of claim 11 wherein the GAS AI polypeptide is selected from a GAS AI-I.
13 (37). The immunogenic composition of claim 11 wherein the GAS AI polypeptide is selected from a GAS AI-2. 14 (38). The immunogenic composition of claim 11 wherein the GAS AI polypeptide is selected from a GAS AI-3.
15 (39). The immunogenic composition of claim 11 wherein the GAS AI polypeptide is selected from a GAS AI-4.
16 (66). The immunogenic composition of any one of claims 8-15 wherein the oligomeric form is a hyperoligomer. ,
17. An immunogenic composition comprising a first and a second Group B Streptococcus (GBS) adhesin island (AI) polypeptide. ,,...„ , P !■&. ,T|be,|;itηmiti;nogenic of claim 17 wherein the first GBS AI polypeptide is
Figure imgf000279_0001
encoded by a GBS AI-1.
19. The immunogenic composition of claim 18 wherein the second GBS AI polypeptide is encoded by a GBS AI-2. 20. The immunogenic composition of claim 18 wherein the first GBS AI polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof.
21. The immunogenic composition of claim 19 wherein the second GBS AI polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof, and wherein the first and the second GBS AI polypeptide are not the same polypeptide.
22. The immunogenic composition of claim 19 wherein the first GBS AI polypeptide is GBS 80 and the second GBS AI polypeptide is GBS 67.
23. An immunogenic composition comprising a first and a second Gram positive bacteria adhesin island (AI) polypeptide. 24. The immunogenic composition of claim 23 wherein the Gram positive bacteria is
Streptococcus, Enterococcus, Staphylococcus, Clostridium, Corynebacterium, or Listeria.
25. The immunogenic composition of claim 23 wherein the first Gram positive bacteria AI polypeptide is a first Group A Streptococcus (GAS) AI polypeptide.
26. The immunogenic composition of claim 25 wherein the first GAS AI polypeptide is a first GAS AM polypeptide.
27. The immunogenic composition of claim 25 wherein the first GAS AI polypeptide is a first GAS AI-2 polypeptide.
28. The immunogenic composition of claim 25 wherein the first GAS AI polypeptide is a first GAS AI-3 polypeptide. 29. The immunogenic composition of claim 25 wherein the first GAS AI polypeptide is a first GAS AI-4 polypeptide.
30. The immunogenic composition of any one of claims 25-29 wherein the second Gram positive bacteria AI polypeptide is a second GAS AI polypeptide.
31. The immunogenic composition of claim 30 wherein the second GAS AI polypeptide is a second GAS AI-I polypeptide.
32. The immunogenic composition of claim 30 wherein the second GAS AI polypeptide is a second GAS AI-2 polypeptide.
33. The immunogenic composition of claim 30 wherein the second GAS AI polypeptide is a second GAS AI-3 polypeptide. 34. The immunogenic composition of claim 30 wherein the second GAS AI polypeptide is a second GAS AI-4 polypeptide.
35. A modified Gram positive bacterium adapted to produce increased levels of AI surface protein. of claim 35 wherein the AI surface protein is in
Figure imgf000280_0001
oligomeric form.
37. The modified Gram positive bacterium of claim 36 wherein the oligomeric form is a hyperoligomer. 38. The modified Gram positive bacterium of any one of claims 35-37 which is a nonpathogenic Gram positive bacterium.
39. The modified Gram positive bacterium of claim 38 wherein the non-pathogenic Gram positive bacterium is Lactococcus lactis.
40. A method for manufacturing an oligomeric adhesin island (AI) surface antigen comprising: culturing a Gram positive bacterium that expresses an oligomeric AI surface antigen and isolating the expressed oligomeric AI surface antigen.
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