US20090317420A1 - Immunogenic compositions for gram positive bacteria such as streptococcus agalactiae - Google Patents

Immunogenic compositions for gram positive bacteria such as streptococcus agalactiae

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US20090317420A1
US20090317420A1 US11658842 US65884205A US2009317420A1 US 20090317420 A1 US20090317420 A1 US 20090317420A1 US 11658842 US11658842 US 11658842 US 65884205 A US65884205 A US 65884205A US 2009317420 A1 US2009317420 A1 US 2009317420A1
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ai
gbs
gas
protein
surface
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John Telford
Guido Grandi
Peter Lauer
Marlrosa Mora
Immaculada Margarit y Ros
Domenico Malone
Guiliano Bensi
Daniela Rinaudo
Vega Masignani
Michelle Barocchi
Rino Rappuloi
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Novartis Vaccines and Diagnostics Inc
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Chiron Corp
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092Streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/0208Specific bacteria not otherwise provided for
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/05Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/08Clostridium, e.g. Clostridium tetani
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/085Staphylococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/095Neisseria
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/381Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus
    • Y02A50/384Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus
    • Y02A50/395Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus the disease being Tick-borne encephalitis [TBE]
    • Y02A50/396Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a virus of the genus Flavivirus the disease being Tick-borne encephalitis [TBE] the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera

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

    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 Lancefield 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. Sci. USA, 10.1073/pnas.182380799) 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-specificity and poor immunogenicity, and so there is a need for effective vaccines against S. agalactiae infection.
  • S. agalactiae is classified as a gram positive bacterium, a collection of about 21 genera of 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. 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).
  • 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-1” or “GBS AI-1”, 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-1 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 FIGS. 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 FIGS. 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-1 comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (“AI-1 proteins”). Specifically, AI-1 includes polynucleotide sequences encoding for two or more of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. One or more of the AI-1 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the AI-1 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF.
  • AI-1 typically resides on an approximately 16.1 kb transposon-like element frequently inserted into the open reading frame for trmA. One or more of the AI-1 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-1 may encode at least one surface protein. Alternatively, AI-1 may encode at least two surface proteins and at least one sortase. Preferably, AI-1 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-1 protein of the composition may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. GBS AI-1 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-1 proteins, AI-1 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. coli).
  • 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.
  • One or more of the 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. 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-1, 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-1 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 present 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-1, 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.
  • It is also an object of the invention to provide further and improved compositions for 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 hyperoligomeric 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, 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.
  • 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 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 (tee6), 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., Mol. 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 tee6 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 microscopy of strain M62724 with antisera specific for the product of tee6 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-1) 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 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 eradicate 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 form 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.
  • Gas Adhesin Island of the invention preferably include a divergently transcribeds 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.
  • 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 FIG. 51A and FIG. 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-1 proteins”). GAS AI-1 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 fimbrial structural subunit. The fimbrial structural subunit (also known as tee6) 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-1 includes polynucleotide sequences encoding for two or more of M6_Spy0157, M6_Spy0158, M6_Spy0159, M6_Spy0160, M6_Spy0161. The GAS AI-1 may also include polynucleotide sequences encoding for any one of CDC SS 410_fimbrial, ISS3650_fimbrial, DSM2071_fimbrial
  • A preferred immunogenic composition of the invention comprises a GAS AI-1 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-1 surface protein in oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising GAS AI-1 surface proteins may be purified or otherwise formulated for use in immunogenic compositions.
  • One or more of the GAS AI-1 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-1 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-1 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 encode for at least one surface protein. Alternatively, GAS AI-1 may encode for at least two surface proteins and at least one sortase. Preferably, GAS AI-1 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-1 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-1 protein of the composition may be selected from the group consisting of M6_Spy0157, M6_Spy0158, M6_Spy0159, M6_Spy0160 M6_Spy0161, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial. GAS AI-1 surface proteins M6_Spy0157 (a fibronectin binding protein), M6_Spy0159 (a collagen adhesion protein, Cpa), M6_Spy0160 (a fimbrial structural subunit, tee6), CDC SS 410_fimbrial (a fimbrial structural subunit), ISS3650_fimbrial (a fimbrial structural subunit), and DSM2071_fimbrial (a fimbrial structural subunit) are preferred GAS AI-1 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-1 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-1 proteins, GAS AI-1 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-1 surface proteins may be used alone, in combination with other GAS AI-1 surface proteins or in combination with other GAS AI surface proteins. Preferably, the immunogenic compositions of the invention include the GAS AI-1 fimbrial structural subunit (tee6) and the GAS AI-1 collagen binding protein. Still more preferably, the immunogenic compositions of the invention include the GAS AI-1 fimbrial structural subunit (tee6).
  • 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”). GAS AI-2 preferably comprises surface proteins, a srtB sortase, a srtC1 sortase and a rofA divergently transcribed transcriptional regulator.
  • Specifically, GAS AI-2 includes polynucleotide sequences encoding for two or more of GAS15, Spy0127, GAS16, GAS17, GAS18, Spy0131, Spy0133, 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 srtC1 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 srtC1 sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO:167) motif. GAS srtC1 may be differentially regulated by rofA.
  • The GAS AI-2 protein of the composition may be selected from the group consisting of GAS15, Spy0127, GAS16, GAS17, GAS18, Spy0131, Spy0133, and GAS20. GAS AI-2 surface proteins GAS15 (Cpa), GAS16 (thought to be a fimbrial protein, M1128), GAS18 (M1_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 UPXTG 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 GAS18 (M1_Spy0130). 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 GAS AI-3may also be used in immunogonic 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 fimbrial protein, and a F2 like fibronectin-binding protein. GAS AI-3 surface proteins may also include a hypothetical surface protein. The fimbrial 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 protein, 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 SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, SpyM30104, Sps0100, Sps0101, Sps0102, Sps0103, Sps0104, Sps0105, Sps0106, orf78, orf79, orf80, orf81, orf82, orf83, orf84, spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, spyM180132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. In one embodiment, GAS AI-3 may include open reading frames encoding for two or more of SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, and SpyM30104. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of Sps0100, Sps0101, Sps0102, Sps0103, Sps0104, Sps0105, and Sps0106. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of orf78, orf79, orf80, orf81, orf82, orf83, and orf84. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, and spyM180132. Alternatively, GAS AI-3 may include open reading frames encoding for two or more of SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and SpyoM01000149. Alternatively, GAS AI-1 may also include polynucleotide sequences encoding for any one of ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
  • One or more of the GAS AI-3 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 (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 SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, SpyM30104, Sps0100, Sps0101, Sps0102, Sps0103, Sps0104, Sps0105, Sps0106, orf78, orf79, orf80, orf81, orf82, orf83, orf84, spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, spyM180132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. GAS AI-3 surface proteins SpyM30098, SpyM30100, SpyM30102, SpyM30104, SPs0100, SPs0102, SPs0104, SPs0106, orf78, orf80, orf82, orf84, spyM180126, spyM180128, spyM180130, spyM180132, SpyoM01000155, SpyoM01000153, SpyoM01000151, SpyoM01000149, 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 SpyM30102, M3_Sps0104, M5_orf82, or spyM180130), 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.
  • Representative examples of the GAS AI-3 fimbrial protein include SpyM30100, M3_Sps0102, M5_orf80, spyM18128, SpyoM01000153, ISS3040_fimbrial, ISS3776_fimbrial, ISS4959_fimbrial.
  • Representative examples of the GAS AI-3 collagen binding protein include SpyM30098, M3_Sps0100, M5_orf78, spyM180126, and SpyoM01000155.
  • Representative examples of the GAS AI-3 fibronectin binding protein PrtF2 include SpyM30104, M3_Sps0106, M5_orf84 and spyM180132, and SpyoM01000149.
  • 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, F1 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 fimbral 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.
  • One or more of the GAS AI-4 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-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_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial. GAS AI-4 surface proteins 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, 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 PrtF1 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 may be 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 AI-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 AI-1 and GAS AI-2; GAS AI-1 and GAS AI-3; GAS AI-1 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.
  • The immunogenic compositions may 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 S. 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 rlrA.
  • A schematic of the organization of a S. pneumoniae AI locus is provided in FIG. 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. pneumoniae AI-a and AI-b. S. pneumoniae strains that comprise AI-a include 14 CSR 10, 19A Hungary 6, 23 F Poland 15, 670, 6B Finland 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. 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 S. 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 S. 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 (“S. pneumoniae AI proteins”). Specifically, S. pneumoniae strain 670 AI includes polynucleotide sequences encoding for two or more of orf1670, orf3670, orf4670, orf5670, orf6670, orf7670, and orf8670.
  • One or more of the S. 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 S. 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 CSR10 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 CSR10 includes polynucleotide sequences encoding for two or more of ORF214CSR, ORF314CSR, ORF414CSR, ORF514CSR, ORF614CSR, ORF714CSR, and ORF814CSR.
  • One or more of the S. pneumoniae AI from 14 CSR10 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 CSR10 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 ORF219AH, ORF319AH, ORF419AH, ORF519AH, ORF619AH, ORF719AH, and ORF819AH.
  • 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.
  • S. pneumoniae AI from 19F Taiwan 14 comprises a series of approximately seven openreading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (“S. pneumoniae AI proteins”). Specifically, S. pneumoniae AI from 19F Taiwan 14 includes polynucleotide sequences encoding for two or more of ORF219FTW, ORF319FTW, ORF419FTW, ORF519FTW, ORF619FTW, ORF719FTW, and ORF819FTW.
  • 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 ORF223FP, ORF323FP, ORF423FP, ORF523FP, ORF623FP, ORF723FP, and ORF823FP.
  • 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, S. pneumoniae AI from 23F Taiwan 15 includes polynucleotide sequences encoding for two or more of ORF223FTW, ORF323FTW, ORF423FTW, ORF523FTW, ORF623FTW, ORF723FTW, and ORF823FTW.
  • 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 ORF26BF, ORF36BF, ORF46BF, ORF56BF, ORF66BF, ORF76BF, and ORF86BF.
  • 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. pneumoniae AI proteins”). Specifically, S. pneumoniae AI from 6B Spain 2 includespolynucleotide sequences encoding for two or more of ORF26BSP, ORF36BSP, ORF46BSP, ORF56BSP, ORF66BSP, ORF76BSP, and ORF86BSP.
  • 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 AI 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 (“S. pneumoniae AI proteins”). Specifically, S. pneumoniae AI from 9V Spain 3 includes polynucleotide sequences encoding for two or more of ORF29VSP, ORF39VSP, ORF49VSP, ORF59VSP, ORF69VSP, ORF79VSP, and ORF89VSP.
  • 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, orf1670, orf3670, orf4670, orf5670, orf6670, orf7670, orf8670, ORF214CSR, ORF314CSR, ORF414CSR, ORF514CSR, ORF614CSR, ORF714CSR, ORF814CSR, ORF219AH, ORF319AH, ORF419AH, ORF519AH, ORF619AH, ORF719AH, ORF819AH, ORF219FTW, ORF319FTW, ORF419FTW, ORF519FTW, ORF619FTW, ORF719FTW, ORF819FTW, ORF223FP, ORF323FP, ORF423FP, ORF523FP, ORF623FP, ORF723FP, ORF823FP, ORF223FTW, ORF323FTW, ORF423FTW, ORF523FTW, ORF623FTW, ORF723FTW, ORF823FTW, ORF26BF, ORF36BF, ORF46BF, ORF56BF, ORF66BF, ORF76BF, ORF86BF, ORF26BSP, ORF36BSP, ORF46BSP, ORF56BSP, ORF66BSP, ORF76BSP, ORF86BSP, ORF29VSP, ORF39VSP, ORF49VSP, ORF59VSP, ORF69VSP, ORF79VSP and, ORF89VSP.
  • 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 selected from two or more of the group consisting of SP0462, SP0463, SP0464, orf3670, orf4670, orf5670, ORF314CSR, ORF414CSR, ORF514CSR, ORF319AH, ORF419AH, ORF519AH, ORF319FTW, ORF419FTW, ORF59FTW, ORF323FP, ORF423FP, ORF523FP, ORF323FW, ORF423FTW, ORF523FTW, ORF36BF, ORF46BF, ORF56BF, ORF36BSP, ORF46BSP, ORF56BSP, ORF39VSP, ORF49VSP, and ORF59VSP.
  • 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 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 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
  • FIG. 1 presents a schematic depiction of Adhesin Island 1 (“AI-1”) comprising open reading frames for GBS 80, GBS 52, SAG0647, SAG0648 and GBS 104.
  • FIG. 2 illustrates the identification of AI-1 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 CJB111; GBS serotype III, strain isolate COH1 and GBS serotype 1a, strain isolate A909). (An AI-1 was not identified in GBS serotype 1b, strain isolate H36B or GBS serotype 1a, strain isolate 515).
  • FIG. 3 presents a schematic depiction of the correlation between AI-1 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).
  • FIG. 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 II, strain isolate NEM316; GBS serotype 1b, strain isolate H36B; GBS serotype V, strain isolate CJB111; GBS serotype U, strain isolate 18RS21; and GBS serotype 1a, strain isolate 515). FIG. 4 also illustrates the identification of AI-2 comprising open reading frames encoding for 01520 (a sortase), 01521, 01522, (a sortase), 01523 (spb1), 01524 and 01525 (or sequences having sequence homology thereto).
  • FIG. 5 presents data showing that GBS 80 binds to fibronectin and fibrinogen in ELISA.
  • FIG. 6 illustrates that all genes in AI-1 are co-transcribed as an operon.
  • FIG. 7 presents schematic depictions of in-frame deletion mutations within AI-1.
  • FIG. 8 presents FACS data showing that GBS 80 is required for surface localization of GBS 104.
  • FIG. 9 presents FACS data showing that sortases SAG0647 and SAG0648 play a semi-redundant role in surface exposure of GBS 80 and GBS 104.
  • FIG. 10 presents Western Blots of the in-frame deletion mutants probed with anti-GBS80 and anti-GBS 104 antisera.
  • FIG. 11: Electron micrograph of surface exposed pili structures in Streptococcus agalactiae containing GBS 80.
  • FIG. 12: PHD predicted secondary structure of GBS 067.
  • FIGS. 13, 14 and 15: Electron micrograph of surface exposed pili structures of strain isolate COH1 of Streptococcus agalactiae containing a plasmid insert encoding GBS 80.
  • FIGS. 16 and 17: Electron micrograph of surface exposed pili structure of wild type strain isolate COH1 of Streptococcus agalactiae.
  • FIG. 18: Alignment of polynucleotide sequences of AI-1 from serotype V, strain isolates 2603 and CJB111; serotype II, strain isolate 18RS21; serotype III, strain isolates COH1 and NEM316; and serotype 1a, strain isolate A909.
  • FIG. 19: Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolates 2603 and CJB111; serotype U, strain isolate 18RS21; serotype 1b, strain isolate H36B; and serotype 1a, strain isolate 515.
  • FIG. 20: Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolate 2603 and serotype III, strain isolate NEM316.
  • FIG. 21: Alignment of polynucleotide sequences of AI-2 from serotype III, strain isolate COH1 and serotype Ia, strain isolate A909.
  • FIG. 22: Alignment of amino acid sequences of AI-1 surface protein GBS 80 from serotype V, strain isolates 2603 and CJB111; serotype 1a, strain isolate A909; serotype III, strain isolates COH1 and NEM316.
  • FIG. 23: Alignment of amino acid sequences of AI-1 surface protein GBS 104 from serotype V, strain isolates 2603 and CJB111; serotype III, strain isolates COH1 and NEM316; and serotype II, strain isolate 18RS21.
  • FIG. 24: Alignment of amino acid sequences of AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJB111; serotype 1a, strain isolate 515; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype III, strain isolate NEM316.
  • FIG. 25: Illustrates that GBS closely associates with tight junctions and cross the monolayer of ME180 cervical epithelial cells by a paracellular route.
  • FIG. 26: Illustrates GBS infection of ME180 cells.
  • FIG. 27: Illustrates that GBS 80 recombinant protein does not bind to epithelial cells.
  • FIG. 28: Illustrates that deletion of GBS 80 does not effect the capacity of GBS strain 2603 V/R to adhere and invade ME180 cervical epithelial cells.
  • FIG. 29: Illustrates binding of recombinant GBS 104 protein to epithelial cells.
  • FIG. 30: Illustrates that deletion of GBS 104 in the GBS strain COH1, reduces the capacity of GBS to adhere to ME180 cervical epithelial cells.
  • FIG. 31: Illustrates that GBS 80 knockout mutant strain partially loses the ability to translocate through an epithelial cell monolayer.
  • FIG. 32: Illustrates that deletion of GBS 104, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cell line.
  • FIG. 33: Illustrates that GBS 104 knockout mutant strain translocates through an epithelial monolayer less efficiently than the isogenic wild type.
  • FIG. 34: Negative stained electron micrographs of GBS serotype III, strain isolate COH1, containing a plasmid insert to over-express GBS 80.
  • FIG. 35: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COH1, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles).
  • FIG. 36: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COH1, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles).
  • FIG. 37: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COH1, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles).
  • FIG. 38: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COH1, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 104 antibodies or preimmune sera (visualized with 10 nm gold particles).
  • FIG. 39: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COH1, 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).
  • FIG. 40: Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COH1, 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). FIG. 41: Illustrates that GBS 80 is necessary for polymer formation and GBS 104 and sortase SAG0648 are necessary for efficient assembly of pili.
  • FIG. 42: Illustrates that GBS 67 is part of a second pilus and that GBS 80 is polymerized in strain 515.
  • FIG. 43: Illustrates that two macro-molecules are visible in Coh1, one of which is the GBS 80 pilin.
  • FIG. 44: Illustrates pilin assembly.
  • FIG. 45: Illustrates that GBS 52 is a minor component of the GBS pilus.
  • FIG. 46: Illustrates that the pilus is found in the supernatant of a bacterial culture.
  • FIG. 47: Illustrates that the pilus is found in the supernatant of bacterial cultures in all phases.
  • FIG. 48: Illustrates that in Coh1, only the GBS 80 protein and one sortase (sag0647 or sag0648) is required for polymerization.
  • FIG. 49: IEM image of GBS 80 staining of a GBS serotype VIII strain JM9030013 that express pili.
  • FIG. 50: IEM image of GBS 104 staining of a GBS serotype VIII strain JM9030013 that express pili.
  • FIG. 51A: Schematic depiction of open reading frames comprising a GAS AI-2 serotype M1 isolate, GAS AI-3 serotype M3, M5, M18, and M49 isolates, a GAS AI-4 serotype M12 isolate, and an GAS AI-1 serotype M6 isolate.
  • FIG. 51B: Amino acid alignment of SrtC1-type sortase of a GAS AI-2 serotype M1 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.
  • FIG. 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-1 serotype M3, S. pyogenes strain MGAS8232 serotype M3, and GAS AI-2 serotype M1.
  • FIG. 53: Amino acid alignment of F-like fibronectin-binding proteins of GAS AI-4 serotype M12 (A735) and S. pyogenes strain MGAS10394 serotype M6.
  • FIG. 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.
  • FIG. 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 MGAS8232 serotype M3, and S. pyogenes M1 GAS serotype M1.
  • FIG. 56: Amino acid alignment of hypothetical proteins of GAS AI-4 serotype M12 (A735), S. pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI-1 serotype M3, GAS AI-3 serotype M5 (Manfredo), and S. pyogenes strain MGAS8232 serotype M3.
  • FIG. 57: Results of FASTA homology search for amino acid sequences that align with the collagen adhesion protein of GAS AI-1 serotype M6 (MGAS10394).
  • FIG. 58: Results of FASTA homology search for amino acid sequences that align with the fimbrial structural subunit of GAS AI-1 serotype M6 (MGAS10394).
  • FIG. 59: Results of FASTA homology search for amino acid sequences that align with the hypothetical protein of GAS AI-2 serotype M1 (SF370).
  • FIG. 60: Specifies pilin and E box motifs present in GAS type 3 and 4 adhesin islands.
  • FIG. 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.
  • FIG. 62: Illustrates that surface exposure is capsule-dependent for GBS 322 but not for GBS 80.
  • FIG. 63: Illustrates the amino acid sequence identity of GBS 59 proteins in GBS strains.
  • FIG. 64: Western blotting of whole GBS cell extracts with anti-GBS 59 antibodies.
  • FIG. 65: Western blotting of purified GBS 59 and whole GBS cell extracts with anti-GBS 59 antibodies.
  • FIG. 66: FACS analysis of GBS strains CJB111, 7357B, 515 using GBS 59 antiserum.
  • FIG. 67: Illustrates that anti-GBS 59 antibodies are opsonic for CJB111 GBS strain serotype V.
  • FIG. 68: Western blotting of GBS strain JM9130013 total extracts.
  • FIG. 69: Western blotting of GBS stain 515 total extracts shows that GBS 67 and GBS 150 are parts of a pilus.
  • FIG. 70: Western blotting of GBS strain 515 knocked out for GBS 67 expression
  • FIG. 71: FACS analysis of GBS strain 515 and GBS strain 515 knocked out for GBS 67 expression using GBS 67 and GBS 59 antiserum.
  • FIG. 72: Illustrates complementation of GBS 515 knocked out for GBS 67 expression with a construct overexpressing GBS 80.
  • FIG. 73: FACS analysis of GAS serotype M6 for spyM60159 surface expression.
  • FIG. 74: FACS analysis of GAS serotype M6 for spyM60160 surface expression.
  • FIG. 75: FACS analysis of GAS serotype M1 for GAS 15 surface expression.
  • FIG. 76: FACS analysis of GAS serotype M1 for GAS 16 surface expression using a first anti-GAS 16 antiserum.
  • FIG. 77: FACS analysis of GAS serotype M1 for GAS 18 surface expression using a first anti-GAS 18 antiserum.
  • FIG. 78: FACS analysis of GAS serotype M1 for GAS 18 surface expression using a second anti-GAS 18 antiserum.
  • FIG. 79: FACS analysis of GAS serotype M1 for GAS 16 surface expression using a second anti-GAS 16 antisera.
  • FIG. 80: FACS analysis of GAS serotype M3 for spyM30098 surface expression.
  • FIG. 81: FACS analysis of GAS serotype M3 for spyM30100 surface expression.
  • FIG. 82: FACS analysis of GAS serotype M3 for spyM30102 surface expression.
  • FIG. 83: FACS analysis of GAS serotype M3 for spyM30104 surface expression.
  • FIG. 84: FACS analysis of GAS serotype M3 for spyM30106 surface expression.
  • FIG. 85: FACS analysis of GAS serotype M12 for 19224134 surface expression.
  • FIG. 86: FACS analysis of GAS serotype M12 for 19224135 surface expression.
  • FIG. 87: FACS analysis of GAS serotype M12 for 19224137 surface expression.
  • FIG. 88: FACS analysis of GAS serotype M12 for 19224141 surface expression.
  • FIG. 89: Western blot analysis of GAS 15 expression on GAS M1 bacteria.
  • FIG. 90: Western blot analysis of GAS 15 expression using GAS 15 immune sera
  • FIG. 91: Western blot analysis of GAS 15 expression using GAS 15 pre-immune sera.
  • FIG. 92: Western blot analysis of GAS 16 expression on GAS M1 bacteria
  • FIG. 93: Western blot analysis of GAS 16 expression using GAS 16 immune sera.
  • FIG. 94: Western blot analysis of GAS 16 expression using GAS 16 pre-immune sera.
  • FIG. 95: Western blot analysis of GAS 18 on GAS M1 bacteria.
  • FIG. 96: Western blot analysis of GAS 18 using GAS 18 immune sera.
  • FIG. 97: Western blot analysis of GAS 18 using GAS 18 pre-immune sera.
  • FIG. 98: Western blot analysis of M6_Spy0159 expression on GAS bacteria.
  • FIG. 99: Western blot analysis of 19224135 expression on M12 GAS bacteria.
  • FIG. 100: Western blot analysis of 19224137 expression on M12 GAS bacteria.
  • FIG. 101: Full length nucleotide sequence of an S. pneumoniae strain 670 AI.
  • FIG. 102: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M1 strain 2580.
  • FIG. 103: Western blot analysis of GAS 15, GAS, 16, and GAS 18 in GAS M1 strain 2913.
  • FIG. 104: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M1 strain 3280.
  • FIG. 105: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M1 strain 3348.
  • FIG. 106: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M1 strain 2719.
  • FIG. 107: Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M1 strain SF370.
  • FIG. 108: Western blot analysis of 19224135 and 19224137 in GAS M12 strain 2728.
  • FIG. 109: Western blot analysis of 19224139 in GAS M12 strain 2728 using antisera raised against SpyM30102.
  • FIG. 110: Western blot analysis of M6_Spy0159 and M6_Spy0160 in GAS M6 strain 2724.
  • FIG. 111: Western blot analysis of M6_Spy 159 and M6_Spy0160 in GAS M6 strain SF370.
  • FIG. 112: Western blot analysis of M6_Spy160 in GAS M6 strain 2724.
  • FIGS. 113-115: Electron micrographs of surface exposed GAS 15 on GAS M1 strain SF370.
  • FIGS. 116-121: Electron micrographs of surface exposed GAS 16 on GAS M1 strain SF370.
  • FIGS. 122-125: Electron micrographs of surface exposed GAS 18 on GAS M1 strain SF370 detected using anti-GAS 18 antisera.
  • FIG. 126: IEM image of a hyperoligomer on GAS M1 strain SF370 detected using anti-GAS 18 antisera.
  • FIGS. 127-132: IEM images of oligomeric and hyperoligomeric structures containing M6_Spy0160 extending from the surface of GAS serotype M6 3650.
  • FIGS. 133A and B: Western blot analysis of L. lactis transformed to express GBS 80 with anti-GBS 80 antiserum.
  • FIG. 134: Western blot analyses of L. lactis transformed to express GBS AI-1 with anti-GBS 80 antiserum.
  • FIG. 135: Ponceau staining of same acrylamide gel as used in FIG. 134.
  • FIG. 136A: Western blot analysis of sonicated pellets and supernatants of cultured L. lactis transformed to express GBS AI-1 polypeptides using anti-GBS 80 antiserum.
  • FIG. 136B: Polyacrylamide gel electrophoresis of sonicated pellets and supernatants of cultured L. lactis transformed to express GBS AI polypeptides.
  • FIG. 137: Depiction of an example S. pneumoniae AI locus.
  • FIG. 138: Schematic of primer hybridization sites within the S. pneumoniae AI locus of FIG. 137.
  • FIG. 139A: The set of amplicons produced from the S. pneumoniae strain TIGR4 AI locus.
  • FIG. 139B: Base pair lengths of amplicons produced from FIG. 139A primers in S. pneumoniae strain TIGR4.
  • FIG. 140: CGH analysis of S. pneumoniae strains for the AI locus.
  • FIG. 141: Amine acid sequence alignment of polypeptides encoded by AI orf2 in S. pneumoniae AI-positive strains.
  • FIG. 142: Amino acid sequence alignment of polypeptides encoded by AI orf 3 in S. pneumoniae AI-positive strains.
  • FIG. 143: Amino acid sequence alignment of polypeptides encoded by AI orf 4 in S. pneumoniae AI-positive strains.
  • FIG. 144: Amino acid sequence alignment of polypeptides encoded by AI orf 5 in S. pneumoniae AI-positive strains.
  • FIG. 145: Amino acid sequence alignment of polypeptides encoded by AI orf 6 in S. pneumoniae AI-positive strains.
  • FIG. 146: Amino acid sequence alignment of polypeptides encoded by AI orf 7 in S. pneumoniae AI-positive strains.
  • FIG. 147: Amino acid sequence alignment of polypeptides encoded by AI orf 8 in S. pneumoniae AI-positive strains.
  • FIG. 148: Diagram comparing amino acid sequences of RrgA in S. pneumoniae strains.
  • FIG. 149: Amino acid sequence comparison of RrgB S. pneumoniae strains.
  • FIG. 150A: Sp0462 amino acid sequence.
  • FIG. 150B: Primers used to produce a clone encoding the Sp0462 polypeptide.
  • FIG. 151A: Schematic depiction of recombinant Sp0462 polypeptide.
  • FIG. 151B: Schematic depiction of full-length Sp0462 polypeptide.
  • FIG. 152A: Western blot probed with serum obtained from S. pneumoniae-infected patients for Sp0462.
  • FIG. 152B: Western blot probed with GBS 80 serum for Sp0462.
  • FIG. 153A: Sp0463 amino acid sequence.
  • FIG. 153B: Primers used to produce a clone encoding the Sp0463 polypeptide.
  • FIG. 154A: Schematic depiction of recombinant Sp0463 polypeptide.
  • FIG. 154B: Schematic depiction of full-length Sp0463 polypeptide.
  • FIG. 155: Western blot detection of recombinant Sp0463 polypeptide.
  • FIG. 156: Western blot detection of high molecular weight Sp0463 polymers.
  • FIG. 157A: Sp0464 amino acid sequence.
  • FIG. 157B: Primers used to produce a clone encoding the Sp0464 polypeptide.
  • FIG. 158A: Schematic depiction of recombinant Sp0464 polypeptide.
  • FIG. 158B: Schematic depiction of full-length Sp0464 polypeptide.
  • FIG. 159: Western blot detection of recombinant Sp0464 polypeptide.
  • FIG. 160: Amplification products prepared for production of Sp0462, Sp0463, and Sp0464 clones.
  • FIG. 161: Opsonic killing by anti-sera raised against L. lactis expressing GBS AI
  • FIG. 162: Schematic depicting GAS adhesin islands GAS AI-1, GAS AI-2, GAS AI-3 and GAS AI-4.
  • FIGS. 163 A-D: Immunoblots of cell-wall fractions of GAS strains with antisera specific for LPXTG proteins of M6_ISS3650 (A), M1_SF370 (B), M5_ISS4883 (C) and M1220010296(D).
  • FIGS. 163 E-H: Immunoblots of cell-wall fractions of deletion mutants M1_SF370Δ128 (E) M1_SF370Δ130 (F) M1_SF370ΔSrtC1 (G) and the M1128 deletion strain complemented with plasmid pAM::128 which contains the M1128 gene (H) with antisera specific for the pilin components of M1_SF370.
  • FIGS. 163 I-N: Immunogold labelling and transmission electron microscopy of: T6 (I) and Cpa (J) in M6_ISS3650; M1128 in M1_SF370 (K) and deletion strain M1_SF370Δ128 (N); M5_orf80 in M5_ISS4883 (L); M12_EftLSL.A in M1220010296 (M). The strains used are indicated below the panels. Bars=200 nm.
  • FIG. 164: Schematic representation of the FCT region from 7 GAS strains
  • FIGS. 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), M1_SF370 stained with anti-M1 (C) or anti-M1128 (D), M5_ISS4883 stained with anti-PrtF (E) or anti-M5_orf80 (F) and M1220010296 with anti-M12 (G) or anti-EftLSL.A (H)
  • FIGS. 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.
  • FIGS. 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.
  • FIGS. 166 H and I Flow cytometry analysis of strain M1_SF370 (H) and the deletion strain M1_SF370Δ128 (1) with T-typing antisera pool T.
  • FIG. 167: Chart describing the number and type of sortase sequences identified within GAS AIs.
  • FIG. 168 A: Immunogold-electronmicroscopy of L. lactis lacking an expression construct for GBS AI-1 using anti-GBS 80 antibodies.
  • FIGS. 168 B and C: Immunogold-electronmicroscopy detects GBS 80 in oligomeric (pilus) structures on surface of L. lactis transformed to express GBS AI-1
  • FIG. 169: FACS analysis detects expression of GBS 80 and GBS 104 on the surface of L. lactis transformed to express GBS AI-1.
  • FIG. 170: Phase contrast microscopy and immuno-electronmicroscopy shows that expression of GBS AI-1 in L. lactis induces L. lactis aggregation.
  • FIG. 171: Purification of GBS pili from L. lactis transformed to express GBS AI-1.
  • islands and portions of the adhesin islands inserted in the pAM401 construct for expression in L. lactis.
  • FIG. 173 A-C: Western blot analysis showing assembly of GAS pili in L. lactis expressing GAS AI-2 (M1) (A), GAS AI-4 (M12) (B), and GAS AI-1 (M6) (C).
  • FIG. 174: FACS analysis of GAS serotype M6 for M6_Spy0157 surface expression.
  • FIG. 175: FACS analysis of GAS serotype M12 for 19224139 surface expression.
  • FIG. 176 A-E: Immunogold electron microscopy using antibodies against M6_Spy0160 detects pili on the surface of M6 strain 2724.
  • FIG. 176 F: Immunogold electron microscopy using antibodies against M6_Spy0159 detects M6_Spy0159 surface expression on M6 strain 2724.
  • FIG. 177 A-C: Western blot analysis of M1 strain SF370 GAS bacteria individually deleted for M1130, SrtC1, or M1128 using anti-M1130 serum (A), anti-M1128 serum (B), and anti-M1126 serum (C).
  • FIG. 178 A-C: Immunogold electron microscopy using antibodies against M1128 to detect surface expression on wildtype strain SF370 bacteria (A), M1128 deleted SF370 bacteria (B), and SrtC1 deleted SF370 bacteria (C).
  • FIG. 179 A-C: FACS analysis to detect expression of M1126 (A), M1128 (B), and M130 (C) on the surface of wildtype SF370 GAS bacteria.
  • FIG. 179 D-F: FACS analysis to detect expression of M1126 (D), M1128 (E), and M1130 (F) on the surface of M1128 deleted SF370 GAS bacteria.
  • FIG. 179 G-I: FACS analysis to detect expression of M1126 (G), M1128 (H), and M1130 (I) on the surface of SrtC1 deleted SF370 GAS bacteria.
  • FIGS. 180 A and B: FACS analysis of wildtype (A) and LepA deletion mutant (B) strains of SF370 bacteria for M1 surface expression.
  • FIG. 181: Western blot analysis detects high molecular weight polymers in S. pneumoniae TIGR4 using anti-RrgB antisera.
  • FIG. 182: Detection of high molecular weight polymers in S. pneumoniae rlrA positive strains.
  • FIG. 183: Detection of high molecular weight polymers in S. pneumoniae TIGR4 by silver staining and Western blot analysis using anti-RrgB antisera.
  • FIG. 184: Deletion of S. pneumoniae TIGR4 adhesin island sequences interferes with the ability of S. pneumoniae to adhere to A549 alveolar cells.
  • FIG. 185: Negative staining of S. pneumoniae strain TIGR4 showing abundant pili on the bacterial surface.
  • FIG. 186: Negative staining of strain TIGR4 deleted for rrgA-srtD adhesin island sequences showing no pili on the bacterial surface
  • bacterial surface.
  • FIG. 188: Negative staining of the negative control TIGR4 mgrA mutant deleted for adhesin island sequences rrgA-srtD showing no pili on the bacterial surface.
  • FIG. 189: Immuno-gold labelling of S. pneumoniae strain TIGR4 grown on blood agar solid medium using α-RrgB (5 nm) and α-RrgC (10 nm). Bar represents 200 nm.
  • FIGS. 190 A and B: Detection of expression and purification of S. pneumoniae RrgA protein by SDS-PAGE (A) and Western blot analysis (B).
  • FIG. 191: Detection of RrgB by antibodies produced in mice.
  • FIG. 192: Detection of RrgC by antibodies produced in mice.
  • FIG. 193: Purification of S. pneumoniae TIGR 4 pili by a cultivation and digestion method and detection of the purified TIGR4 pili.
  • FIG. 194: Purification of S. pneumoniae TIGR 4 pili by a sucrose gradient centrifugation method and detection of the purified TIGR4 pili.
  • FIG. 195: Purification of S. pneumoniae TIGR 4 pili by a gel filtration method and detection of the purified TIGR4 pili.
  • FIG. 196: Alignment of full length S. pneumoniae adhesin island sequences from ten S. pneumoniae strains.
  • FIG. 197 A: Schematic of GBS AI-1 coding sequences.
  • FIG. 197 B: Nucleotide sequence of intergenic region between AraC and GBS 80 (SEQ ID NO: 273.
  • FIG. 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.
  • FIG. 198: Table comparing the percent identity of surface proteins encoded by a serotype M6 (harbouring a GAS AI-1) adhesin island relative to other GAS serotypes harbouring an adhesin island.
  • FIG. 199: Table comparing the percent identity of surface proteins encoded by a serotype M1 (harbouring a GAS AI-2) adhesin island relative to other GAS serotypes harbouring an adhesin island.
  • FIG. 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.
  • FIG. 201: Table comparing the percent identity of surface proteins encoded by a serotype M12 (harbouring a GAS AI-1) adhesin island—relative to other GAS serotypes harbouring an adhesin island.
  • FIG. 202: GBS 80 recombinant protein does not bind to epithelial cells.
  • FIG. 203: Deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade ME180 cervical epithelial cells.
  • FIG. 205: Deletion of GBS 104 protein, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cells
  • FIG. 206: GBS 104 knockout mutant strains of bacteria translocate through an epithelial monolayer less efficiently that the isogenic wild type strain.
  • FIG. 207: GBS 80 knockout mutant strains of bacteria partially lose the ability to translocate through an epithelial monolayer.
  • FIG. 208: GBS adherence to HUVEC endothelial cells.
  • FIG. 209: Strain growth rate of wildtype, GBS 80-deleted, or GBS 104 deleted COH1 GBS.
  • FIG. 210: Binding of recombinant GBS 104 protein to epithelial cells by FACS analysis.
  • FIG. 211: Deletion of GBS 104 protein in the GBS strain COH1 reduces the ability of GBS to adhere to ME180 cervical epithelial cells.
  • FIG. 212: COH1 strain GBS overexpressing GBS 80 protein has an impaired capacity to translocate through an epithelial monolayer.
  • FIG. 213: Scanning electron microscopy shows that overexpression of GBS 80 protein on COH1 strain GBS enhances the capacity of the COH1 bacteria to form microcolonies on epithelial cells.
  • FIG. 214: Confocal imaging shows that overexpression of GBS 80 proteins on COH1 strain GBS enhances the capacity of the COH1 bacteria to form microcolonies on epithelial cells.
  • FIG. 215: Detection of GBS 59 on the surface of GBS strain 515 by immuno-electron microscopy.
  • FIG. 216: Detection of GBS 67 on the surface of GBS strain 515 by immuno-electron microscopy.
  • FIG. 217: GBS 67 binds to fibronectin.
  • FIG. 218: Western blot analysis shows that deletion of both GBS AI-2 sortase genes abolishes assembly of the pilus.
  • FIG. 219: FACS analysis shows that deletion of both GBS AI-2 sortase genes abolishes assembly of the pilus.
  • FIG. 220 A-C: Western blot analysis shows that GBS 59, GBS 67, and GBS 150 form high molecular weight complexes.
  • FIG. 221 A-C: Western blot analysis shows that GBS 59 is required for polymer formation of GBS 67 and GBS 150.
  • FIG. 222: FACS analysis shows that GBS 59 is required for surface exposure of GBS 67.
  • FIG. 223: Summary Western blots for detection of GBS 59, GBS 67, or GBS 150 in GBS 515 and GBS 515 mutant strain.
  • FIG. 224: Description of GBS 59 allelic variants.
  • GBS 59.
  • FIGS. 226 A and B: Results of FACS analysis for surface expression of GBS 59 using antibodies specific for different GBS 59 isoforms.
  • FIGS. 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.
  • FIG. 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.
  • FIG. 229: Expected immunogenicity coverage of different combinations of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 across strains of GBS bacteria.
  • FIG. 230: GBS 59 opsonophagocytic activity is comparable to that of a mixture of GBS 80, GBS 104, GBS 322 and GBS 67.
  • FIG. 231 A-C: Schematic presentation of example hybrid GBS AIs.
  • FIG. 232: Schematic presentation of an example hybrid GBS AI.
  • FIGS. 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 AI.
  • FIG. 234 A-E Hybrid GBS AI cloning strategy.
  • FIG. 235: High magnification of S. pneumoniae strain TIGR4 pili double labeled with α-RrgB (5 nm) and α-RrgC (10 nm). Bar represents 100 nm.
  • FIG. 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 200 nm.
  • FIG. 237: Variability in GBS 67 amino acid sequences between strains 2603 and H36B.
  • FIG. 238: Strain variability in GBS 67 amino acid sequences of allele I (2603).
  • FIG. 239: Strain 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-1 mutants from GBS strain isolate 2603.
  • TABLE 4: GAS AI-1 sequences from M6 isolate (MGAS10394).
  • TABLE 5: GAS AI-2 sequences from M1 isolate (SF370).
  • TABLE 6: GAS AI-3 sequences from M3 isolate (MGAS315).
  • TABLE 7: GAS AI-3 sequences from M3 isolate (SSI-1).
  • 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).
  • 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 spyM60159.
  • TABLE 19: Summary of FACS values for surface expression of spyM60160.
  • 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 SpyM30098.
  • TABLE 26: Summary of FACS values for surface expression of SpyM30100.
  • TABLE 27: Summary of FACS values for surface expression of SpyM30102 in M3 serotypes.
  • TABLE 28: Summary of FACS values for surface expression of SpyM30102 in M6 serotypes.
  • TABLE 29: Summary of FACS values for surface expression of SpyM30104 in M3 serotypes.
  • TABLE 30: Summary of FACS values for surface expression of SpyM30104 in an M12 serotype.
  • TABLE 31: Summary of FACS values for surface expression of SPs0106 in M3 serotypes.
  • TABLE 32: Summary of FACS values for surface expression of SPs0106 in an M12 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.
  • serotype.
  • TABLE 38: S. pneumoniae strain 670 AI sequences.
  • TABLE 39: Percent identity comparison of S. pneumoniae strains AI sequences.
  • TABLE 40: FACS analysis of L. lactis and GBS bacteria strains expressing GBS AI-1.
  • 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-1.
  • 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-1
  • TABLE 47: Anti-GBS 80 IgA Antibodies Detected in Mouse Tissues Following Immunization with L. lactis Expressing GBS AI-1
  • 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 GBS 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, Vols. I-IV (D. M. Weir and C. C. 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, N.Y.
  • 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
  • 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-1”, or “GBS AI-1”, within the genomes of several Group B Streptococcus serotypes and isolates. AI-1 comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (“AI-1 proteins”). Specifically, AI-1 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-1 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-1 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • A schematic of AI-1 is presented in FIG. 1. AI-1 typically resides on an approximately 16.1 kb transposon-like element frequently inserted into the open reading frame for trmA. One or more of the AI-1 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-1 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. AI-1 may encode for at least one surface protein. Alternatively, AI-1 may encode for at least two surface exposed proteins and at least one sortase. Preferably, AI-1 encodes for at least three surface exposed proteins and at least two sortases. The AI-1 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
  • 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 (aspartate and glutamate), (2) basic (lysine, arginine, histidine), (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 aspartate 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 L 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
  • 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 FIG. 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(X10)K; SEQ ID NO: 146), (YPKN(X9)K, SEQ ID NO: 147), (YPK(X7)K; SEQ ID NO: 148), (YPK(X11)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 (ME180) are presented in FIG. 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 ME180 cells is also shown in the contrast images in FIG. 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-1 surface protein GBS 104 can bind epithelial cells such as ME180 human cervical cells, A549 human lung cells and Caco2 human intestinal cells (See
  • of GBS to adhere to ME180 cervical epithelial cells. (See FIGS. 30 and 211). Deletion of GBS 104 also reduces the capacity of GBS to invade J774 macrophage-like cells. (See FIGS. 32 and 205). Deletion of GBS 104 also causes GBS to translocate through epithelial monolayers less efficiently. See FIG. 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 FIG. 207. Deletion of either GBS 80 or GBS 104 in COH1 cells diminishes adherence to HUVEC endothelial cells. See FIG. 208. Deletion of GBS 80 or GBS 104 in COH1 does not, however, affect growth of COH1 either with ME1180 cells or in incubation medium (IM). See FIG. 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 FIG. 202. Furthermore, deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade ME180 cervical epithelial cells. See FIG. 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 FIGS. 5 and 204, and Example 1, GBS 80, one of the AI-1 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 FIGS. 27 and 28), removal of GBS 80 from a wild type strain decreases the ability of that strain to translocate through an epithelial cell layer (see FIG. 31).
  • GBS 80 may also be involved in formation of biofilms. COH1 bacteria overexpressing GBS 80 protein have an impaired ability to translocate through an epithelial monolayer. See FIG. 212. These COH1 bacteria overexpressing GBS 80 form microcolonies on epithelial cells. See FIGS. 213 and 214. These microcolonies may be the initiation of biofilm development.
  • AI 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-1, 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 fimbrae 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 Corynebacterium diphtheriae (SpaA, SpaD, and SpaH). See, Ton-That et al. (2003) Molecular Microbiology 50(4):1429-1438 and Ton-That et al.
  • 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 catalyzed 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 FIG. 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-1 polypeptides of the immunogenic compositions comprise an E box motif. Some examples of E box motifs in the AI-1 polypeptides may include the amino acid sequences YxLxExxxxxGY (SEQ ID NO: 153), YxLxExxPxGY (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, FIG. 34 presents electron micrographs of GBS serotype III, strain isolate COH1 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 shown in FIGS. 11, 16 and 17, which reveal the presence of pilus structures in wild type COH1 Streptococcus agalactiae. Other examples of these electron
  • clinical isolate of S. agalactiae, JM9030013. (See FIG. 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 FIGS. 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. FIG. 61 provides FAC analysis of GBS 80 surface levels on bacterial strains COH1 and JM9130013 using an anti-GBS 80 antisera. Immunogold electron microscopy of the COH1 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. FIG. 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 Coh1 bacterial clones that overexpress GBS 80, but lack GBS 104, or one of the AI-1 sortases sag0647 or sag0648. However, pili are not formed in Coh1 bacterial clones that overexpress GBS 80 and lack both sag0647 and sag0648. Thus, for example, it appears that at least GBS 80 and a sortase, sag0647 or sag0648, may be necessary for pili formation. (See FIG. 48.) Overexpression of GBS 80 in GBS strain 515, which lacks an AI-1, 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 FIG. 42.) Overexpression of GBS 80 in GBS strain 515 cell knocked out for GBS 67 expression also apparently polymerizes GBS 80 into pili. (See FIG. 72.)
  • While GBS 80 appears to be required for GBS AI-1 pili formation, GBS 104 and sortase SAG0648 appears to be important for efficient AI-1 pili assembly. For example, high-molecular structures are not assembled in isogenic COH1 strains which lack expression of GBS 80 due to gene disruption and are less efficiently assembled in isogenic COH1 strains which lack the expression of GBS 104 (see FIG. 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 COH1 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 FIG. 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 FIGS. 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 FIG. 50.)
  • antisera on total cell extracts of Coh1 and a GBS 52 null mutant Coh1 reveal a shift in detected proteins in the Coh1 wild type strain relative to, the GBS 52 null mutant Coh1 strain. The shifted proteins were also detected in the wild type Coh1 bacteria with an anti-GBS 52 antisera, indicating that the GBS 52 may be present in the pilus. (See FIG. 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-1 surface proteins, such as GBS 80, GBS 104 and GBS 52. The surface proteins may be isolated from Gram positive 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-1”) 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
  • 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 Sep. 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 Coh1. (See FIG. 46.) These pili are found in the supernatant at all growth phases of the cultured Coh1 bacteria. (See FIG. 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
  • 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-1 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 FIG. 197 A, which is a schematic showing the organization of GBS AI-1 and FIG. 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 FIG. 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 FIG. 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 as 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 non-pathogenic Gram positive bacteria, such as Streptococcus 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,
  • human host subject and are not associated with human pathogenesis. 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 non-pathogenic 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 Adhesin 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 FIGS. 133A 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-1 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 FIG. 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 FIG. 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 FIGS. 136A and B. See also FIG. 171, which shows purification of GBS pili from whole extracts of L. lactis expressing the GBS AI-1 following sonication and gel filtration on a Sephacryl HR 400 column.
  • GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences expressed the GBS AI-1 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 COH1 and JM9130013, which naturally express GBS AI-1. See FIG. 169 for FACS analysis data for L. lactis transformed with GBS AI-1 and wildtype JM9130013 bacteria using anti-GBS 80 and GBS 104 antisera. Table 40 provides the results of FACS analysis of transformed L. lactis, COH1, 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 L. lactis and GBS bacteria strains
    expressing GBS AI-1
    Anti-GBS 104
    Anti-GBS 80 antiserum antiserum
    GBS AI-1 transformed 298 251
    L. lactis
    GBS COH1 305 305
    GBS JM9130013 461 355

    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-1, confirming the results of the FACS analysis. See FIGS. 168 B and C. Interestingly, this expression of GBS pili on the surface of the L. lactis induced L. lactis aggregation. See FIG. 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-1 was protective in a subsequent challenge with GBS. Female mice were immunized with L. lactis transformed with GBS AI-1. 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-1 (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-1).
  • TABLE 43
    Protection of Mice Immunized with L. lactis expressing GBS AI-1
    Immunization Survival
    Route Antigen Alive/Treated Survival % % Range p value
    Intraperitoneum Recombinant GBS 80 16/18 89 80-100 <0.001
    Subcutaneous LL-AI 1 10 E9 40/49 82 70-90  <0.001
    LL-AI 1 10 E10 50/60 83 60-100 <0.001
    PBS  4/30 13 0-30
    LL 10 E9  3/57 5 0-20
    Intranasal LL-AI 1 10 E9 22/60 37  0-100 0.02
    LL-AI 1 10 E10 31/49 63 30-90  <0.001
    LL 10 E9  2/27 7 0-20
  • Table 51 provides further evidence that immunization of mice with L. lactis transformed with GBS AI-1 is protective against GBS.
  • TABLE 51
    Further Protection of Mice Immunized with L. lactis
    expressing GBS AI-1
    Immunization Alive/ Survival %
    Antigen route Treated (Pval <0.0000001)
    Recombinant GBS 80 IP 48/50 92
    Recombinant GBS 80 SC 21/30 70
    L. lactis + AI1 106 cfu SC  6/66 9
    L. lactis + AI1 107 cfu SC 47/70 73
    L. lactis + AI1 108 cfu SC 116/153 76
    L. lactis + AI1 109 cfu SC  98/118 83
    L. lactis + AI1 1010 cfu SC 107/129 83
    L. lactis 1010 cfu SC  4/83 5
    PBS SC  6/110 5
    L. lactis + AI1 1010 cfu IN 51/97 52
    L. lactis 1011 cfu IN  1/40 7
    PBS IN  0/37 0
  • Protection of immunized mice with L. lactis expressing the GBS AI-1 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 L. lactis expressing the GBS AI-1 as described above. Mice immunized with L. lactis expressing the GBS AI-1 have anti-GBS 80 antibody titers, 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 L. lactis transformed to express the GBS AI-1 have significantly higher serum anti-GBS 80 antibody titers than mice intranasally immunized with L. lactis transformed to express the GBS AI-1.
  • TABLE 46
    Antibody Responses against GBS 80 in Serum of Mice
    Immunized with L. lactis Expressing GBS AI-1
    Ab Titre Obtained Following
    Subcutaneous Intranasal Intraperitoneal
    Antigen Immunization Immunization Immunization
    LL 10 E9 0 0
    LL-AI 1 10 E9 14000 50
    LL-AI 1 10 E10 25000 406
    Recombinant GBS 80 120000
  • Anti-GBS 80 antibodies of the IgA isotype were specifically detected in various body fluids of the mice subcutaneously or intranasally immunized with L. lactis expressing the GBS AI-1.
  • TABLE 47
    Anti-GBS 80 IgA Antibodies Detected in Mouse Tissues
    Following Immunization with L. lactis Expressing GBS AI-1
    Anti-GBS 80 IgA
    Antibodies Detected in
    Antigen Immunization route Serum Vaginal Wash Nasal Wash
    LL 10 E9 0 0 0
    LL-AI 1 Subcutaneous 0 25 20
    LL-AI 1 Intranasal 140 0 150
    GBS 80 Intraperitoneal 60 0
  • Furthermore, opsonophagocytosis assays also demonstrated that at least some of the antiserum produced against the L. lactis expressing GBS AI 1 is opsonic for GBS. See FIG. 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-1 and AI-2, i.e., a hybrid GBS AI. By way of example, a hybrid GBS AI may be a GBS AI-1 with a replacement of the GBS 104 gene with a GBS 67 gene. A schematic of such a hybrid GBS AI is depicted in FIG. 231 A. A hybrid GBS AI may alternatively be a GBS AI-1 with a replacement of the GBS 52 gene with a GBS 59 gene. See the schematic at FIG. 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-1 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 FIG. 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-1 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 FIG. 234 A-F.
  • Applicants have prepared a hybrid GBS AI having a GBS AI-1 sequence with a substitution of a GBS 67 coding sequence for the GBS 104 gene as depicted in FIG. 231 A. Transformation of L. lactis with the hybrid GBS AI-1 resulted in L. lactis expression of high molecular weight polymers containing the GBS 80 and GBS 67 proteins. See FIG. 233 A, which provides Western blot analysis of L. lactis transformed with the hybrid GBS AI depicted in FIG. 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 FIG. 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.
  • 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 FIG. 1, AI-1 includes the surface exposed proteins of GBS 80, GBS 52 and GBS 104 and the sortases SAG0647 and SAG0648. AI-1 typically appears as an insertion into the 3′ end of the trmA gene.
  • In addition to the open reading frames encoding the AI-1 proteins, AI-1 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-1 may also include a sequence encoding a rho independent transcriptional terminator (see hairpin structure in FIG. 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-1 within several GBS serotypes is depicted in FIG. 2. AI-1 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 CJB111; GBS serotype III, strain isolate COH1 and GBS serotype 1a, strain isolate A909. (Percentages shown are amino acid identity to the 2603 sequence). (An AI-1 was not identified in GBS serotype 1b, strain isolate H36B or GBS serotype 1a, strain isolate 515).
  • An alignment of AI-1 polynucleotide sequences from serotype V, strain isolates 2603 and CJB111; serotype II, strain isolate 18RS21; serotype III, strain isolates COH1 and NEM316; and serotype 1a, strain isolate A909 is presented in FIG. 18. An alignment of amino acid sequences of AI-1 surface protein GBS 80 from serotype V, strain isolates 2603 and CJB111; serotype 1a, strain isolate A909; serotype III, strain isolates COH1 and NEM316 is presented in FIG. 22. An alignment of amino acid sequences of AI-1 surface protein GBS 104 from serotype V, strain isolates 2603 and CJB111; serotype III, strain isolates COH1 and NEM316; and serotype II, strain isolate 18RS21 is presented in FIG. 23. Preferred AI-1 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 COH1), 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 FIG. 30.
  • 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-1 and AI-2 within the GBS serotype V, strain isolate 2603 is shown in FIG. 3. (Homology percentages in S FIG. 3 represent amino acid identity of the AI-2 proteins to the AI-1 proteins). Alignments of AI-2 polynucleotide sequences are presented in FIGS. 20 and 21 (FIG. 20 includes sequences from serotype V, strain isolate 2603 and serotype III, strain isolate NEM316. FIG. 21 includes sequences from serotype III, strain isolate COH1 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 CJB111; serotype 1a, strain isolate 515; serotype II, strain isolate 18RS21; serotype Ib, strain isolate H36B; and serotype III, strain isolate NEM316 is presented in FIG. 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)K; SEQ ID NO: 160), (PKN(X8)K; SEQ ID NO: 161), or (PK(X10)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),
  • (SEQ ID NO: 152).
  • As shown in FIG. 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 RofA like protein (for example rogB). As in AI-1, 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 FIG. 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-1 surface proteins, AI-2 surface protein GBS 67 appears to be conserved in GBS serotypes where the AI-1 surface proteins are disrupted or missing.
  • For example, as discussed above and in FIG. 2, the AI-1 GBS 80 surface protein is fragmented in GBS serotype II, strain isolate 18RS21. Within AI-2 for this same sequence, as shown in FIG. 4, the GBS 67 surface protein has 99% amino acid sequence homology with the corresponding sequence in strain isolate 2603. Similarly, the AI-1 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 FIGS. 237-239.
  • Unlike for GBS 67, amino acid sequence identity of GBS 59 is variable across different GBS strains. As shown in FIGS. 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 CJB111, 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 CJB111, NEM316, and 515. The second isoform appears to include the GBS 59 protein of GBS strains 18RS21, 2603, and H36B. (See FIGS. 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 FIG. 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-cjb111) 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
  • 226B.
  • Also, GBS 59 is opsonic only against GBS strains expressing a homologous GBS 59 protein. See FIG. 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 CJB111, 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. FIG. 64 shows detection of high molecular weight GBS 59 polymers in whole extracts of GBS strains CJB111, 7357B, COH31, D1363C, 5408, 1999, 5364, 5518, and 515 using antiserum raised against GBS 59 of GBS strain CJB111. FIG. 65 also shows detection of these high molecular weight GBS 59 polymers in whole extracts of GBS strains D136C, 515, and CJB111 with anti-GBS 59 antiserum. (See also FIG. 220 A for detection of GBS 59 high molecular weight polymers in strain 515.) FIG. 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. FIG. 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 CJB111, DK1, DK8, Davis, 515, 2986, 5551, 1169, and 7357B by FACS analysis using mouse antiserum raised against GBS 59 of GBS CJB111. FACS analysis did not detect surface expression of GBS 59 in GBS strains SMU071, JM9130013, and COH1, which do not contain a GBS 59 gene. (See FIG. 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 FIG. 215.
  • GBS 67 and GBS 150 also appear to be included in high molecular weight structures, or pili. FIG. 69 shows that anti-GBS 67 and anti-GBS 150 immunoreact with high molecular weight structures in whole GBS strain 515 extracts. (See also FIGS. 220 B and C.) It is also notable in FIG. 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 BS 59 in CBS strain 515. GBS 59 of CBS stain 515 is of a different isotype than GBS 59 of GBS stain 2603. See FIG. 63, which illustrates that the homology of these two CBS 59 polypeptides is 48%, and FIG. 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. FIG. 70 provides Western blots showing that higher molecular weight structures in GBS strain 515 total
  • 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-hybridize 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 FIG. 216. GBS 67 binds to fibronectin. See FIG. 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 (FIG. 221 A, lane 3), GBS 67 (FIG. 221B, lane 3), and GBS 150 (FIG. 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 (FIG. 221 A, lane 2) and GBS 150 (FIG. 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 (FIG. 221 A, lane 4) and GBS 67 (FIG. 221 B, lane 4). See also FIG. 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 FIG. 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 FIG. 218, which provides Western blot analysis of strain 515 bacteria lacking Srt1, Srt2, or both Srt1 and Srt2. Only deletion of both Srt1 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 FIG. 219.
  • As shown in FIG. 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 FIG. 4 are amino acid identity to the COH1 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, spb1, 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
  • 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 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-2 surface proteins, such as GBS 59, GBS 67 and GBS 150. The surface proteins may be isolated from Gram positive 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.
  • 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 (tee6), 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., Mol. 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 tee6 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 microscopy of strain M62724 with antisera specific for the product of tee6 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-1) and forms long covalently linked pilus structures.
  • 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 eradicate 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 FIG. 51A and FIG. 162. In all strains identified so far, the adhesin island region is flanked by highly conserved open reading frames M1123 and M1136. 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-1”, within the genome Group A Streptococcus serotypes and isolates. 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-1 proteins”). GAS AI-1 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
  • 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-1 includes open reading frames encoding for two or more (i.e., 2, 3, 4 or 5) of M6_Spy0157, M6_Spy0158, M6_Spy0159, M6_Spy0160, M6_Spy0161.
  • Applicants have also identified open reading frames encoding fimbrial structural subunits in other GAS bacteria harbouring an AI-1. These open reading frames encode fimbrial structural subunits CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial. A GAS AI-1 may comprise a polynucleotide encoding any one of CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
  • As discussed above, the hyper-oligomeric pilus structure of GAS AI-1 appears to be responsible for the T-antigen type 6 classification, and GAS AI-1 corresponds to the FCT region previously identified for tee6. As in GAS AI-1, the tee6 FCT region includes open reading frames encoding for a collagen adhesion protein (cpa, capsular polysaccharide adhesion) and a fibronectin binding protein (prtF1). Immunoblots of tee6, a GAS AI-1 fimbrial structural subunit corresponding to M6_Spy160, 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-1 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 PrtF1 recognize only a single molecular species with electrophoretic mobility corresponding to its predicted molecular mass, indicating that PrtF1 may not be associated with the oligomeric pilus structure. A preferred immunogenic composition of the invention comprises a GAS AI-1 surface protein which may be formulated or purified in an oligomeric (pilus) form. In a preferred embodiment, the oligomeric form is a hyperoligomer. Another preferred immunogenic composition of the invention comprises a GAS AI-1 surface protein which has been isolated in an oligomeric (pilus) form. The oligomer or hyperoligomeric pilus structures comprising the GAS AI-1 surface proteins may be purified or otherwise formulate for use in immunogenic compositions.
  • One or more of the GAS AI-1 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-1 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
  • One or more of the GAS AI-1 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
  • 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-1 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. GAS AI-1 may encode for at least one surface protein. Alternatively, GAS AI-1 may encode for at least two surface exposed proteins and at least one sortase. Preferably, GAS AI-1 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-1 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-1 surface protein such as M6_Spy0157, M6_Spy0159, 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 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 1 (“GAS AI-1”) 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-”) 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-1 proteins, GAS AI-1 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, Spy0127, GAS16, GAS17, GAS18, Spy0131, Spy0133, 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 (pilis) 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, fibronectin, or collagen.
  • 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 LPXRG 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 GAS15, GAS16, or GAS18. 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-1”), 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
  • 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 SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, SpyM30104, SPs0100, SPs0101, SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf78, orf79, orf80, orf81, orf82, orf83, orf84, spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, spyM180132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and SpyoM01000149. 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 SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, and SpyM30104. 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 SPs0100, SPs0101, SPs0120, SPs0103, SPs0104, SPs0105, and SPs0106. 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, orf80, orf81, 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 spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, and spyM180132. 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 SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and SpyoM01000149.
  • 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 ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. A GAS AI-3 may comprise a polynucleotide encoding any one of ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
  • 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 epithelial cell surface. AI surface proteins 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 SpyM30098, SpyM30100, SpyM30102, SpyM30104, SPs0100, SPs0102, SPs0104, SPs0106, orf78, orf80, orf82, orf84, spyM180126, spyM180128, spyM180130, spyM180132, SpyoM01000155, SpyoM01000153, SpyoM01000151, 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 SpyM30098, SpyM30100, SpyM30102, and SpyM30104. In another embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SPs0100, SPs0102, SPs0104, and SPs0106. 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 spyM180126, spyM180128, spyM180130, and spyM180132. In a further embodiment, the invention includes a composition comprising oligomeric, pilus-like structures comprising an AI surface protein such as SpyoM01000155, SpyoM01000153, SpyoM01000151, 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 subunits may be covalently associated via an LPXTG motif, preferably, via the threonine amino acidresidue.
  • 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-1”), 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 635_fimbrial, 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 invention may effect the ability of the GAS bacteria to adhere to and invade epithelial cells. AIsurface 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 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 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, 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-1”), 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.
  • In addition to the open reading frames encoding the GAS AI-4 proteins, GAS AI-4 may alsoinclude 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 to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasingexpression 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 non-pathogenic Gram positive bacteria, such as Streptococcus 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 pathogenesis. 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 non-pathogenic 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-1, AI-2, and AI-4 adhesin islands. Briefly, the pAM401 is a promoterless high-copy plasmid. The entire pili gene clusters of an M6 (AI-1), M1 (AI-2), and M12 (AI-4) bacteria were inserted into the pAM401 construct. The gene clusters were transcribed under the control their own (M6, M1, or M12) promoter or the GBS promoter that successfully initiated expression of the GBS AI-1 adhesin islands in L. lactis, described above. FIG. 172 provides a schematic depiction of GAS M6 (AI-1),
  • inserted in the pAM401 construct.
  • Each of the L. lactic transformed with one of the M6, M1, 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. FIGS. 173 A-C provide results of Western blot analysis of surface protein-enriched extracts of L. lactis transformed with M6 (FIG. 173 A), M1 FIG. 173 B), or M12 (FIG. 173 C) adhesin island gene clusters using antibodies that bind to the fimbrial structural subunit encoded by each cluster. FIG. 173A at lanes 3 and 4 shows detection of high molecular structures in L. lactis transformed with an adhesin island pilus gene cluster from an M1 AI-2 using an antibody that binds to fimbrial structural subunit Spy0128. 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 M12 AI-4 using an antibody that binds to fimbrial structural subunit EftLSL.A. FIG. 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 FIGS. 173 A-C, “p1” 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 L. 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.
  • 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.
  • 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 orf1670, orf3670, orf4670, orf5670, orf6670, orf7670, orf8670.
  • 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
  • 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 orf3670, orf4670, or orf5670. 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 ORF214CSR, ORF314CSR, ORF414CSR, ORF514CSR, ORF614CSR, ORF714CSR, ORF814CSR.
  • 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.
  • 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 LPXRG 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 LPXRG 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.
  • 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 ORF219AH, ORF319AH, ORF419AH, ORF519AH, ORF619AH, ORF719AH, ORF819AH.
  • 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.
  • 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 orf319AH, orf419AH, or orf519AH. 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)
  • ORF819FTW.
  • 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 S. 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 ORF. 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 S. 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 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 19F Taiwan 14 AI surface protein such as orf319FTW, orf419FTW, or orf519FTW. 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,
  • 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 ORF223FP, ORF323FP, ORF423FP, ORF523FP, ORF623FP, ORF723FP, and ORF823FP.
  • 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
  • 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 23F Poland 16 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen.
  • The S. pneumoniae strain 23F 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 AI 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 orf323FP, orf423FP, or orf523FP. 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.
  • 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 ORF223FTW, ORF323FTW, ORF423FTW, ORF523FTW, ORF623FTW, ORF723FTW, ORF823FTW.
  • 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 23F 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 23F 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
  • 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 orf323FTW, orf423FTW, or orf523FTW. 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.
  • S. 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 ORF26BF, ORF36BF, ORF46BF, ORF56BF, ORF66BF, ORF76BF, ORF86BF.
  • 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.
  • 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 orf36BF, orf46BF, or orf56BF. 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.
  • 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 ORF26BSP, ORF36BSP, ORF46BSP, ORF56BSP, ORF66BSP, ORF76BSP, and ORF86BSP.
  • 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.
  • 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 orf36BSP, orf46BSP, or orf56BSP. 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 ORF29VSP, ORF39VSP, ORF49VSP, ORF59VSP, ORF69VSP, ORF79VSP, and ORF89VSP.
  • 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 AI surface protein which has been isolated in an oligomeric (pilis) form.
  • One or more of the S. pneumoniae strain 9V 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 S. 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 9V 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 orf39VSP, orf49VSP, or orf59VSP. 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 9V 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 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 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 as 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 non-pathogenic Gram positive bacteria, such as Streptococcus 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 pathogenesis. 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 non-pathogenic 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 express an Adhesin Island surface protein. Preferable, 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.
  • FIGS. 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 compositions 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 (i.e., 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% (i.e., 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
  • 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-1 are of the same AI subtype. If two AIs are of a different subtype, the AIs have different numerical designations. For example, AI-1 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-1, AI-3, and AI-4, etc.
  • For example, the invention includes an immunogenic composition comprising one or more GBS AI-1 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-1 proteins may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. Preferably, the GBS AI-1 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-1, 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-1 proteins may be selected from the group consisting of M6_Spy0156, M6_Spy0157, M6_Spy0158, M6_Spy0159, M6_Spy0160, M6_Spy0161, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial. Preferably, the GAS AI-1 proteins are selected from the group consisting of M6_Spy0157, M6_Spy0159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
  • The GAS AI-2 proteins may be selected from the group consisting of Spy0124, GAS15, Spy0127, GAS16, GAS17, GAS18, Spy0131, Spy0133, and GAS20. Preferably, the GAS AI-2 proteins are selected from the group consisting of GAS15, GAS16, and GAS18.
  • The GAS AI-3 proteins may be selected from the group consisting of SpyM30097, SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, SpyM30104, SPs0099, SPs0100, SPs0101, SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf77, orf78, orf79, orf80, orf81, orf82, orf83, orf84, spyM180125, spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, spyM180132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. In one embodiment the GAS AI-3 proteins are selected from the group consisting of SpyM30097, SpyM30098, SpyM30099, SpyM30100, SpyM30101, SpyM30102, SpyM30103, and SpyM30104. In another embodiment, the GAS AI-3 proteins are selected from the group consisting of SPs0099, SPs0100, SPs0101, 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, orf80, orf81, orf82, orf83, and orf84. In a further embodiment, the GAS AI-3 proteins are selected from the group consisting of spyM180125, spyM180126, spyM180127, spyM180128, spyM180129, spyM180130, spyM180131, and spyM180132. In yet another embodiment the GAS AI-3 proteins are selected from the group consisting of SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, 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-AI-4 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 Orf1670, Orf3670, Orf4670, Orf5670, Orf6670, Orf7670, and Orf8670. Preferably, the S. pneumonaie strain 670 AI proteins include Orf3670, Orf4670, or Orf5670.
  • The S. pneumonaie from 19A Hungary 6 AI proteins may be selected from the group consisting of ORF219AH, ORF319AH, ORF419AH, ORF519AH, ORF619AH, ORF719AH, or ORF819AH.
  • The S. pneumonaie from 6B Finland 12 AI proteins may be selected from the group consisting of ORF26BF, ORF36BF, ORF46BF, ORF56BF, ORF66BF, ORF76BF, or ORF86BF.
  • The S. pneumonaie from 6B Spain 2 AI proteins may be selected from the group consisting of ORF26BSP, ORF36BSP, ORF46BSP, ORF56BSP, ORF66BSP, ORF76BSP, or ORF86BSP.
  • The S. pneumonaie from 9V Spain 3 AI proteins may be selected from the group consisting of ORF29VSP, ORF39VSP, ORF49VSP, ORF59VSP, ORF69VSP, ORF79VSP, or ORF89VSP.
  • The S. pneumonaie from 14 CSR 10 AI proteins may be selected from the group consisting of ORF214CSR, ORF314CSR, ORF414CSR, ORF514CSR, ORF614CSR, ORF714CSR, or ORF814CSR.
  • The S. pneumonaie from 19F Taiwan 14 AI proteins may be selected from the group consisting of ORF219FTW, ORF319FTW, ORF419FTW, ORF519FTW, ORF619FTW, ORF719FTW, or ORF819FTW.
  • The S. pneumonaie from 23F Taiwan 15 AI proteins may be selected from the group consisting of ORF223FTW, ORF323FTW, ORF423FTW, ORF523FTW, ORF623FTW, ORF723FTW, or ORF823FTW.
  • The S. pneumonaie from 23F Poland 16 AI proteins may be selected from the group consisting of ORF223FP, ORF323FP, ORF423FP, ORF523FP, ORF623FP, ORF723FP, or ORF823FP.
  • 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, 97, 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 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-1 protein or an AI-2 protein. For example, the first AI protein may be a GBS AI-1 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 COH1 and the GBS 80 amino acid sequence from GBS serotype 1a, 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 COH1, the GBS 104 amino acid sequence from GBS serotype 1a, 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
    GBS 80 GBS 104
    Serotype Strains cGH % AA identity cGH % AA identity
    Ia 090 + 99.79 + 100.00
    A909 + 100.00 + 100.00
    515
    DK1
    DK8
    Davis
    Ib 7357b + 100.00 +
    H36B
    II 18RS21 + 100.00
    DK21
    III NEM316 + 100.00 + 100.00
    COH31 + 100.00 +
    D136 + 100.00 +
    M732 + 100.00 + 99.88
    COH1 + 99.79 + 99.88
    M781 + 99.79 + 99.88
    No type CJB110 + 99.37 + 100.00
    1169NT
    V CJB111 + 100.00 + 100.00
    2603 + 100.00 + 100.00
    VIII JM130013 + 99.79 + 100.00
    SMU014 + 100.00 +
    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 67 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 1b, 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 spb1. The amino acid sequence of spb1 from GBS serotype III, strain isolate COH1 is greater than 90% homologous to the spb1 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 CJB111 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 AI 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 FIG. 2), the sequence for GBS 80 in GBS serotype 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).
  • Further, 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.
  • TABLE 15
    Antigen surface exposure of GBS 80, GBS 322, GBS 104, and GBS 67
    GBS strains Type GBS 80 GBS 322 GBS 104 GBS 67
    DK1* Ia 0 nd 237 478
    DK8* 0 213 151 475
    Davis* 0 86 271 430
    515* 0 227 262 409
    090 0 0 0 0
    A909 0 0 0 0
    2986 0 0 157 397
    5551 0 36 384 485
    2177 Ib 477 323 328 66
    H36B* 0 105 518 444
    7357b- 91 102 309 316
    2129 57 71 132 0
    5518 31 nd 60 28
    COH1 III 305 130 305 0
    D136C 16 460 226 406
    COH31 0 479 71 273
    M732 105 292 101 0
    M781 65 224 136 0
    1998 95 288 205 350
    5376 165 76 156 0
    5435 93 88 100 0
    18RS21 II 0 471 50 103
    DK21* 0 342 419 331
    3050 43 188 289 460
    5401 170 135 494 618
    2141 0 76 0 69
    CJB111 V 365 58 355 481
    2603 62 293 100 105
    5364 454 463 379 394
    2110 0 11 345 589
    2274 IV 113 161 465 484
    1999 0 55 492 453
    2210 0 0 363 574
    2928 VII 0 0 0 0
    SMU071 VIII 556 170 393 79
    JM9130013 587 133 436 83
    2189 0 0 0 0
    5408 0 0 159 433
    CJB110 NT 71 587 169 245
    1169* 0 213 371 443
    Δ Mean >100 9/40 22/38 32/40 25/40
    22% 58% 80% 62%
  • 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.
  • 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 1b, strain isolate H36B). As shown in FIGS. 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. SACS 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. FIG. 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. FIG. 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 FIGS. 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 comprising 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
    GBS 80 GBS 104 GBS 67 GBS 59 GBS 322
    5-fold shift in 17/70 14/70 49/70 46/70 33/70
    fluorescence 24% 20% 70% 66% 47%
    by FACS
  • 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
    5-fold shift in
    fluorescence
    by FACS GBS 80 GBS 104 GBS 67 GBS 59 GBS 322
    Ia + Ib + III  4/36 2/36 22/36 20/36 18/36
    II + V 11/25 9/25 21/25 21/25 13/25
    Others 2/9 3/9  6/9 5/9 2/9
  • 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 FIG. 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 FIG. 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 prtF2 protein (such as the 19224141 protein from GAS serotype M12, strain isolate A735). As previously discussed (and depicted in FIG. 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_Spy0159 (from M6 isolate (MGAS10394), which comprises an AI-1) or GAS15 (from M1 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_Spy0160 from M6 strain 10394, SPy0128 from M1 strain SF370, SpyM30100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM180128 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 M12 strain 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-1, AI-2, AI-3, and AI-4 representative strains (i.e., M6_Spy0160 from M6 strain 10394, SPy0128 from M1 strain SF370, SpyM30100 from M3 strain 315, SPs012 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM180128 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 M12 strain 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
    AI-1 AI-2 AI-3 AI-4
    M6-10394 M1-370 M3-315 M5-Manfredo M18-8232 M12-A735
    AI-1 M6-10394 100%  23% 25% 23% 24% 26%
    M6-10394
    M6-SS-410
    56% M23
    DSM2071
    AI-2 M1-370 23% 100%  40% 41% 38% 40%
    AI-3 M3-315 25% 40% 100%  64% 67% 61%
    M3-315
    M3-SSI-1
    M5-Manfredo 23% 39% 64% 100%  60% 65%
    M5-Manfredo
    M44-3776
    M77-4959
    M18-8232 24% 38% 67% 60% 90% 62%
    M18-8232
    M49-591
    AI-4 M12-A735 26% 40% 61% 65% 62% 97-100%
    M12-735
    M12-SS-635
    M50-4538
  • 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 M1 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 M1 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. FIGS. 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-1, 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.
  • 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-1 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-1 genes. (A schematic of the GBS mutants is presented in FIG. 7). FACS analysis of the various mutants comparing mean shift values using anti-GBS 80 versus anti-GBS 104 antibodies is presented in FIG. 8. Removal of the GBS 80 operon prevented surface exposure of GBS 104; removal of the
  • 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.
  • Pili structures that comprise GBS 104 appear to be of a lower molecular weight than pili structures lacking GBS 104. FIG. 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 A-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 suface proteins may participate in the formation of a pilus sturcture 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 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 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 as 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 non-pathogenic Gram positive bacteria, such as Streptococcus 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.
  • Adhesion 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 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 Corynebacterium adhesin island protein.
  • Examples of AI sequences in several Gram positive bacteria are discussed further below.
  • Streptococcus pyogenes (GAS)
  • 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.
  • 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 (tee6), 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., Mol. 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 tee6 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 tee6 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 FIG. 163A, 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 microscopy of strain M6 ISS3650 with antisera specific for the product of tee6 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 anitgens recognized in the original Lancefield serotyping system, is located within a GAS Adhesion Island (GAS AI-1) and forms long covalently linked pilus structures. See FIG. 163I.
  • In addition to the tee6 gene, the FCT region in M6_ISS3650 (GAS AI-1) contains two other genes (prtF1 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 PrtF1 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. 163A, 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, M18 and M49 have a similar organization whereas those of M6, M1 and M12 differ. See FIG. 164. As discussed below, these four FCT regions correlate to four GAS Adhesin Island types (AI-1, 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 (M1_SF370, M5_ISS4883 and M1220010296) 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 M1 strain SF370, there are three predicted surface proteins (Cpa (also referred to as M1126 and GAS 15), M1128 (a fimbrial protein also referred to as Spy0128 and GAS 16), and M1130 (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 M1 strain SF370 with antiserum specific for M1128 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 M1130 revealed abundant surface staining and occasional structures extending from the surface of M1 strain SF370 bacteria (FIG. 163S).
  • The M1128 protein appears to be necessary for polymerization of Cpa and M1130 proteins. If the M1128 gene in M1_SF370 was deleted, Western blot analysis using antibodies that hybridize to Cpa and M1130 no longer detected high molecular weight ladders comprising the Cpa and M1130 proteins (FIG. 163 E). See also FIGS. 177 A-C which provide the results of Western blot analysis of the M1128 (Δ128) deleted bacteria using anti-M1130 antiserum (FIG. 177 A), anti-M1128 antiserum (FIG. 177 B), and anti-M1126 antiserum (FIG. 177
  • SF370, could not be detected by any of the three antisera in Δ128 bacteria. If the Δ128 bacteria were transformed with a plasmid containing the gene for M1128, Western blot analysis using antisera specific for Cpa and M1130 again detected high molecular weight ladders (FIG. 163 H).
  • In agreement with the Western blot analysis, immunoelectron microscopy failed to detect pilus assembly on the Δ128 strain SF370 bacteria using M1128 antisera (FIG. 178 B). Although Δ128 SF370 bacteria were unable to form pili, M1126 (cpa) and M1130, which contain sortase substrate motifs, were present on the bacteria's surface. FACS analysis of the M1128 deleted (Δ128) strain SF370 bacteria also detected both M1126 and M1130 on the surface of the Δ128 strain SF370 bacteria. See FIGS. 179 D and F, which show a shift in fluorescence when antibodies immunoreactive to M1126 and M1130 are used on Δ128 bacteria. As expected, virtually no shift in fluorescence is observed when antibodies immunoreactive to M1128 are used with the Δ128 bacteria (FIG. 179 E).
  • By contrast, deletion of the M1130 gene did not effect polymerization of M1128 (FIG. 163 F). See also FIGS. 177 A-C, which provide Western blot analysis results of the M1130 deleted (Δ130) strain SF370 bacteria using anti-M1130 (FIG. 177 A), anti-M1128 (FIG. 177 B), and anti-M1126 antiserum (FIG. 177 C). The anti-M1128 and anti-M1126 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 M1126 and M1128 polypeptides in the absence of M1130. As expected, the Western blot probed with antiserum immunoreactive with M1130 did not detect any proteins for the Δ130 bacteria (FIG. 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 srtC1 gene from the FCT region of M1_SF370 abolished polymerization of all three proteins and assembly of pili (FIG. 163 G). See also FIGS. 177 A-C, which provide Western blot analysis of the SrtC1 deleted (ΔSrtC1) strain SF370 bacteria using anti-M1130 (FIG. 177 A), anti-M1128 (FIG. 177 B), and anti-M1126 antiserum (FIG. 177 C). None of the three antisera immunoreacted with high molecular weight structures (pili) in the ΔSrtC1 bacteria. Confirming that deletion of the SrtC1 gene abrogates pilus assembly in strain SF370, immunoelectron microscopy using antisera against M1128 failed to detect pilus formation on the bacteria surface. See FIG. 178 C. Although no assembled pili were detected on ΔSrtC1 SF370, M1128 proteins could be detected on the surface of SF370. Thus, it appeared that SrtC1 deletion prevented pilus assembly on the
  • cell wall. FACS analysis of the ΔSrtC1 strain SF370 confirmed that deletion of SrtC1 does not eliminate cell surface expression of M1126, M1128 or M1130. See FIG. 179 G-I, which show a shift in fluorescence when antibodies immunoreactive to M1126 (FIG. 179 G), M1128 (FIG. 179 H), and M1130 (FIG. 179 I) are used to detect cell surface protein expression on ΔSrtC1 bacteria. Thus, SrtC1 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, Spy0127, 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 M1 expression. See FIG. 180, which provides a FACS analysis of the wildtype (A) and ΔLepA mutant (B) SF370 bacteria using M1 antisera. No shift in fluorescence is observed for the ΔLepA mutant bacteria in the presence of M1 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 AI-3). Antisera against three of the four products of the FCT region (GAS AI-3) of M5_ISS4883 (Cpa, M5_orf80, M5_orf82) stained high molecular weight ladders in Western blot analysis (FIG. 163 C). Long pili were visible when antisera against M5_orf80 was used in immunogold staining followed by electron microscopy (FIG. 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 M12-20010296 (Cpa, EftLSL.A, Orf2) stained high molecular weight ladders in Western blot analysis (FIG. 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, M1128, 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 Lancefield 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 FIG. 165, which provides a FACS analysis of bacteria harboring each of the FCT types that had or had not been treated with trypsin (6). Following treatment, surface expression of the
  • specific for the pilus proteins, the bacteria's respective M proteins, or surface proteins not associated with the pili (FIG. 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 Lancefield 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, and Y) could detect recombinant proteins for each of the major pilis components (T6, M1128, 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-1)(FIG. 166 B). Pool T specifically recognized M1128 (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-1) (FIG. 166 E) and identified M1128 as antigen T1 (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 M1128 gene from M1_SF370 abolishes pilus formation, the pool T sera stained whole M1_SF370 bacteria (FIG. 166 H) but failed to stain M1_SF370 bacteria lacking the M1128 gene (FIG. 166 I).
  • 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
  • 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 AI 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-1 and AI-2; GAS AI-1 and GAS AI-3; GAS AI-1 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 FIG. 162. In all strains, the AI region is flanked by the highly conserved open reading frames M1-123 and M1-136. 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 M1, 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.
  • 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. FIG. 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-1 have a SrtB type sortase, all GAS strains and serotypes thus far characterized as an AI-2 have SrtB and SrtC1 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-1”)
  • A GAS Adhesin Island within M6 serotype (MGAS10394) is outlined in Table 4 below. This GAS adhesin island 1 (“GAS AI-1”) comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator.
  • GAS AI-1 surface proteins include Spy0157 (a fibronectin binding protein), Spy0159 (a collagen adhesion protein) and Spy0160 (a fimbrial structural subunit). Preferably, each of these GAS AI-1 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-1 includes a srt 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-1 sequences from M6 isolate (MGAS10394)
    Sortase substrate
    AI-1 sequence sequence or sortase
    identifier type functional description
    M6_Spy0156 Transcriptional regulator (rofA)
    M6_Spy0157 LPXTG Fibronectin-binding protein
    M6_Spy0158 Reverse transcriptase
    M6_Spy0159 LPXSG Collagen adhesion protein
    M6_Spy0160 LPXTG Fimbrial structural subunit
    M6_Spy0161 srtB Sortase
  • M6_Spy0160 appears to be present on the surface of GAS as part of oligomeric (pilus) structures. FIGS. 127-132 present electron micrographs of GAS serotype M6 strain 3650 immunogold stained for M6_Spy0160 using anti-M6_Spy0160 antiserum. Oligomeric or
  • GAS in each of these figures, indicating the presence of multiple M6_Spy0160 polypeptides in the oligomeric or hyperoligomeric structures. FIG. 176 A-F present electron micrographs of GAS M6 strain 2724 immunogold stained for M6_Spy0160 using anti-M6_Spy0160 antiserum (FIGS. 176 A-E) or immunogold stained for M6_Spy0159 using anti-M6_Spy0159 antiserum (FIG. 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_Spy0159 is also detected on the surface of the M6 strain 2724 GAS.
  • FACS analysis has confirmed that the GAS AI-1 surface proteins spyM60159 and spyM60160 are indeed expressed on the surface of GAS. FIG. 73 provides the results of FACS analysis for surface expression of spyM60159 on each of GAS serotypes M6 2724, M6 3650, and M6 2894. A shift in fluorescence is observed for each GAS serotype when anti-spyM60159 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-spyM60159 antiserum, and the difference in fluorescence value between the pre-immune and anti-spyM60159 antiserum.
  • TABLE 18
    Summary of FACS values for surface expression of spyM6_0159
    2724 3650 2894
    Pre- Anti- Pre- Anti- Pre- Anti-
    immune spyM6_0159 Change immune spyM6_0159 Change immune spyM6_0159 Change
    134.84 427.48 293 149.68 712.62 563 193.86 597.8 404
  • FIG. 74 provides the results of FACS analysis for surface expression of spyM60160 on each of GAS serotypes M6 2724, M6 3650, and M6 2894. In the presence of anti-spyM60160 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-spyM60160 antiserum, and the change in fluorescence value between the pre-immune and anti-spyM60160 antiserum.
  • TABLE 19
    Summary of FACS values for surface expression of spyM6_0160
    2724 3650 2894
    Pre- Anti- Pre- Anti- Pre- Anti-
    immune spyM6_0160 change immune spyM6_0160 change immune spyM6_0160 change
    117.12 443.24 326 128.57 776.39 648 125.87 621.17 495
  • Surface expression of M6_Spy0159 and M6_Spy0160 on M6 serotype GAS has also been confirmed by Western blot analysis. FIG. 98 shows that while pre-immune sera (Pα-0159) does not detect expression of M6_Spy0159 in GAS serotype M6, anti-M6_Spy0159 immune sera (Iα-0159) is able to detect M6_Spy0159 protein in both total GAS M6 extracts (M6 tot) and GAS M6 fractions enriched for cell surface proteins (M6 surf prot). The M6_Spy0159 proteins detected in the
  • molecular weight structures, indicating that M6_Spy0159 may be in an oligomeric (pilus) form.
  • FIG. 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_Spy0160 may be in an oligomeric (pilus) form.
  • FIGS. 110 and 111 both further verify the presence of M6_Spy0159 and M6_Spy0160 in higher molecular weight structures on the surface of GAS. FIG. 110 provides a Western blot performed to detect M6_Spy0159 and M6_Spy0160 in GAS M6 strain 2724 extracts enriched for surface proteins. Antiserum raised against either M6_Spy0159 (Anti-159) or M6_Spy0160 (Anti-160) cross-hybridizes with high molecular weight structures (pili) in these extracts. FIG. 111 provides a similar Western blot that verifies the presence of M6_Spy0159 and M6_Spy0160 in high molecular weight structures in GAS M6 strain 3650 extracts enriched for surface proteins.
  • SpyM60157 (a fibronectin-binding protein) may also be expressed on the surface of GAS serotype M6 bacteria. FIG. 174 shows the results of FACS analysis for surface expression of spyM60157 on M6 strain 3650. A slight shift in fluorescence is observed, which demonstrates that some spyM60157 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 M1 serotype (SF370) is outlined in Table 5 below. This GAS adhesin island 2 (“GAS AI-2”) comprises surface proteins, a SrtB sortase, a SrtC1 sortase and a RofA divergently transcribed transcriptional regulator.
  • GAS AI-2 surface proteins include GAS 15 (Cpa), Spy0128 (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), VVXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
  • GAS AI-2 includes a srtB type sortase and a srtC1 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 SrtC1 sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO: 167) motif. GAS SrtC1 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 M1 isolate (SF370)
    Sortase
    substrate
    AI-2 sequence sequence or
    identifier sortase type functional description
    SPy0124 rofA regulatory protein
    GAS15 VVXTG cpa
    (not annotated in SF370)
    SPy0127 LepA putative signal peptidase I
    SPy0128 (GAS16) EVXTG hypothetical protein (fimbrial)
    SPy0129 (GAS17) srtC1 sortase
    SPy0130 (GAS18) LPXTG hypothetical protein
    SPy0131 conserved hypothetical protein
    SPy0133 conserved hypothetical protein
    SPy0135 (GAS20) srtB sortase (putative fimbrial-
    associated protein)
  • GAS 15, GAS 16, and GAS 18 appear to be present on the surface of GAS as part of oligomeric (pilus) structures. FIGS. 113-115 present electron micrographs of GAS serotype M1 strain SF370 immunogold stained for GAS 15 using anti-GAS 15 antiserum. FIGS. 116-121 provide electron micrographs of GAS serotype M1 strain SF370 immunogold stained for GAS 16 using anti-GAS 16 antiserum. FIGS. 122-125 present electron micrograph of GAS serotype M1 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.
  • FIG. 126 reveals a hyperoligomer on the surface of a GAS serotype M1 strain SF370 bacterium immunogold stained for GAS 18. This long hyperogliomeric 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. FIG. 75 provides the results of FACS analysis for surface expression of GAS 15 on each of GAS serotypes M1 2719, M1 2580, M1 3280, M1 SF370, M1 2913, and M1 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
    Pre- Anti-GAS Pre- Anti-GAS Pre- Anti-GAS
    immune 15 Change immune 15 Change immune 15 Change
    2719 2580 3280
    159.46 712.71 553 123.9  682.84 559 217.02 639.69 423
    SF370 2913 3348
    201.93 722.68 521 121.41 600.45 479 152.09 446.41 294
  • FIGS. 76 and 79 provide the results of FACS analysis for surface expression of GAS 16 on each of GAS serotypes M1 2719, M1 2580, M1 3280, M1 SF370, M1 2913, and M1 3348. The FACS data in FIG. 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,
  • 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
    Pre- Anti-GAS Pre- Anti-GAS Pre- Anti-GAS
    immune 16 Change immune 16 Change immune 16 Change
    2719 2580 3280
    233.27 690.09 457 133.82 732.29 598 264.47 649.43 385
    SF370 2913 3348
    237.2  727.46 490 138.52 588.04 450 180.56 420.93 240
  • The FACS data in FIG. 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:
    GCTACAACAGTTCACGGGGAGACTGTTGTAAACGGAGCCAAACTAACAGTTACAAAAAACCTTGATTTAGTTAAT
    AGCAATGCATTAATTCCAAATACAGATTTTACATTTAAAATCGAACCTGATACTACTGTCAACGAAGACGGAAAT
    AAGTTTAAAGGTGTAGCTTTGAACACACCGATGACTAAAGTCACTTACACCAATTCAGATAAAGGTGGATCAAAT
    ACGAAAACTGCAGAATTTGATTTTTCAGAAGTTACTTTTGAAAAACCAGGTGTTTATTATTACAAAGTAACTGAG
    GAGAAGATAGATAAAGTTCCTGGTGTTTCTTATGATACAACATCTTACACTGTTCAAGTTCATGTCTTGTGGAAT
    GAAGAGCAACAAAAACCAGTAGCTACTTATATTGTTGGTTATAAAGAAGGTAGTAAGGTGCCAATTCAGTTCAAA
    AATAGCTTAGATTCTACTACATTAACGGTGAAGAAAAAAGTTTCAGGTACCGGTGGAGATCGCTCTAAAGATTTT
    AATTTTGGTCTGACTTTAAAAGCAAATCAGTATTATAAGGCGTCAGAAAAAGTCATGATTGAGAAGACAACTAAA
    GGTGGTCAAGCTCCTGTTCAAACAGAGGCTAGTATAGATCAACTCTATCATTTTACCTTGAAAGATGGTGAATCA
    ATCAAAGTCACAAATCTTCCAGTAGGTGTGGATTATGTTGTCACTGAAGACGATTACAAATCAGAAAAATATACA
    ACCAACGTGGAAGTTAGTCCTCAAGATGGAGCTGTAAAAAATATCGCAGGTAATTCAACTGAACAAGAGACATCT
    ACTGATAAAGATATGACCATTACTTTTACAAATAAAAAAGATTT

    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
    Pre- Anti-GAS Pre- Anti-GAS Pre- Anti-GAS
    immune 16 Change immune 16 Change immune 16 Change
    2719 2580 3280
    141.55 650.22 509 119.57 672.35 553 209.18 666.71 458
    SF370 2913 3348
    159.92 719.32 559 115.97 585.9  470 146.1  414.01 268


    each of GAS serotypes M1 2719, M1 2580, M1 3280, M1 SF370, M1 2913, and M1 3348. The antiserum used to obtain the FACS data in each of FIGS. 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
    Pre- Anti-GAS Pre- Anti-GAS Pre- Anti-GAS
    immune 18 Change immune 18 Change immune 18 Change
    2719 2580 3280
    135.68 327.98 192 116.32 379.41 263 208.12 380.84 173
    SF370 2913 3348
    185.39 438.23 253 119.95 373.32 253 147.12 266.51 119
  • TABLE 24
    Summary of FACS values for surface expression of GAS 18 using a second antisera
    Pre- Anti-GAS Pre- Anti-GAS Pre- Anti-GAS
    immune 18 Change immune 18 Change immune 18 Change
    2719 2580 3280
    150.4 250.39 100 139.18 386.38 247 253.38 347.72 94
    SF370 2913 3348
    188.64 373.11 184 124.94 384.82 260 168.8 213.65 45
  • Surface expression of GAS 15, GAS 16, and GAS 18 on M1 serotype GAS has also been confirmed by Western blot analysis. FIG. 89 shows that while pre-immune sera does not detect GAS M1 expression of GAS 15, anti-GAS 15 immune sera is able to detect GAS 15 protein in both total GAS M1 extracts and GAS M1 proteins enriched for cell surface proteins. The GAS15 proteins detected in the M1 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that GAS 15 may be in an oligomeric (pilus) form. FIG. 90 also shows the results of Western blot analysis of M1 serotype GAS using anti-GAS 15 antisera. Again, the lanes that contain GAS M1 extracts enriched for surface proteins (M1 prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 15. FIG. 91 provides an additional Western blot identical to that of FIG. 90, but that was probed with preimmune sera. As expected, no proteins were detected on this membrane.
  • FIG. 92 provides a Western blot that was probed for GAS 16 protein. While pre-immune sera does not detect GAS M1 expression of GAS 16, anti-GAS 16 immune sera is able to detect GAS
  • the M1 extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 16 may be in an oligomeric (pilus) form. FIG. 93 also shows the results of Western blot analysis of M1 serotype GAS using anti-GAS 16 antisera. The lanes that contain total GAS M1 protein (M1 tot new and M1 tot old) and the lane that contains GAS M1 extracts enriched for surface proteins (M1 prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 16. FIG. 94 provides an additional Western blot identical to that of FIG. 93, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
  • FIG. 95 provides a Western blot that was probed for GAS 18 protein. While pre-immune sera does not detect GAS M1 expression of GAS 18, anti-GAS 18 immune sera is able to detect GAS 18 protein in GAS M1 extracts enriched for cell surface proteins. The GAS 18 proteins detected in the M1 extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 18 may be in an oligomeric (pilus) form. FIG. 96 also shows the results of Western blot analysis of M1 serotype GAS using anti-GAS 18 antisera. The lane that contains GAS M1 extracts enriched for surface proteins (M1 prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 18. FIG. 97 provides an additional Western blot identical to that of FIG. 96, but that was probed with pre-immune sera. As expected, no proteins were detected on this membrane.
  • FIGS. 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 M1 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 FIGS. 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 (Iα-16), and GAS 18 (Iα-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 M1.
  • FIG. 107 provides a similar Western blot performed to detect GAS 15, GAS 16, and GAS 18 proteins in a GAS serotype M1 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
  • 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)
    Sortase
    substrate
    AI-3 sequence sequence or
    identifier sortase type functional description
    SpyM3_0097 Negative transcriptional regulator (Nra)
    SpyM3_0098 VPXTG putative collagen binding protein (Cpb)
    SpyM3_0099 LepA putative signal peptidase I
    SpyM3_0100 QVXTG conserved hypothetical protein (fimbrial)
    SpyM3_0101 SrtC2 sortase
    SpyM3_0102 LPXAG hypothetical protein
    SpyM3_0103 putative multiple sugar metabolism
    regulator
    SpyM3_0104 LPXTG protein F2 like fibronectin-binding protein
  • TABLE 7
    GAS AI-3 sequence from M3 isolate (SSI-1)
    Sortase
    Substrate
    AI-3 sequence sequence or
    identifier sortase type functional description
    SPs0099 Negative transcriptional regulator (Nra)
    SPs0100 VPXTG putative collagen binding protein (Cpb)
    SPs0101 LepA putative signal peptidase I
    SPs0102 QVXTG conserved hypothetical protein (fimbrial)
    SPs0103 SrtC2 sortase
    SPs0104 LPXAG hypothetical protein
    SPs0105 putative multiple sugar metabolism
    regulator
    SPs0106 LPXTG protein F2 like fibronectin-binding protein
  • TABLE 10
    GAS AI-3 sequences from M5 isolate (Manfredo)
    Sortase
    AI-3 substrate
    sequence sequence or
    identifier sortase type Functional description
    orf77 Negative transcriptional regulator (Nra)
    orf78 VPXTG putative collagen binding protein (Cpb)
    orf79 LepA putative signal peptidase I
    orf80 QVXTG conserved hypothetical protein (fimbrial)
    orf81 SrtC2 sortase
    orf82 LPXAG hypothetical protein
    orf83 putative multiple sugar metabolism regulator
    orf84 LPXTG protein F2 like fibronectin-binding protein
  • TABLE 8
    GAS AI-3 sequences from M18 isolate (MGAS8232)
    Sortase
    substrate
    AI-3 sequence
    sequence or sortase
    identifier type Functional description
    spyM18_0125 Negative transcriptional regulator (Nra)
    (N-terminal fragment)
    spyM18_0126 VPXTG putative collagen binding protein (Cpb)
    spyM18_0127 LepA putative signal peptidase I
    spyM18_0128 QVXTG conserved hypothetical protein (fimbrial)
    spyM18_0129 SrtC2 sortase
    spyM18_0130 LPXAG hypothetical protein
    spyM18_0131 putative multiple sugar metabolism regulator
    spyM18_0132 LPXTG protein F2 like fibronectin-binding protein
  • TABLE 44
    GAS AI-3 sequences from M49 isolate (591)
    Sortase
    substrate
    sequence
    AI-3 sequence or sortase
    identifier type Functional description
    SpyoM01000156 Negative transcriptional regulator (Nra)
    SpyoM01000155 VPXTG collagen binding protein (Cpa)
    SpyoM01000154 LepA putative signal peptidase I
    SpyoM01000153 QVXTG conserved hypothetical protein (fimbrial)
    SpyoM01000152 SrtC2 sortase
    SpyoM01000151 LPXAG hypothetical protein
    SpyoM01000150 MsmRL
    SpyoM01000149 LPXTG protein F2 like fibronectin-binding protein
  • A schematic of AI-3 serotypes M3, M5, M18, and M49 is shown in FIG. 51A. Each contains an open reading frame encoding a SrtC2-type sortase of nearly identical amino acid sequence. See FIG. 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. FIG. 60 indicates the amino acid sequence of the pilin motif and E-box of each of GAS AI-3 serotype M3 MGAS315 (SpyM30104/21909640), GAS AI-3 serotype M3 SSI (Sps0106/28895018), GAS AI-3 serotype M18 (SpyM180132/19745307), and GASAI-3 serotype M5 (orf84).
  • FACS analysis has confirmed that the GAS AI-3 surface proteins SpyM30098, SpyM3010, SpyM30102, and SpyM30104 are expressed on the surface of GAS. FIG. 80 provides the results of FACS analysis for surface expression of SpyM30098 on each of GAS
  • anti-SpyM30098 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-SpyM30098 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM30098 antiserum.
  • TABLE 25
    Summary of FACS values for surface expression of SpyM3_0098
    2721 3135
    Pre- Anti- Pre- Anti-
    immune spyM3_0098 Change immune spyM3_0098 Change
    117.85 249.51 132 99.17 277.21 178
  • FIG. 81 provides the results of FACS analysis for surface expression of SpyM30100 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM30100 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-SpyM30100 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM30100 antiserum.
  • TABLE 26
    Summary of FACS values for surface expression of SpyM3_0100
    2721 3135
    Pre- Anti- Pre- Anti-
    immune spyM3_0100 Change immune spyM3_0100 Change
    110.31 181.91 72 97.87 250.01 152
  • FIG. 82 provides the results of FACS analysis for surface expression of SpyM30102 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM30102 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-SpyM30102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM30102 antiserum.
  • TABLE 27
    Summary of FACS values for surface expression of SpyM3_0102
    in M3 serotypes
    2721 3135
    Pre- Anti- Pre- Anti-
    immune spyM3_0102 Change immune spyM3_0102 Change
    109.86 155.26 45 100.02 112.58 13
  • FIG. 82 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM30102 identified in a different GAS serotype, M6. FACS analysis conducted with the SpyM30102 antisera was able to detect surface expression of the homologous SpyM30102 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-SpyM30102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM30102 antiserum.
  • TABLE 28
    Summary of FACS values for surface expression of SpyM3_0102
    in M6 serotypes
    2724 3650 2894
    Pre- Anti- Pre- Anti- Pre- Anti-
    immune spyM3_0102 Change immune spyM3_0102 Change immune spyM3_0102 Change
    146.59 254.03 107 162.56 294.03 131 175.49 313.69 138
  • SpyM30102 is also homologous to pilin antigen 19224139 of GAS serotype M12. Antisera raised against SpyM30102 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 FIG. 109 at the lane labelled M12 2728 surf prot.
  • FIG. 83 provides the results of FACS analysis for surface expression of SpyM30104 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM30104 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-SpyM30104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM30104 antiserum.
  • TABLE 29
    Summary of FACS values for surface expression of SpyM3_0104
    in M3 serotypes
    2721 3135
    Pre- Anti- Pre- Anti-
    immune spyM3_0104 Change immune spyM3_0104 Change
    128.45 351.65 223 105.1 339.88 235
  • FIG. 83 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM30104 identified in a different GAS serotype, M12. FACS analysis conducted with the SpyM30104 antisera was able to detect surface expression of the homologous SpyM30104 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-SpyM30104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM30104 antiserum.
  • TABLE 30
    Summary of FACS values for surface expression of
    SpyM3_0104 in an M12 serotype
    2728
    Pre-immune Anti-spyM3_0104 Change
    198.57 288.75 90
  • FIG. 84 provides the results of FACS analysis for surface expression of SPs0106 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SPs0106 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-SPs0106 antiserum, and the difference in fluorescence value between the pre-immune and anti-SPs0106 antiserum.
  • TABLE 31
    Summary of FACS values for surface expression of SPs_0106
    in M3 serotypes
    2721 3135
    Pre- Pre-
    immune Anti-SPs_0106 Change immune Anti-SPs_0106 Change
    116 463.28 347 103.02 494.27 391
  • FIG. 84 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SPs0106 identified in a different GAS serotype, M12. FACS analysis
  • SPs0106 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-SPs0106 antiserum, and the difference in fluorescence value between the pre-immune and anti-SPs0106 antiserum.
  • TABLE 32
    Summary of FACS values for surface expression of
    SPs_0106 in an M12 serotype
    2728
    Pre-immune Anti-SPs_0106 Change
    304.01 254.64 −49
  • (4) Adhesin Island Sequence within M12: 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 P2 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 sequences from M12 isolate (A735)
    AI-4 Sortase substrate
    sequence sequence or sortase
    identifier type Functional description
    19224133 RofA regulatory protein
    19224134 LPXTG protein F
    SrtB SrtB (stop codon*)
    19224135 VPXTG Cpa
    19224136 LepA
    19224137 QVXTG EftLSL.A (fimbrial)
    19224138 SrtC2 EftLSL.B
    19224139 LPXAG Orf2
    19224140 MsmRL
    19224141 LPXTG protein F2
  • A schematic of AI-4 serotype M12 is shown in FIG. 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
  • sequences.
  • Other proteins encoded by the open reading frames of the AI-4 serotype M12 are homologous to proteins encoded by other known adhesin islands in S. pyogenes, as well as the GAS AI-3 serotype M5 (Manfredo). 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-1 serotype M3 (28810257), S. pyogenes MGAS8232 serotype M3 (19745301), and GAS AI-2 serotype M1 (GAS15). The amino acid sequence of the AI-4 serotype M12 cpa shares a high degree of homology with other cpa proteins.
  • FIG. 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 MGAS10394 serotype M6 (50913503).
  • FIG. 54 is an amino acid sequence alignment that illustrates that the F2-like fibronectin-binding protein of AI-4 serotype M12 (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).
  • FIG. 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 M1 GAS serotype M1 (13621428).
  • FIG. 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-1 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. FIG. 60 indicates the amino acid sequence of the pilin motif and E-box in AI-4 serotype M12.
  • FACS analysis has confirmed that the GAS AI-4 surface proteins 19224134, 19224135, 19224137, and 19224141 are expressed on the surface of GAS. FIG. 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
    Pre-immune Anti-19224134 Change
    137.8 485.32 348
  • FIG. 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 preimmune and anti-19224134 antiserum.
  • TABLE 34
    Summary of FACS values for surface expression of 19224134 in M6 serotypes
    2724 3650 2894
    Pre- Anti- Pre- Anti- Pre- Anti-
    immune 19224134 Change immune 19224134 Change immune 19224134 Change
    123.58 264.59 141 140.82 262.64 122 135.4 307.25 172
  • FIG. 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
    2728
    Pre-immune Anti-19224135 Change
    151.38 471.95 321
  • FIG. 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
    2728
    Pre-immune Anti-19224137 Change
    140.44 433.25 293
  • FIG. 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 M12 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.
  • TABLE 37
    Summary of FACS values for surface expression of
    19224141 in an M12 serotype
    2728
    Pre-immune Anti-19224141 Change
    147.02 498 351
  • 19224139 (designated as orf2) may also be expressed on the surface of GAS serotype M12 bacteria. FIG. 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. FIG. 99 shows that while pre-immune era (Pα-4135) does not detect GAS M12 expression of 19224135, anti-19224135 immune sera (Iα-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 FIG. 108, which provides a further Western blot showing that anti-19224135 antiserum (Anti-19224135) immunoreacts 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. FIG. 100 shows that while pre-immune sera (Pα-4137) does not detect GAS M12 expression of 19224137, anti-19224137 immune sera (Iα-4137) is able to detect 19224137 protein in both total GAS M12 extracts (M12 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 FIG. 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
  • 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 FIG. 137. The locus comprises open reading frames encoding a transcriptional regulator (rlrA), cell wall surface proteins (rrgA, rrgB, rrgC), and sortases (srtB, srtC, srtD). FIG. 137 also indicates the S. pneumoniae strain TIGR4 gene name corresponding to each of these open reading frames.
  • Tables 9 and 38 identify the genomic location of each of these open reading frames in S. pneumoniae strains TIGR4 and 670, respectively.
  • TABLE 9
    S. pneumoniae AI sequences from TIGR4
    Synonym (AI Sequence
    Genomic Location Strand Length PID Identifier) Functional description
    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 . . . 445696 + 279 15900382 SP0466 sortase
    445791 . . . 446576 + 261 15900383 SP0467 sortase
    446563 . . . 447414 + 283 15900384 SP0468 sortase
  • TABLE 38
    S. pneumoniae strain 670 AI sequences
    Genomic AI Sequence
    Location Strand Identifier\ Functional description
    4383-5645 Orf1_670 IS1167, transposase
    5910-7439 Orf2_670 transcriptional regulator, putative
     7934-10606 + Orf3_670 cell wall surface anchor family
    protein
    10839-12773 + Orf4_670 cell wall surface anchor family
    protein
    12796-14001 + Orf5_670 cell wall surface anchor family
    protein
    14327-15241 + Orf6_670 sortase, putative
    15336-16121 + Orf7_670 sortase, putative
    16108-16959 + Orf8_670 sortase, putative
  • The full-length nucleotide sequence of the S. pneumoniae strain 670 AI is also shown in FIG. 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 FIG. 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
    Pri-
    mer Reverse
    Pair Forward Primer Sequence Primer Sequence
    1 ACTTTCTAATGAGTTGTTTAGGCG AGCGACAAGCCACTGTAT
    CATATT
    2 CTGGTCGATAACTCCTTCAATCTT GTACGACAAAAGTGTGGC
    TTGTT
    3 GAATGCGATATTCAGGACCAACTA ATCTCACTGAGTTAATCC
    GTTCAC
    4 TGTATACAAGTGTGTCATTGCCAG CATCTTCACCTGTTCTCA
    CATTTT
    5 GCGGTCTTTAGTCTTCAAAAACA CAAGAGAAAAACACAGAG
    CCATAA
    6 TTGCTTAAGTAAGAGAGAAAGGAGC CAGGAGTATAGTGTCCGC
    TTTCTT
    7 GGCAATGTTGACTTTATGAAGGTG TATCAGCATCCCTTTATC
    TTCAAAC
    8 TGAGATTTTCTCGTTTCTCTTAGC AATAGACGATGGGTATTG
    ATCATGT
    9 CCGACGAACTTTGATGATTTATTG ACCAACAGACGATGACTG
    TTAATC
    10 AATGACTTTGAGCCTGTCTTGAT TTCTACAATTTCCTGGCC
    ATTATC
    11 GCCATTTGGATCAGCTAAAAGTT TTTTTCAACCCACTACAG
    TTGACA

    These primers hybridized along the entire length of the AI locus to generate amplification products representative of sequences throughout the locus. See FIG. 138, which is a schematic of the location where each of these primers hybridizes to the S. pneumoniae AI locus. FIG. 139A provides the set of amplicons obtained from amplification of the AI locus in S. pneumoniae strain TIGR4. FIG. 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, 9V 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 CSR10), 9V Spain 3 (9VSP3), 19F Taiwan 14 (19FTW14), 23F Taiwan 15 (19FTW15), and 23F Poland 16 (23FP16). See FIG. 140.
  • The AI locus has been sequenced for each of these strains and the nucleotide and encoded amino acid sequence 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 FIG. 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 identity comparison of S. pneumoniae strains AI sequences
    S. pneumoniae S. pneumoniae
    strain 670 from TIGR4
    polypeptide polypeptide Shared identity of polypeptides
    Orf1_670 SP0460  99.3% identity in 422 aa overlap
    Orf2_670 SP0461 100.0% identity in 509 aa overlap
    Orf3_670 SP0462  83.2% identity in 895 aa overlap
    Orf4_670 SP0463  47.9% identity in 678 aa overlap
    Orf5_670 SP0464  99.7% identity in 393 aa overlap
    Orf6_670 SP0466 100.0% identity in 279 aa overlap
    Orf7_670 SP0467  94.2% identity in 260 aa overlap
    Orf8_670 SP0468  91.5% identity in 283 aa overlap

    FIGS. 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
    % Identity
    Encoded Groups of % Identity in Between
    Putative Role of Polypeptide by Orf Homology Group Groups
    RlrA, transcriptional regulator 2 1 group (10 strains) 100
    RrgA, cell wall surface protein 3 2 groups (6 + 4) 98-100 83
    RrgB, cell wall surface protein 4 2 groups (6 + 4) 99-100 48
    RrgC, cell wall surface protein 5 2 groups (6 + 4) 99-100 97
    SrtB, putative sortase 6 2 groups (7 + 3) 99-100 97
    SrtC, putative sortase 7 2 groups (6 + 4) 95-100 93
    SrtD, putative sortase 8 2 groups (6 + 4) 99-100 92
  • 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 FIG. 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 FIGS. 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 FIG. 182, which shows detection of high molecular weight structures expressed by S. pneumoniae strains that comprise the adhesin island
  • microscopy and negative staining detects the presence of pili extending from the surface of S. pneumoniae. See FIG. 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 FIG. 186. See also FIG. 235, which provides an electron micrograph of S. 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 FIG. 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 FIG. 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 FIG. 184.
  • The Sp0463 (S. pneumoniae TIGR4 rrgB) adhesion island polypeptide is expressed in oligomeric form. Whole cell extracts were analyzed by Western blot using a Sp0463 antiserum. The antiserum cross-hybridized with high molecular weight Sp0463 polymers. See FIG. 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 FIG. 137. Immunogold labelling of S. pneumoniae TIGR4 using RrgB antiserum confirms the presence of RrgB in pili. FIG. 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 FIG. 234 at arrows; FIG. 234 is a close up of a pilus in FIG. 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 S. pneumoniae TIGR4. See FIG. 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 FIG. 183.
  • A detailed diagram of the amino acid sequence comparisons of the RrgA protein in the ten S. pneumoniae strains is shown in FIG. 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, Sp0462 (rrgA), Sp0463 (rrgB), and Sp0464 (rrgC), have been cloned and expressed. See examples 15-17. A polyacrylamide gel showing successful recombinant expression of RrgA is provided in FIG. 190A. Detection of the RrgA protein, which is expressed in pET21b with a histidine tag, is also shown by Western blot analysis in FIG. 190B, using an anti-histidine tag antibody.
  • Antibodies that detect RrgB and RrgC antibodies have been produced in mice. See FIGS. 191 and 192, which show detection of RrgB and RrgC, respectively, using the raised antibodies.
  • SrtB type sortases have been identified in several S. pneumoniae clinical isolates, demonstrating conservation of a SrtB type sortase across these isolates.
  • Recombinantly 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 sequence 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
  • 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-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).
  • GBS80
  • 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 FIGS. 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
    ATGAAATTATCGAAGAAGTTATTGTTTTCGGCTGCTGTTTTAACAATGGTGGCGGGGTCAACTGTTGAACCAGTA
    GCTCAGTTTGCGACTGGAATGAGTATTGTAAGAGCTGCAGAAGTGTCACAAGAACGCCCAGCGAAAACAACAGTA
    AATATCTATAAATTACAAGCTGATAGTTATAAATCGGAAATTACTTCTAATGGTGGTATCGAGAATAAAGACGGC
    GAAGTAATATCTAACTATGCTAAACTTGGTGACAATGTAAAAGGTTTGCAAGGTGTACAGTTTAAACGTTATAAA
    GTCAAGACGGATATTTCTGTTGATGAATTGAAAAAATTGACAACAGTTGAAGCAGCAGATGCAAAAGTTGGAACG
    ATTCTTGAAGAAGGTGTCAGTCTACCTCAAAAAACTAATGCTCAAGGTTTGGTCGTCGATGCTCTGGATTCAAAA
    AGTAATGTGAGATACTTGTATGTAGAAGATTTAAAGAATTCACCTTCAAACATTACCAAAGCTTATGCTGTACCG
    TTTGTGTTGGAATTACCAGTTGCTAACTCTACAGGTACAGGTTTCCTTTCTGAAATTAATATTTACCCTAAAAAC
    GTTGTAACTGATGAACCAAAAACAGATAAAGATGTTAAAAAATTAGGTCAGGACGATGCAGGTTATACGATTGGT
    GAAGAATTCAAATGGTTCTTGAAATCTACAATCCCTGCCAATTTAGGTGACTATGAAAAATTTGAAATTACTGAT
    AAATTTGCAGATGGCTTGACTTATAAATCTGTTGGAAAAATCAAGATTGGTTCGAAAACACTGAATAGAGATGAG
    CACTACACTATTGATGAACCAACAGTTGATAACCAAAATACATTAAAAATTACGTTTAAACCAGAGAAATTTAAA
    GAAATTGCTGAGCTACTTAAAGGAATGACCCTTGTTAAAAATCAAGATGCTCTTGATAAAGCTACTGCAAATACA
    GATGATGCGGCATTTTTGGAAATTCCAGTTGCATCAACTATTAATGAAAAAGCAGTTTTAGGAAAAGCAATTGAA
    AATACTTTTGAACTTCAATATGACCATACTCCTGATAAAGCTGACAATCCAAAACCATCTAATCCTCCAAGAAAA
    CCAGAAGTTCATACTGGTGGGAAACGATTTGTAAAGAAAGACTCAACAGAAACACAAACACTAGGTGGTGCTGAG
    TTTGATTTGTTGGCTTCTGATGGGACAGCAGTAAAATGGACAGATGCTCTTATTAAAGCGAATACTAATAAAAAC
    TATATTGCTGGAGAAGCTGTTACTGGGCAACCAATCAAATTGAAATCACATACAGACGGTACGTTTGAGATTAAA
    GGTTTGGCTTATGCAGTTGATGCGAATGCAGAGGGTACAGCAGTAACTTACAAATTAAAAGAAACAAAAGCACCA
    GAAGGTTATGTAATCCCTGATAAAGAAATCGAGTTTACAGTATCACAAACATCTTATAATACAAAACCAACTGAC
    ATCACGGTTGATAGTGCTGATGCAACACCTGATACAATTAAAAACAACAAACGTCCTTCAATCCCTAATACTGGT
    GGTATTGGTACGGCTATCTTTGTCGCTATCGGTGCTGCGGTGATGGCTTTTGCTGTTAAGGGGATGAAGCGTCGT
    ACAAAAGATAAC
    SEQ ID NO: 2
    MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG
    EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
    SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG
    EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
    EIAELLKGMTLVKNQDATDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
    PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
    GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG
    GIGTAIFVAIGAAVMAFAVKGMKRRTKDN
  • 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
    SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG
    EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
    EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
    PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
    GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG
  • GBS 80 contains an amino acid motif indicative of a cell wall anchor: SEQ ED 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 ID NO: 6
    MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG
    EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
    SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG
    EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
    EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
    PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
    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
    TINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVK
    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
    EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
    PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
    GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG
    GIGTAIFVAIGAAVMAFAVKGMKRRTKDN
    SEQ ID NO: 8
    AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKK
    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.
  • 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-1 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 μog 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 t=36-37), and typically had a gestation period of 21 days. Within 48 hours of birth, the pups were challenged via I.P. 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 50 μl 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 CD1 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 I.P. with a 70-90% lethal dose of GBS serotype III COH1. 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.
  • TABLE 1
    Immunization
    %
    Antigen Alive/total Survival Fisher's exact test
    PBS (neg control) 13/80 16%
    GBS (whole cell) 54/65 83% P < 0.00000001
    GBS80 (intact) 62/70 88% P < 0.00000001
    GBS80 (fragment) SEQ ID 7 35/64 55% P = 0.0000013
    GBS80 (fragment) SEQ ID 8 13/67 19% P = 0.66
  • TABLE 2
    Passive Maternal Immunization
    %
    Antigen Alive/total Survival Fisher's exact test
    PBS (neg control) 12/42 28%
    GBS (whole cell) 48/52 92% P < 0.00000001
    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 = 1
  • 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
    SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEP K TDKDVKKLGQDDAGYTIG
    EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
    EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
    PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
    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
    MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDG
    EVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
    SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIG
    EEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
    EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRK
    PEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
    GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTG
    GIGTAIFVAIGAAVMAFAVKGMKRRTKDN
  • GBS 104
  • amino acid sequences of GBS 104 sequenced from serotype V isolated strain 2603 are set forth below as SEQ ID NOS 10 and 11:
  • SEQ ID NO.10
    ATGAAAAAGAGACAAAAAATATGGAGAGGGTTATCAGTTACTTTACTAATCCTGTCCCAAATTCCATTTGGTATA
    TTGGTACAAGGTGAAACCCAAGATACCAATCAAGCACTTGGAAAAGTAATTGTTAAAAAAACGGGAGACAATGCT
    ACACCATTAGGCAAAGCGACTTTTGTGTTAAAAAATGACAATGATAAGTCAGAAACAAGTCACGAAACGGTAGAG
    GGTTCTGGAGAAGCAACCTTTGAAAACATAAAACCTGGAGACTACACATTAAGAGAAGAAACAGCACCAATTGGT
    TATAAAAAAACTGATAAAACCTGGAAAGTTAAAGTTGCAGATAACGGAGCAACAATAATCGAGGGTATGGATGCA
    GATAAAGCAGAGAAACGAAAAGAAGTTTTGAATGCCCAATATCCAAAATCAGCTATTTATGAGGATACAAAAGAA
    AATTACCCATTAGTTAATGTAGAGGGTTCCAAAGTTGGTGAACAATACAAAGCATTGAATCCAATAAATGGAAAA
    GATGGTCGAAGAGAGATTGCTGAAGGTTGGTTATCAAAAAAAATTACAGGGGTCAATGATCTCGATAAGAATAAA
    TATAAAATTGAATTAACTGTTGAGGGTAAAACCACTGTTGAAACGAAAGAACTTAATCAACCACTAGATGTCGTT
    GTGCTATTAGATAATTCAAATAGTATGAATAATGAAAGAGCCAATAATTCTCAAAGAGCATTAAAAGCTGGGGAA
    GCAGTTGAAAAGCTGATTGATAAAATTACATCAAATAAAGACAATAGAGTAGCTCTTGTGACATATGCCTCAACC
    ATTTTTGATGGTACTGAAGCGACCGTATCAAAGGGAGTTGCCGATCAAAATGGTAAAGCGCTGAATGATAGTGTA
    TCATGGGATTATCATAAAACTACTTTTACAGCAACTACACATAATTACAGTTATTTAAATTTAACAAATGATGCT
    AACGAAGTTAATATTCTAAAGTCAAGAATTCCAAAGGAAGCGGAGCATATAAATGGGGATCGCACGCTCTATCAA
    TTTGGTGCGACATTTACTCAAAAAGCTCTAATGAAAGCAAATGAAATTTTAGAGACACAAAGTTCTAATGCTAGA
    AAAAAACTTATTTTTCACGTAACTGATGGTGTCCCTACGATGTCTTATGCCATAAATTTTAATCCTTATATATCA
    ACATCTTACCAAAACCAGTTTAATTCTTTTTTAAATAAAATACCAGATAGAAGTGGTATTCTCCAAGAGGATTTT
    ATAATCAATGGTGATGATTATCAAATAGTAAAAGGAGATGGAGAGAGTTTTAAACTGTTTTCGGATAGAAAAGTT
    CCTGTTACTGGAGGAACGACACAAGCAGCTTATCGAGTACCGCAAAATCAACTCTCTGTAATGAGTAATGAGGGA
    TATGCAATTAATAGTGGATATATTTATCTCTATTGGAGAGATTACAACTGGGTCTATCCATTTGATCCTAAGACA
    AAGAAAGTTTCTGCAACGAAACAAATCAAAACTCATGGTGAGCCAACAACATTATACTTTAATGGAAATATAAGA
    CCTAAAGGTTATGACATTTTTACTGTTGGGATTGGTGTAAACGGAGATCCTGGTGCAACTCCTCTTGAAGCTGAG
    AAATTTATGCAATCAATATCAAGTAAAACAGAAAATTATACTAATGTTGATGATACAAATAAAATTTATGATGAG
    CTAAATAAATACTTTAAAACAATTGTTGAGGAAAAACATTCTATTGTTGATGGAAATGTGACTGATCCTATGGGA
    GAGATGATTGAATTCCAATTAAAAAATGGTCAAAGTTTTACACATGATGATTACGTTTTGGTTGGAAATGATGGC
    AGTCAATTAAAAAATGGTGTGGCTCTTGGTGGACCAAACAGTGATGGGGGAATTTTAAAAGATGTTACAGTGACT
    TATGATAAGACATCTCAAACCATCAAAATCAATCATTTGAACTTAGGAAGTGGACAAAAAGTAGTTCTTACCTAT
    GATGTACGTTTAAAAGATAACTATATAAGTAACAAATTTTACAATACAAATAATCGTACAACGCTAAGTCCGAAG
    AGTGAAAAAGAACCAAATACTATTCGTGATTTCCCAATTCCCAAAATTCGTGATGTTCGTGAGTTTCCGGTACTA
    ACCATCAGTAATCAGAAGAAAATGGGTGAGGTTGAATTTATTAAAGTTAATAAAGACAAACATTCAGAATCGCTT
    TTGGGAGCTAAGTTTCAACTTCAGATAGAAAAAGATTTTTCTGGGTATAAGCAATTTGTTCCAGAGGGAAGTGAT
    GTTACAACAAAGAATGATGGTAAAATTTATTTTAAAGCACTTCAAGATGGTAACTATAAATTATATGAAATTTCA
    AGTCCAGATGGCTATATAGAGGTTAAAACGAAACCTGTTGTGACATTTACAATTCAAAATGGAGAAGTTACGAAC
    CTGAAAGCAGATCCAAATGCTAATAAAAATCAAATCGGGTATCTTGAAGGAAATGGTAAACATCTTATTACCAAC
    ACTCCCAAACGCCCACCAGGTGTTTTTCCTAAAACAGGGGGAATTGGTACAATTGTCTATATATTAGTTGGTTCT
    ACTTTTATGATACTTACCATTTGTTCTTTCCGTCGTAAACAATTG
    SEQ ID NO. 11
    MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVE
    GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE
    NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV
    VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
    SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR
    KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV
    PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFWGNIR
    PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
    EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY
    DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
    LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN
    LKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL
  • 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
  • example of such a GBS 104 fragment is set forth below as SEQ ID NO 12.
  • SEQ ID NO 12
    GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKK
    TDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGR
    REIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGEAVE
    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 cytoplasmic 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
    EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY
    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
  • 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
    GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTK E
    NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV
    VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
    SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR
    KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV
    PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATK QIKTHGEPTTLYFNGNIR
    PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
    EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY
    DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
    LGAKFQLQIEKDESGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN
    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 ID NO. 11
    MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVE
    GSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE
    NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVV
    VLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
    SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNAR
    KKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV
    PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIR
    PKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
    EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTY
    DVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
    LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTN
    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.
  • SEQ ID NO: 15
    ATGAGAAAATACCAAAAATTTTCTAAAATATTGACGTTAAGTCTTTTTTGTTTGTCGCAAATACCGCTTAATACC
    AATGTTTTAGGGGAAAGTACCGTACCGGAAAATGGTGCTAAAGGAAAGTTAGTTGTTAAAAAGACAGATGACCAG
    AACAAACCACTTTCAAAAGCTACCTTTGTTTTAAAAACTACTGCTCATCCAGAAAGTAAAATAGAAAAAGTAACT
    GCTGAGCTAACAGGTGAAGCTACTTTTGATAATCTCATACCTGGAGATTATACTTTATCAGAAGAAACAGCGCCC
    GAAGGTTATAAAAAGACTAACCAGACTTGGCAAGTTAAGGTTGAGAGTAATGGAAAAACTACGATACAAAATAGT
    GGTGATAAAAATTCCACAATTGGACAAAATCAGGAAGAACTAGATAAGCAGTATCCCCCCACAGGAATTTATGAA
    GATACAAAGGAATCTTATAAACTTGAGCATGTTAAAGGTTCAGTTCCAAATGGAAAGTCAGAGGCAAAAGCAGTT
    AACCCATATTCAAGTGAAGGTGAGCATATAAGAGAAATTCCAGAGGGAACATTATCTAAACGTATTTCAGAAGTA
    GGTGATTTAGCTCATAATAAATATAAAATTGAGTTAACTGTCAGTGGAAAAACCATAGTAAAACCAGTGGACAAA
    CAAAAGCCGTTAGATGTTGTCTTCGTACTCGATAATTCTAACTCAATGAATAACGATGGCCCAAATTTTCAAAGG
    CATAATAAAGCCAAGAAAGCTGCCGAAGCTCTTGGGACCGCAGTAAAAGATATTTTAGGAGCAAACAGTGATAAT
    AGGGTTGCATTAGTTACCTATGGTTCAGATATTTTTGATGGTAGGAGTGTAGATGTCGTAAAAGGATTTAAAGAA
    GATGATAAATATTATGGCCTTCAAACTAAGTTCACAATTCAGACAGAGAATTATAGTCATAAACAATTAACAAAT
    AATGCTGAAGAGATTATAAAAAGGATTCCGACAGAAGCTCCTAAAGCTAAGTGGGGATCTACTACCAATGGATTA
    ACTCCAGAGCAACAAAAGGAGTACTATCTTAGTAAAGTAGGAGAAACATTTACTATGAAAGCCTTCATGGAGGCA
    GATGATATTTTGAGTCAAGTAAATCGAAATAGTCAAAAAATTATTGTTCATGTAACTGATGGTGTTCCTACGAGA
    TCATATGCTATTAATAATTTTAAACTGGGTGCATCATATGAAAGCCAATTTGAACAAATGAAAAAAAATGGATAT
    CTAAATAAAAGTAATTTTCTACTTACTGATAAGCCCGAGGATATAAAAGGAAATGGGGAGAGTTACTTTTTGTTT
    CCCTTAGATAGTTATCAAACACAGATAATCTCTGGAAACTTACAAAAACTTCATTATTTAGATTTAAATCTTAAT
    TACCCTAAAGGTACAATTTATCGAAATGGACCAGTGAAAGAACATGGAACACCAACCAAACTTTATATAAATAGT
    TTAAAACAGAAAAATTATGACATTTTTAATTTTGGTATCGATATATCTGGTTTTAGACAAGTTTATAATGAGGAG
    TATAAGAAAAATCAAGATGGTACTTTTCAAAAATTGAAAGAGGAAGCTTTTAAACTTTCAGATGGAGAAATCACA
    GAACTAATGAGGTCGTTCTCTTCCAAACCTGAGTACTACACCCCTATCGTAACTTCAGCCGATACATCTAACAAT
    GAAATTTTATCTAAAATTCAGCAACAATTTGAAACGATTTTAACAAAAGAAAACTCAATTGTTAATGGAACTATC
    GAAGATCCTATGGGTGATAAAATCAATTTACAGCTTGGTAATGGACAAACATTACAGCCAAGTGATTATACTTTA
    CAGGGAAATGATGGAAGTGTAATGAAGGATGGTATTGCAACTGGTGGGCCTAATAATGATGGTGGAATACTTAAG
    GGGGTTAAATTAGAATACATCGGAAATAAACTCTATGTTAGAGGTTTGAATTTAGGAGAAGGTCAAAAAGTAACA
    CTCACATATGATGTGAAACTAGATGACAGTTTTATAAGTAACAAATTCTATGACACTAATGGTAGAACAACATTG
    AATCCTAAGTCAGAGGATCCTAATACACTTAGAGATTTTCCAATCCCTAAAATTCGTGATGTGAGAGAATATCCT
    ACAATAACGATTAAAAACGAGAAGAAGTTAGGTGAAATTGAATTTATAAAAGTTGATAAAGATAATAATAAGTTG
    CTTCTCAAAGGAGCTACGTTTGAACTTCAAGAATTTAATGAAGATTATAAACTTTATTTACCAATAAAAAATAAT
    AATTCAAAAGTAGTGACGGGAGAAAACGGCAAAATTTCTTACAAAGATTTGAAAGATGGCAAATATCAGTTAATA
    GAAGCAGTTTCGCCGGAGGATTATCAAAAAATTACTAATAAACCAATTTTAACTTTTGAAGTGGTTAAAGGATCG
    ATAAAAAATATAATAGCTGTTAATAAACAGATTTCTGAATATCATGAGGAAGGTGACAAGCATTTAATTACCAAC
    ACGCATATTCCACCAAAAGGAATTATTCCTATGACAGGTGGGAAAGGAATTCTATCTTTCATTTTAATAGGTGGA
    GCTATGATGTCTATTGCAGGTGGAATTTATATTTGGAAAAGGTATAAGAAATCTAGTGATATGTCCATCAAAAAA
    GAT
    SEQ ID NO: 16
    MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQNKPLSKATFVLKTTAHPESKIEKVT
    AELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
    DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDK
    QKPLDVVFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
    DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEA
    DDILSQVNRNSQKIIVHVTDGVPTRSYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
    PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEE
    YKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
    EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGEGQKVT
    LTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
    LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLIEAVSPEDYQKITNKPILTFEVVKGS
    IKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK
    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
  • 17.
  • SEQ ID NO: 17
    MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
    NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAP
    EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
    DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
    GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
    HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
    DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
    TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
    SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
    PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
    LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
    ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
    EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
    GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
    NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
    LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
    EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
    THIPPKGIIPMTGGKGILS
  • 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
    MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
    NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAP
    EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
    DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
    GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
    HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
    DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
    TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
    SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
    PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
    LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
    ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
    EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
    GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
    NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
    LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
    EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
    THIPPKGI
  • 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 motif, 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 ID NO: 16
    MRKYQKFSKILTLSLFGLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
    NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAP
    EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
    DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
    GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
    HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
    DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAP K A K WGSTTNGL
    TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
    SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
    PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPV K EHGTPTKLYINS
    LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
    ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
    EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
    GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
    NPKSEDPNTLRDFPIP K IRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
    LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
    EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
    THIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK
    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
    MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
    NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLS E ETAP
    EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
    DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
    GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
    HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
    DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
    TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
    SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
    PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
    LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
    ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
    EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
    GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
    NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
    LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
    E AVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
    THIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK
    D
  • Predicted secondary structure for the GBS 067 amino acid sequence is set forth in FIG. 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 (pfam05738). 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
  • collagen, fibronectin, and fibrinogen, discussed above.
  • Because applicants have identified GBS 67 as a surface exposed protein on GBS and because GBS 67 may be involved in GBS adhesion the immunogenicity of the GBS 67 protein was examined in mice. The results of an immunization assay with GBS 67 are set forth in Table 48, below.
  • TABLE 48
    GBS 67 Protects Mice in an Immunization Assay
    Challenge GBS 67 immungen
    GBS strain % PBS immunogen FACS
    (serotype) dead/treated survival dead/treated % survival Δmean
    3050 (II) 0/30 100 29/49 41 460
    CJB111 (V) 76/185 59 143/189 24 481
    7357 b (Ib) 34/56  39 65/74 12 316
  • As shown in Table 48, immunization with GBS 67 provides a substantially improved survival rate for challenged mice relative to negative control, PBS, immunized mice. These results indicate that GBS 67 may comprise an immunogenic composition of the invention.
  • GBS 59
  • The following offers examples of GBS 59 fragments. Nucleotide and amino acid sequences of GBS 59 sequenced from serotype V isolated strain 2603 are set forth below as SEQ ID NOS: 125 and 126. The GBS 59 polypeptide of SEQ ID NO: 126 is referred to as SAG1407.
  • SEQ ID NO: 125
    ttaagcttcctttgattggcgtcttttcatgataactactgctccaagca
    taatgcttaaaccaataattgtgaaaagaattgtaccaataccacctgtt
    tgtgggattgttacctttttattttctacacgtgtcgcatctttttggtt
    gctgttagcaacgtagtcaatgttaccacctgttatgtatgacccttgat
    taactacaaacttaatattacctgccaacttagcaaatcctgctggagca
    agtgtttcttcaaggttgtaagtaccgtctgcaagacctqtaacttcaaa
    ttgaccttgatcgtttgaagtgtaggtaatggctctagccttatctgtta
    tccactcataagctgtacgagcctcaatgaaggctgcatcgtaatctgct
    tgtttagttttgataagttcttttgcagtaattcctttttcacctttttg
    gtctgttgcagacaacttgttataagcagcgatagcttcatctaaagcta
    ttttcttagcagctaaagttttttgaccttctgattgatctgctttaaga
    gcaaggtatttacctgctgagtttttcacaacgaattgtgcaccagccaa
    acggtcaccttgttcattagttttgacaaatttcttaccatgagtttcaa
    cttttggttcagttgggttcaatggtgttgggttatcagaatctttggta
    ttggtaatggttactttaccattttctagatttattgcacttccgtaacc
    agaaacacgttctgagatcatgtatgatttgttttctagaccagtgaatt
    tacccgagaagttaccagatacttcaaatttgataccatttccaaggtcg
    attgtacctttagatgtttttgtcaatgatactgaagcaacagttttatc
    tttatctttcaatgtgtaaacaacgtttacaccatcaggtgcaattccgt
    cagaccaagttttagcaactgttacttcaccctttgaaggtgtaacagga
    agttcagtcaagtctttacctggtttgttaccatacgacaatttgatatc
    attggattctggattatcaataattgcttgaccattaacagtagcactat
    aagtcaatgtaaattcaatatcagctgttttagctgctttttccaatttg
    cccaatccatcagctgtgaattttaatgtgaaaccacgggcatcaatgct
    aagttcatagtctgtatccttagcaaaagtttctgtagttcctgaagctt
    taaggctaacagttgaacccattgtcaaaccatttgacattatatctgtc
    caaaccaagttttcgtatttagaacctttgtgaatttttgttttaacttc
    ataaggaacaactttaccgatttcagcagtagcagttgctttgtcacgtg
    cataattaccataatttgcgccagctgtcaaaagtctattaacatctgtc
    aatgctgtcaaatcgtttgttttagcaaagtttttatcaatttctggttt
    ttcttcagtgttctttggataaacatgggcatcagcaacaacaccatctt
    catttaccaatggaagagtgatgttaactggaaccgcttttgaagcagcc
    aggagggaaccattattgttgtaagtagattttgatttaacttcaacaat
    tttaaactcgcctttcaatcctttggtgttgaaaacaagtccagtatctc
    cctctggtgtcaatccagacacggcctcatcaatatttactgttatttca
    ggagtaccatctttattaattaaggctggtgttaatttgttaccttcttt
    tgccttaacatattgcactttaccacttttatcttctttcaaagctaaag
    caaagaacgcaccttcgatttctttagatccctcgccaaagtaaccagca
    aggtcagaaatagctccacctttgtagtcttttccgttaagacctgtagt
    tcctgggaagttacttttgttaagatttgattcggtttgcaaaatcttgt
    gcaaagtcactgtattagttgttgcttcatccgcaaaggctggtgcaact
    gagagcaatgacgttaaagtcagtaacaatgccgagaacattgcaaaata
    tttgttgattcttttcat
    SEQ ID NO: 126
    MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLN
    KSNFPGTTGLNGKDYKGGAISDLAGYFGEGSKEIEGAFFALALKEDKSGK
    VQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
    LKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGVVADAHVY
    PKNTEEKPEIDKNFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAEI
    GKVVPYEVKTKIHKGSKYENLVWTDIMSNGLTMGSTVSLKASGTTETFAK
    DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII
    DNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNVV
    YTLKDKDKTVASVSLTKTSKGTIDLGNGIKFEVSGNFSGKFTGLENKSYM
    ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKT
    NEQGDRLAGAQFVVKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYN
    KLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT
    SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNI
    DYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKRR
    QSKEA
  • Nucleotide and amino acid sequences of GBS 59 sequenced from serotype V isolated strain CJB111 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
    ATGAAAAAAATCAACAAATGTCTTACAATGTTCTCGACACTGCTATTGAT
    CTTAACGTCACTATTCTCAGTTGCACCAGCGTTTGCGGACGACGCAACAA
    CTGATACTGTGACCTTGCACAAGATTGTCATGCCACAAGCTGCATTTGAT
    AACTTTACTGAAGGTACAAAAGGTAAGAATGATAGCGATTATGTTGGTAA
    ACAAATTAATGACCTTAAATCTTATTTTGGCTCAACCGATGCTAAAGAAA
    TCAAGGGTGCTTTCTTTGTTTTCAAAAATGAAACTGGTACAAAATTCATT
    ACTGAAAATGGTAAGGAAGTCGATACTTTGGAAGCTAAAGATGCTGAAGG
    TGGTGCTGTTCTTTCAGGGTTAACAAAAGACAATGGTTTTGTTTTTAACA
    CTGCTAAGTTAAAAGGAATTTACCAAATCGTTGAATTGAAAGAAAAATCA
    AACTACGATAACAACGGTTCTATCTTGGCTGATTCAAAAGCAGTTCCAGT
    TAAAATCACTCTGCCATTGGTAAACAACCAAGGTGTTGTTAAAGATGCTC
    ACATTTATCCAAAGAATACTGAAACAAAACCACAAGTAGATAAGAACTTT
    GCAGATAAAGATCTTGATTATACTGACAACCGAAAAGACAAAGGTGTTGT
    CTCAGCGACAGTTGGTGACAAAAAAGAATACATAGTTGGAACAAAAATTC
    TTAAAGGCTCAGACTATAAGAAACTGGTTTGGACTGATAGCATGACTAAA
    GGTTTGACGTTCAACAACAACGTTAAAGTAACATTGGATGGTGAAGATTT
    TCCTGTTTTAAACTACAAACTCGTAACAGATGACCAAGGTTTCCGTCTTG
    CCTTGAATGCAACAGGTCTTGCAGCAGTAGCAGCAGCTGCAAAAGACAAA
    GATGTTGAAATCAAGATCACTTACTCAGCTACGGTGAACGGCTCCACTAC
    TGTTGAAATTCCAGAAACCAATGATGTTAAATTGGACTATGGTAATAACC
    CAACGGAAGAAAGTGAACCACAAGAAGGTACTCCAGCTAACCAAGAAATT
    AAAGTCATTAAAGACTGGGCAGTAGATGGTACAATTACTGATGCTAATGT
    TGCAGTTAAAGCTATCTTTACCTTGCAAGAAAAACAAACGGATGGTACAT
    GGGTGAACGTTGCTTCACACGAAGCAACAAAACCATCACGCTTTGAACAT
    ACTTTCACAGGTTTGGATAATGCTAAAACTTACCGCGTTGTCGAACGTGT
    TAGCGGCTACACTCCAGAATACGTATCATTTAAAAATGGTGTTGTGACTA
    TCAAGAACAACAAAAACTCAAATGATCCAACTCCAATCAACCCATCAGAA
    CCAAAAGTGGTGACTTATGGACGTAAATTTGTGAAAACAAATCAAGCTAA
    CACTGAACGCTTGGCAGGAGCTACCTTCCTCGTTAAGAAAGAAGGCAAAT
    ACTTGGCACGTAAAGCAGGTGCAGCAACTGCTGAAGCAAAGGCAGCTGTA
    AAAACTGCTAAACTAGCATTGGATGAAGCTGTTAAAGCTTATAACGACTT
    GACTAAAGAAAAACAAGAAGGCCAAGAAGGTAAAACAGCATTGGCTACTG
    TTGATCAAAAACAAAAAGCTTACAATGACGCTTTTGTTAAAGCTAACTAC
    TCATATGAATGGGTTGCAGATAAAAAGGCTGATAATGTTGTTAAATTGAT
    CTCTAACGCCGGTGGTCAATTTGAAATTACTGGTTTGGATAAAGGCACTT
    ATGGCTTGGAAGAAACTCAAGCACCAGCAGGTTATGCGACATTGTCAGGT
    GATGTAAACTTTGAAGTAACTGCCACATCATATAGCAAAGGGGCTACAAC
    TGACATCGCATATGATAAAGGCTCTGTAAAAAAAGATGCCCAACAAGTTC
    AAAACAAAAAAGTAACCATCCCACAAACAGGTGGTATTGGTACAATTCTT
    TTCACAATTATTGGTTTAAGCATTATGCTTGGAGCAGTAGTTATCATGAA
    AAAACGTCAATCAGAGGAAGCTTAA
    SEQ ID NO: 128
    MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFD
    NFTEGTKGKNDSDYVGKQINDLKSYFGSTDAKEIKGAFFVFKNETGTKFI
    TENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
    NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNF
    ADKDLDYTDNRKDKGVVSATVGDKKEYIVGTKILKGSDYKKLVWTDSMTK
    GLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
    DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEI
    KVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEH
    TFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE
    PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAV
    KTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANY
    SYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
    DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTIL
    FTIIGLSIMLGAVVIMKKRQSEEA


    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
    MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLN
    KSNFPGTTGLNGKDYKGGAISDLAGYFGEGSKEIEGAFFALALKEDKSGK
    VQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
    LKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGVVADAHVY
    P K NTEEKPEID K NFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAEI
    GKVVPYEVKTKIHKGSKYENLVWTDIMSNGLTMGSTVSLKASGTTETFAK
    DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII
    DNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNVV
    YTLKDKDKTVASVSLTKTSKGTIDLGNGIKFEVSGNFSGKFTGLENKSYM
    ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEP K VETHG K KFVKT
    NEQGDRLAGAQFVVKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYN
    KLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT
    SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNI
    DYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKRR
    QSKEA
    SEQ ID NO: 128
    MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFD
    NFTEGTKGKNDSDYVGKQINDLKSYFGSTDAKEIKGAFFVFKNETGTKFI
    TENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
    NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYP K NTETKPQVD K NF
    ADKDLDYTDNRKDKGVVSATVGDKKEYIVGTKILKGSDYKKLVWTDSMTK
    GLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
    DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEI
    KVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEH
    TFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE
    PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAV
    KTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANY
    SYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
    DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTIL
    FTIIGLSIMLGAVVIMKKRQSEEA
  • 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.
  • SEQ ID NO: 126
    MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLN
    KSNFPGTTGLNGKDYKGGAISDLAGYFGEGSKEIEGAFFALALKEDKSGK
    VQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
    LKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGVVADAHVY
    PKNTEEKPEIDKNFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAEI
    GKVVPYEVKTKIHKGSKYENLVWTDIMSNGLTMGSTVSLKASGTTETFAK
    DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII
    DNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNVV
    YTLKDKDKTVASVSLTKTSKGTIDLGNGIKFEVSGNFSGKFTGLENKSYM
    ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKT
    NEQGDRLAGAQFVVKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYN
    KLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT
    SNDQGQFEVTGLADGTYNLE E TLAPAGFAKLAGNIKFVVNQGSYITGGNI
    DYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKRR
    QSKEA
    SEQ ID NO: 128
    MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFD
    NFTEGTKGKNDSDYVGKQINDLKSYFGSTDAKEIKGAFFVFKNETGTKFI
    TENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
    NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNF
    ADKDLDYTDNRKDKGVVSATVGDKKEYIVGTKILKGSDYKKLVWTDSMTK
    GLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
    DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEI
    KVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEH
    TFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE
    PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAV
    KTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANY
    SYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLE E TQAPAGYATLSG
    DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTIL
    FTIIGLSIMLGAVVIMKKRQSEEA
  • Female mice were immunized with either SAG1407 (SEQ ID NO: 126) or BO1575 (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
    Challenge GBS 59 PBS
    GBS strain Survival Survival
    (serotype) Dead/treated (%) Dead/treated (%) FACS
    CJB111 (V)*  7/20 65 41/49 16 493
    18RS21 (II)** 18/30 40 39/40 2.5 380
    *immunized with BO1575
    **immunized with SAG1407
  • Opsonophagocytosis assays also demonstrated that antibodies against BO1575 are opsonic for GBS serotype V, strain CJB111. See FIG. 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
    ATGAAACAAACATTAAAACTTATGTTTTCTTTTCTGTTGATGTTAGGGAC
    TATGTTTGGAATTAGCCAAACTGTTTTAGCGCAAGAAACTCATCAGTTGA
    CGATTGTTCATCTTGAAGCAAGGGATATTGATCGTCCAAATCCACAGTTG
    GAGATTGCCCCTAAAGAAGGGACTCCAATTGAAGGAGTACTCTATCAGTT
    GTACCAATTAAAATCAACTGAAGATGGCGATTTGTTGGCACATTGGAATT
    CCCTAACTATCACAGAATTGAAAAAACAGGCGCAGCAGGTTTTTGAAGCC
    ACTACTAATCAACAAGGAAAGGCTACATTTAACCAACTACCAGATGGAAT
    TTATTATGGTCTGGCGGTTAAAGCCGGTGAAAAAAATCGTAATGTCTCAG
    CTTTCTTGGTTGACTTGTCTGAGGATAAAGTGATTTATCCTAAAATCATC
    TGGTCCACAGGTGAGTTGGACTTGCTTAAAGTTGGTGTGGATGGTGATAC
    CAAAAAACCACTAGCAGGCGTTGTCTTTGAACTTTATGAAAAGAATGGTA
    GGACTCCTATTCGTGTGAAAAATGGGGTGCATTCTCAAGATATTGACGCT
    GCAAAACATTTAGAAACAGATTCATCAGGGCATATCAGAATTTCCGGGCT
    CATCCATGGGGACTATGTCTTAAAAGAAATCGAGACACAGTCAGGATATC
    AGATCGGACAGGCAGAGACTGCTGTGACTATTGAAAAATCAAAAACAGTA
    AGAGTAACGATTCAAAATAAAAAAGTTCCGACACCTAAAGTGCCATCTCG
    AGGAGGTCTTATTCCCAAAACAGGTGAGCAACAGGCAATGGCACTTGTAA
    TTATTGGTGGTATTTTAATTGCTTTAGCCTTACGATTACTATCAAAACAT
    CGGAAACATCAAAATAAGGAT
    SEQ ID NO: 21
    MKQTLKLMESFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQL
    EIAPKEGTPIEGVLYQLYQLKSTEDGDLLAHWNSLTITELKKQAQQVFEA
    TTNQQGKATFNQLPDGIYYGLAVKAGEKNRHVSAFLVDLSEDKVIYPKII
    WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPIRVKNGVHSQDIDA
    AKHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVTIEKSKTV
    TVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH
    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
    MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQL
    EIAPKEGTPIEGVLYQLYQLKSTEDGDLLAHWNSLTITELKKQAQQVFEA
    TTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYP K II
    WSTGELDLL K VGVDGDTKKPLAGVVFELYEKNGRTPIRVKNGVHSQDIDA
    AKHLFTDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVTIEKSKTV
    TVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH
    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
    MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQL
    EIAPKEGTPIEGVLYQLYQLKSTEDGDLLAHWNSLTITELKKQAQQVFEA
    TTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII
    WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPIRVKNGVHSQDIDA
    AKHLETDSSGHIRISGLIHGDYVLK E IETQSGYQIGQAETAVTIEKSKTV