WO2009027768A2 - Immunogenic compositions for gram positive bacteria - Google Patents

Abstract

The invention relates to the identification of a new adhesion islands within the genomes of several Gram positive Streptococcus serotypes and isolates. Adhesion island polypeptides of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against GAS, GBS, and S pneumococcal infections.

Description

IMMUNOGENIC COMPOSITIONS FOR GRAM POSITIVE BACTERIA

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 πsk of transmitting the disease to their babies. Risk of neonatal infection is associated with low serotype specific maternal antibodies and high titers are believed to be protective. In addition, invasive GBS disease is increasingly recognized in elderly adults with underlying disease such as diabetes and cancer. The "B" in "GBS" refers to the Lancefϊeld classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate, designated A to O, that could be serologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains can be divided into at least 9 serotypes (Ia, Ib, Ia/c, II, III, IV, V, VI, VII and VIII) based on the structure of their polysaccharide capsule. In the past, serotypes Ia, Ib, II, and III were equally prevalent in normal vaginal carriage and early onset sepsis in newborns. Type V GBS has emerged as an important cause of GBS infection in the USA, however, and strains of types VI and VIII have become prevalent among Japanese women.

The genome sequence of a serotype V strain 2603 V/R has been published (See Tettehn et al. (2002) Proc. Natl Acad. Sci. USA, 2002 Sep 17;99(19): 12391-6) 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 lmmunogenicity, 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 5 agalactiae (GBS), S. pyogenes (GAS), S. pneumoniae, S" mutans), Enterococcus (such as E. faecalis and E. faecium), Closti idiuni (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-I," "GBS AI-I," or "PI-I" within the genomes of several Group B Streptococcus serotypes and isolates This adhesin island is thought to encode surface proteins which are important in the bacteria's virulence In addition, Applicants have discovered that surface proteins within GBS Adhesin Islands form a previously unseen pilus structure on the surface of GBS bacteria Ammo acid sequences encoded by such GBS Adhesin Islands may be used in immunogenic compositions for the treatment or prevention of GBS infection

A preferred immunogenic composition of the invention comprises an AI-I surface protein, such as GBS 80, which may be formulated or purified in an oligomeπc (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-ohgomeπc structures comprising GBS 80 which appear to cover portions of the surface of the bacteria and stretch far out into the supernatant. These hyper-oligomeπc pilus structures comprising a GBS AI surface protein may be purified or otherwise formulated for use in immunogenic compositions GBS AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("AI-I proteins"). Specifically, AI-I includes polynucleotide sequences encoding for two or more of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. One or more of the AI-I polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the AI-I open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF

AI-I typically resides on an approximately 16 1 kb transposon-hke element frequently inserted into the open reading frame for trmA. One or more of the AI-I surface protein sequences typically include an LPXTG motif (e g , SEQ ID NO 122) or other sortase substrate motif The AI surface proteins of the invention may affect the ability of the GBS bacteria to adhere to and invade epithelial cells. AI surface proteins may also affect the ability of GBS to translocate through an epithelial cell layer Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface. AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen

The sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins AI-I may encode at least one surface protein Alternatively, AI-I may encode at least two surface proteins and at least one sortase Preferably, AI-I encodes for at least three surface proteins and at least two sortases One or more of the surface proteins may include an LPXTG motif or other sortase substrate motif The GBS Al-I protein of the composition may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648 GBS Al-I surface proteins GBS 80 and GBS 104 are preferred for use in the immunogenic compositions of the invention

In addition to the open reading frames encoding the AI-I proteins, AI-I may also include a divergently transcribed transcriptional regulator such as araC (ι 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," "GBS AI-2, " or "PI-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 (or PI-2a). 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 (or PI-2b). 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 {e.g. , SEQ ID NO: 122) or other sortase substrate motif. These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins. AI-2 may encode for at least one surface protein Alternatively, AI-2 may encode for at least two surface proteins and at least one sortase. Preferably, AI-2 encodes for at least three surface proteins and at least two sortases. One or more of the surface proteins may include an LPXTG motif. The AI-2 protein of the composition may be selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405,

SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525. AI-2 surface proteins GBS 67, GBS 59, and 01524 are preferred AI-2 proteins for use in the immunogenic compositions of the invention. GBS 67 or GBS 59 is particularly preferred.

GBS AI-2 may also include a divergently transcribed transcriptional regulator such as a RofA like protein (for example rogB). As in AI-I, rogB is thought to regulate the expression of the AI-2 operon

The GBS AI proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against GBS infection. For example, the invention may include an immunogenic composition comprising one or more GBS AI-I proteins and one or more GBS AI-2 proteins.

The immunogenic compositions may also be selected to provide protection against an increased range of GBS serotypes and strain isolates. For example, the immunogenic composition may comprise a first and second GBS AI protein, wherein a full length polynucleotide sequence encoding for the first GBS AI protein is not present in a genome comprising a full length polynucleotide sequence encoding for the second GBS AI protein. In addition, each antigen selected for use in the immunogenic compositions will preferably be present in the genomes of multiple GBS serotypes and strain isolates. Preferably, each antigen is present in the genomes of at least two (ι e , 3, 4, 5, 6, 7, 8, 9, 10, or more) GBS strain isolates. More preferably, each antigen is present in the genomes of at least two (i.e., at least 3. 4. 5 or more) GBS serotypes.

Within GBS AI-I, Applicants have found that Group B Streptococcus surface exposure of GBS 104 is dependent on the concurrent expression of GBS 80. It is thought that GBS 80 is involved in the transport or localization of GBS 104 to the surface of the bacteria. The two proteins may be oligomerized or otherwise chemically or physically associated. It is possible that this association involves a conformational change in GBS 104 that facilitates its transition to the surface of the GBS bacteria. In addition, one or more AI sortases may also be involved in this surface localization and chemical or physical association. Similar relationships are thought to exist within GBS AI-2. The compositions of the invention may therefore include at least two AI proteins, wherein the two AI proteins are physically or chemically associated. Preferably, the two AI proteins form an oligomer. Preferably, one or more of the AI proteins are in a hyper-oligomeric form. In one embodiment, the associated AI proteins may be purified or isolated from a GBS bacteria or recombinant host cell.

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. pneumoniae, S. mutans), Enterococcus (such as E. faecalis and E. faecium), Clostridium (such as C. difficile), Listeria (such as L monocytogenes) and Corynebacterium (such as C. diphtheria). Preferably, the Gram positive adhesin island surface proteins are in oligomeric or hyperologimeric form.

For example, Applicants have identified adhesin islands within the genomes of several Group A Streptococcus serotypes and isolates. These adhesion islands are thought to encode surface proteins which are important in the bacteria's virulence, and Applicants have obtained the first electron micrographs revealing the presence of these adhesin island proteins in hyperoligomeric pilus structures on the surface of Group A Streptococcus.

Group A Streptococcus is a human specific pathogen which causes a wide variety of diseases ranging from pharyngitis and impetigo through life threatening invasive disease and necrotizing fasciitis. In addition, 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 inappropriate 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 Pathogenesis, 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, trypsm-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-protem 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 (sevapharma cz/en) The gene coding for one form of T-antigen, T-type 6, from an M6 strain of GAS (D741) has been cloned and characterized and maps to an approximately 11 kb highly variable pathogenicity island Schneewind et al , J Bacteriol (1990) 172(6) 3310 - 3317 This island is known as the Fibronectin-binding, Collagen-binding T-antigen (FCT) region because it contains, in addition to the T6 coding gene (teeό), members of a family of genes coding for Extra Cellular Matrix (ECM) binding proteins Bessen et al , Infection & Immunity (2002) 70(3) 1159-1167 Several of the protein products of this gene family have been shown to directly bind either fibronectin and/or collagen See Hanski et al , Infection & Immunity (1992) 60(12) 5119-5125, Talay et al , Infection & Immunity (1992( 60(9) 3837 3844, Jaffe et al (1996) 21(2) 373-384, Rocha et al , Adv Exp Med Biol (1997) 418 737-739, Kreikemeyer et al , J Biol Chem (2004) 279(16) 15850-15859, Podbielski et al , MoI Microbiol (1999) 31(4) 1051- 64, and Kreikemeyer et al , Int J Med Microbiol (2004) 294(2-3) 177-88 In some cases direct evidence for a role of these proteins in adhesion and invasion has been obtained

Applicants raised antiserum against a recombinant product of the teeό gene and used it to explore the expression of T6 in M6 strain 2724 In immunoblot of mutanolysin extracts of this strain, the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used This pattern of high molecular weight products is similar to that observed in immunoblots of the protein components of the pih identified in Streptococcus agalactiae (described above) and previously in Corynebactenum dφhtheriae Electron microscopy of strain M6_2724 with antisera specific for the product of teeό revealed abundant surface staining and long pilus like structures extending up to 700 nanometers from the bacterial surface, revealing that the T6 protein, one of the antigens recognized in the original Lancefield serotyping system, is located within a GAS Adhesin Island (GAS AI-I) and forms long covalently linked pilus structures

Applicants have identified at least six 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 trypsm-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-oligomeπc 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 pill structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysacchaπde 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 ohgomeπc, or hyperoligomeπc (pilus) form The immunogenic compositions of the invention may include one or more GAS AI surface proteins The invention also includes combinations of GAS AJ 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 oligomeπc (pilus) form In a preferred embodiment, the oligomeπc 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 (e g , 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 fimbπal 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 51 A and FIG 162 "GAS Adhesin Island- 1 or "GAS AI-I" comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases (' GAS AI-I proteins") GAS AI-I preferably comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator GAS AI-I surface proteins may include a fibronectin binding protein, a collagen adhesion protein and a fimbria! structural subunit The fimbria! structural subunit (also known as teeό) is thought to form the shaft portion of the pilus like structure, while the collagen adhesion protein (Cpa) is thought to act as dn accessory protein facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule

Specifically, GAS AI-I includes polynucleotide sequences encoding for two or more of M6_SpyO157, M6_Spy0158, M6_SpyO159, M6_Spy0160, M6_SpyO161 The GAS AI-I may also include polynucleotide sequences encoding for any one of CDC SS 410_fimbπal, ISS3650_fimbπal, DSM2071_fimbπal

A preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which may be formulated or purified in an ohgomeπc (pilus) form In a preferred embodiment, the oligomeπc form is a hyperoligomer The immunogenic composition of the invention may alternatively comprise an isolated GAS AI-I surface protein in oligomeπc (pilus) form The oligomer or hyperoligomeπc pilus structures comprising GAS AI-I surface proteins may be purified or otherwise formulated for use in immunogenic compositions

One or more of the GAS AI-I polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the GAS AI-I open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF

One or more of the GAS AI-I surface proteins typically include an LPXTG motif (e g , 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-I 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-I preferably includes a srtB sortase GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO 166), particularly where the motif is followed by a serine

The GAS AI 1 protein of the composition may be selected from the group consisting of M6_SpyO157, M6_Spy0158, M6_SpyO159, M6_Spy0160 M6_SpyO161, CDC SS 410_fimbπal, ISS3650_ftmbπal, and

DSM2071_fimbπal GAS AM surface proteins M6_SpyO157 (a fibronectin binding protein), M6_SpyO159 (a collagen adhesion protein, Cpa), M6_Spy0160 (a fimbπal structural subunit, teeό), CDC SS 410_fimbπal (a fimbπal structural subunit), ISS3650_fimbπal (a fimbπal structural subunit), and DSM2071_fimbπal (a fimbrial structural subunit) are preferred GAS AI-I proteins for use in the immunogenic compositions of the invention The fimbrial structural subunit teeβ and the collagen adhesion protein Cpa are preferred GAS AI -1 surface proteins Preferably, each of these GAS AI-I surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID

NO 122) or LPXSG (SEQ ID NO 134) (conservative replacement of threonine with serine)

In addition to the open reading frames encoding the GAS AI-I proteins, GAS AI-I may also include a divergently transcribed transcriptional regulator such as rofA (i e , the transcriptional regulator is located near or adjacent to the GAS AI protein open reading frames, but it transcribed in the opposite direction)

The GAS AI-I surface proteins may be used alone, in combination with other GAS AI-I surface proteins or in combination with other GAS AI surface proteins Preferably, the immunogenic compositions of the invention include the GAS AI 1 fimbrial structural subunit (teeό) and the GAS AI-I collagen binding protein Still more preferably, the immunogenic compositions of the invention include the GAS AI-I fimbrial structural subunit (teeό) A second GAS adhesion island, "GAS Adhesin Island-2" or "GAS AI-2," has also been identified in GAS serotypes Amino acid sequences encoded by the open reading frames of GAS AI-2 may also be used in immunogenic compositions for the treatment or prevention of GAS infection A preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which may be formulated or purified in an oligomeπc (pilus) form In a preferred embodiment, the oligomeπc form is a hyperoligomer A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-

2 surface protein in oligomeπc (pilus) form The oligomer or hyperoligomeπc 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 srtCl sortase and a rofA divergently transcribed transcriptional regulator Specifically, GAS AI-2 includes polynucleotide sequences encoding for two or more of GAS15, SpyO127,

GAS16, GAS17, GAS18, SpyO131, SpyO133, and GAS20

One or more of the GAS AI-2 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the GAS AI-2 open reading frames may be replaced by a sequence having sequence homology (sequence identity) to the replaced ORF One or more of the GAS AI-2 surface proteins typically include an LPXTG motif (e g , SEQ ID NO 122) or other sortase substrate motif These sortase proteins are thought to be involved in the secretion and anchoring of the LPXTG containing surface proteins GAS AI-2 may encode for at least one surface protein Alternatively, GAS AI- 2 may encode for at least two surface proteins and at least one sortase Preferably, GAS AI-2 encodes for at least three surface proteins and at least two sortases One or more of the surface proteins may include an LPXTG motif GAS AI-2 preferably includes a srtB sortase and a srtCl sortase As discussed above, GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO 166), particularly where the motif is followed by a serine GAS srtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO 167) motif GAS srtCl may be differentially regulated by rofA

The GAS AI-2 protein of the composition may be selected from the group consisting of GAS15, SpyO127, GAS16, GAS17, GAS18, SpyOBl, SpyO133, and GAS20 GAS AI-2 surface proteins GAS15 (Cpa), GAS16 (thought to be a fimbπal protein, Ml_128), GAS18 (M l_SpyO130), and GAS20 are preferred for use in the immunogenic compositions of the invention GAS 16 is thought to form the shaft portion of the pilus like structure, while GAS 15 (the collagen adhesion protein Cpa) and GAS 18 are thought to act as accessory proteins facilitating the formation of the pilus structure, exposed on the surface of the bacterial capsule Preferably, each of these GAS AI-2 surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO 122), VVXTG (SEQ ID NO 135), or EVXTG (SEQ ID NO 136)

In addition to the open reading frames encoding the GAS AI-2 proteins, GAS AI-2 may also include a divergently transcribed transcriptional regulator such as rofA (ι 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 fimbπal protein (GAS 16), the GAS AI-2 collagen binding protein (GAS 15) and GAS 18 (Ml_SpyO13O) More preferably, the immunogenic compositions of the invention include the GAS AI-2 fimbrial protein (GAS 16)

A third GAS adhesion island, "GAS Adhesin Island-3" or "GAS AI-3," has also been identified in numerous GAS serotypes Amino acid sequences encoded by the open reading frames of GAS AI-3 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-3 surface protein which may be formulated or purified in an oligomeπc (pilus) form In J preferred embodiment, the ohgomeπc form is a hyperoligomer A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI- 3 surface protein in oligomeπc (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 fimbπal protein, and a F2 like fibronectin-binding protein GAS AI-3 surface proteins may also include a hypothetical surface protein The fimbria! 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 fimbπal 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 SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SpsOlOO, SpsOlOl, Sps0102, Sps0103, SpsO104 Sps0105, SpsOlOβ, orf78, orf79, orf80, orf81, orf82, orf83, orf84, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_O13O, spyM18_0131, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, SpyoM01000149, ISS3040_fimbπal, ISS3776_fimbπal, and lSS4959_fimbπal In one embodiment, GAS AI-3 may include open reading frames encoding for two or more of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104 Alternatively, GAS AI-3 may include open reading frames encoding for two or more of SpsOlOO, SpsOlOl, Sps0102, Sps0103 Sps0104, Sps0105, and SpsOlOβ Alternatively, GAS AI-3 may include open reading frames encoding for two or more of orf78, orf79, orf80, orfδl, orf82, orf83, and orf84 Alternatively, GAS AI-3 may include open reading frames encoding for two or more of spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, and spyM18_0132 Alternatively, GAS AI-3 may include open reading frames encoding for two or more of SpyoM01000156, SpyoMO 1000155, SpyoM01000154, SpyoMO 1000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and SpyoM01000149 Alternatively, GAS AI-I may also include polynucleotide sequences encoding for any one of ISS3040_fimbπal, ISS3776_fimbπal, and ISS4959_fimbπal

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 (e g , 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 Al-3 preferably includes a srtC2 type sortase GAS srtC2 type sortases may preferably anchor surface proteins with a QVPTG (SEQ ID NO 140) motif, particularly when the motif is followed by a hydrophobic region and a charged C terminus tail GAS SrtC2 may be differentially regulated by Nra

The GAS AI-3 protein of the composition may be selected from the group consisting of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SpsOlOO, SpsOlOl,

Sps0102, Sps0103, Sps0104, SpsOlOS, SpsOlOό, orf78, orf79, orf80, orfδl, orf82, orf83, orf84, spyMJ 8_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_0131, spyM18_O132, SpyoMO 1000156,

SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoMO 1000151, SpyoM01000150,

SpyoMO 1000149, ISS3040_fimbπal, ISS3776_fimbπal, and ISS4959_fimbπal GAS AI-3 surface proteins SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPsOlOo, orf78, orf80, orf82, orf84, spyM18_0126, spyM18_0128, spyM18_0130, spyM18_0132, SpyoM01000155, SpyoM01000153,

SpyoM01000151, SpyoM01000149, ISS3040_fimbπal, ISS3776_fimbπal, and ISS4959_fimbπal 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 fimbπal protein, the GAS Al-3 collagen binding protein, the GAS AI-3 surface protein (such as SpyM3_0102, M3_Sps0104, M5_orf82, or spyM18_0130), and fibronectm binding protein PrtF2 More preferably, the immunogenic compositions of the invention include the GAS AI-3 Fimbπal 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 fimbπal protein

Representative examples of the GAS AI-3 fimbπal protein include SpyM3_0100, M3_Sps0102, M5_orf80, spyM18_128, SpyoM01000153, ISS3040_fimbπal, ISS3776_fimbrial, ISS4959_fimbπal

Representative examples of the GAS AI-3 collagen binding protein include SpyM3_0098, M3_Sps0100, M5_orf 78, spyM18_0126, and SpyoM01000155

Representative examples of the GAS AI-3 fibronectm binding protein PrtF2 include SpyM3_0104, M3_Sps0106, M5_orf84 and spyM18_0132, 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 oligomeπc (pilus) form In a preferred embodiment, the ohgomeπc form is a hyperoligomer A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI- 4 surface protein in oligomeπc (pilus) form The oligomer or hyperoligomeπc pilus structures comprising GAS AI- 3 surface proteins may be purified or otherwise formulated for use in immunogenic compositions The oligomeπc or hyperoligomeπc 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 fimbπal protein, Fl and F2 like fibronectin-binding proteins, and a capsular polysaccharide adhesion protein (cpa) GAS AI-4 surface proteins may also include a hypothetical surface protein in an open reading frame (orf)

The fimbrial 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 ot 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_fimbπal, 20020069_fimbπal, CDC SS 635_fimbπal, ISS4883_fimbπal, lSS4538_fimbπal 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 (e g , 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_iϊmbπal, CDC SS 635_fimbπal, ISS4883_fϊmbπal, and ISS4538_fimbπal GAS AI-4 surface proteins 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fϊmbπal, 20020069_fimbrιal, CDC SS 635_fimbπal, ISS4883_fimbπal, ISS4538_fimbπal 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 (ι 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_fimbπal, 20020069_fimbπal, CDC SS 635_fimbπal, ISS4883_fimbπal, or ISS4538_fϊmbπal), the GAS AI-4 collagen binding protein, the GAS AI-4 surface protein (such as M12 isolate A735 orf 2), and fibronectin binding protein PrtFl and PrtF2 More preferably, the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein, the GAS AI-4 collagen binding protein, and the GAS AI-4 surface protein Still more preferably, the immunogenic compositions of the invention include the GAS AI-4 fimbrial protein A fifth GAS adhesion island, "GAS Adhesin Island-5 or ' GAS AI-5, ' has also been identified in GAS serotypes Ammo acid sequences encoded by the open reading frames of GAS AI-5 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-5 surface protein which may be formulated or purified in an oligomeric (pilus) form In a preferred embodiment, the oligomeric form is a hyperohgomer A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI 5 surface protein in oligomeric (pilus) form The oligomer or hyperoligomeπc pilus structures comprising GAS AI- 5 surface proteins may be purified or otherwise formulated for use in immunogenic compositions

GAS AI-5 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-5 proteins") GAS AI-5 preferably comprises surface proteins, a srtB sortase, a srtCl sortase and a rofA divergently transcribed transcriptional regulator

Specifically, GAS AI-5 includes polynucleotide sequences encoding for two or more of MGAS10270_Spy0108, MGAS10270_Spy0109, MGAS10270_Spy0110, MGAS10270_Spy0111, MGAS10270_Spy0112, MGAS10270_Spy0113, MGAS10270_Spy0114, MGAS10270_Spy0115,

MGAS10270_Spy0116, and MGAS10270_Spy0117 One or more of the GAS AI-5 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-5 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-5 surface proteins typically include an LPXTG motif (e g , 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-5 may encode for at least one surface protein Alternatively, GAS AI- 5 may encode for at least two surface proteins and at least one sortase Preferably, GAS AI-5 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-5 preferably includes a srtB sortase and a srtCl sortase As discussed above, GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO 166), particularly where the motif is followed by a serine GAS srtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO 167) motif GAS srtCl may be differentially regulated by rofA

The GAS AI-5 protein of the composition may be selected from the group consisting of MGAS10270_Spy0108, MGAS 10270_Spy0109, MGAS 10270_Spy0110, MGAS10270_Spy0111,

MGAS10270_Spy0112, MGAS10270_Spy0113, MGAS10270_Spy0114, MGAS10270_Spy0115,

MGAS 10270_Spy0116, and MGAS10270_Spy0117 GAS AI 5 surface proteins are preferred for use in the immunogenic compositions of the invention Preferably, each of these GAS AI-5 surface proteins includes a sortase substrate motif In addition to the open reading frames encoding the GAS AI 5 proteins, GAS AI 5 may also include a divergently transcribed transcriptional regulator such as rofA (ι 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-5 surface proteins may be used alone, in combination with other GAS AI-5 surface proteins or in combination with other GAS AI surface proteins A sixth GAS adhesion island, "GAS Adhesin Island-6" or "GAS AI-6," has also been identified in GAS serotypes Amino acid sequences encoded by the open reading frames of GAS AI-6 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 Al-6 surface protein which may be formulated or purified in an oligomeric (pilus) form In <i preferred embodiment, the oligomeπc form is a hyperoligomer A preferred immunogenic composition of the invention alternatively comprises an isolated GAS AI-

6 surface protein in oligomeric (pilus) form The oligomer or hyperoligomeπc pilus structures comprising GAS AI- 6 surface proteins may be purified or otherwise formulated for use in immunogenic compositions

GAS AI-6 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-6 proteins") GAS AI-6 preferably comprises surface proteins, a srtB sortase, a srtCl sortase and a rofA divergently transcribed transcriptional regulator Specifically, GAS AI-6 includes polynucleotide sequences encoding for two or more of

MGAS10750_Spy0113, MGAS10750_Spy0114, MGAS10750_Spy0115, MGAS10750_Spy0116,

MGAS10750_Spy0117, MGAS10750_Spy0118, MGAS10750_Spy0119, and MGAS 10750_Spy0120

One or more of the GAS AI-6 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-6 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-6 surface proteins typically include an LPXTG motif (e g , 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-6 may encode for at least one surface protein Alternatively, GAS AI-

6 may encode for at least two surface proteins and at least one sortase Preferably, GAS AI-6 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 6 preferably includes a srtB sortase and a srtCl sortase As discussed above, GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO 166), particularly where the motif is followed by a serine GAS srtCl sortase may preferentially anchor surface proteins with a V(PAV)PTG (SEQ ID NO 167) motif GAS srtCl may be differentially regulated by rofA The GAS AI-6 protein of the composition may be selected from the group consisting of Specifically, GAS

Al-6 includes polynucleotide sequences encoding for two or more of MGAS10750_Spy0113, MGAS10750_Spy0114, MGAS10750_Spy0115, MGAS10750_Spy0116, MGAS10750_Spy0117,

MGAS10750_Spy0118, MGAS10750_Spy0119, and MGAS10750_Spy0120 GAS AI-6 surface proteins are preferred for use in the immunogenic compositions of the invention Preferably, each of these GAS AI-6 surface proteins includes a sortase substrate motif

In addition to the open reading frames encoding the GAS AI-6 proteins, GAS AI-6 may also include a divergently transcribed transcriptional regulator such as rofA (ι 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-6 surface proteins may be used alone, in combination with other GAS AI-6 surface proteins or in combination with other GAS AI surface proteins

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-I 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-I and GAS AI-2, GAS AI-I and GAS AI-3, GAS Al-I and GAS AI-4, GAS AI-2 and GAS AI-3, GAS Al-2 and GAS AI-4, and GAS AI 3 and GAS AI-4 Preferably the combination includes fimbπal 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 (ι 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 (ι 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 ohgomeπc (pilus) form In a preferred embodiment, the oligomeπc form is a hyperohgomer A preferred immunogenic composition of the invention alternatively comprises an isolated S pneumoniae surface protein in oligomeπc (pilus) form The oligomer or hyperohgomeπc 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 (e g , 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 5 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 An example of a transcriptional regulator found in S pneumoniae AI sequences is rlrA

A schematic of the organization of a 5 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)

5 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, 19 A Hungary 6, 23 F Poland 16, 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

5 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 ("5 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 5 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 5 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 ("5 pneumoniae AI proteins") Specifically, S pneumoniae strain 670 AI includes polynucleotide sequences encoding for two or more of orfl_670, orf3_670, orf4_670, orf5_670, orf6_670, orf7_670, and orf8_670

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

5 pneumoniae AI from 14 CSRlO comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("5 pneumoniae AI proteins") Specifically, S pneumoniae Al from 14 CSRlO includes polynucleotide sequences encoding for two or more of ORF2_14CSR, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF6_14CSR, ORF7_14CSR, and ORF8.14CSR

One or more of the 5 pneumoniae AI from 14 CSRlO polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the S pneumoniae AI from 14 CSRlO open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

S pneumoniae AI from 19A Hungary 6 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S pneumoniae AI proteins") Specifically, S pneumoniae AI from 19A Hungary 6 includes polynucleotide sequences encoding for two or more of ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF6_19AH, ORF7_19AH, and ORF8_19AH

One or more of the 5 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 5 pneumoniae AI from 19F Taiwan 14 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 19F Taiwan 14 includes polynucleotide sequences encoding for two or more of ORF2_19FTW, ORF3_19FTW, ORF4_19FTW, ORF5_19FTW, ORF6_19FTW, ORF7_19FTW, and ORF8_19FTW One or more of the 5 pneumoniae AI from 19F Taiwan 14 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the S pneumoniae AI from 19F Taiwan 14 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

S pneumoniae AI from 23F Poland 16 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S pneumoniae AI proteins") Specifically, S pneumoniae AI from 23F Poland 16 includes polynucleotide sequences encoding for two or more of ORF2.23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6.23FP, ORF7_23FP, and ORF8_23FP One or more of the S. pneumoniae AI from 23F Poland 16 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 23F Poland 16 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF. S. pneumoniae AI from 23F Taiwan 15 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("5. pneumoniae AI proteins"). Specifically, S. pneumoniae Al from 23F Taiwan 15 includes polynucleotide sequences encoding for two or more of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5J23FTW, ORF6_23FTW, ORF7_23FTW, and

ORF8_23FTW. One or more of the S. pneumoniae AI from 23F Taiwan 15 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the 5. 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 ("5. pneumoniae AI proteins"). Specifically, 5. pneumoniae AI from 6B Finland 12 includes polynucleotide sequences encoding for two or more of ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, and ORF8_6BF.

One or more of the S. pneumoniae AI from 6B Finland 12 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 6B Finland 12 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.

S. pneumoniae AI from 6B Spain 2 comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("S. pneumoniae AJ proteins"). Specifically, S. pneumoniae AI from 6B Spain 2 includes polynucleotide sequences encoding for two or more of ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP, and ORF8J5BSP.

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 5. 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 ("5. pneumoniae AI proteins"). Specifically, 5. pneumoniae AI from 9V Spain 3 includes polynucleotide sequences encoding for two or more of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP, and ORF8_9VSP.

One or more of the S. pneumoniae AI from 9V Spain 3 polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. Alternatively, one or more of the S. pneumoniae AI from 9V Spain 3 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.

One or more of the S. pneumoniae AI surface proteins typically include an LPXTG motif (e.g., 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, 5 pneumoniae AI encodes for at least three surface proteins and at least two sortases One or more of the surface proteins may include an LPXTG motif

The S pneumoniae AI protein of the composition may be selected from the group consisting of SP0462, SP0463 SP0464, SP0465, SP0466 SP0467, SP0468, orfl_670, orO_670 orf4_670, orf5_670, orf6_670, orf7_670, orf8_670, ORF2_14CSR, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF6_14CSR, ORF7_14CSR, ORF8_14CSR, ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF6.19AH, ORF7_19AH, ORF8_19AH, ORF2_19FTW, ORF3_19FTW, ORF4_19FTW ORF5_19FTW, ORF6_19FTW, ORF7_19FTW, ORF8_19FTW, ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, ORF8_23FP, ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, ORF8_23FTW, ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, ORF8_6BF, ORF2_6BSP, ORF3_6BSP, ORF4.6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP, ORF8_6BSP, ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5.9VSP, ORF6_9VSP, ORF7_9VSP and, ORF8_9VSP

S pneumoniae AI surface proteins are preferred proteins for use in the immunogenic compositions of the invention In one embodiment, the compositions of the invention comprise combinations of two or more S pneumoniae AI surface proteins Preferably such combinations are selected from two or more of the group consisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF3_19FTW, ORF4_19FTW, ORF5_19FTW, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF3_9VSP, ORF4_9VSP, and ORF5_9VSP In addition to the open reading frames encoding the S pneumoniae AI proteins, S pneumoniae AI may also include a transcriptional regulator

The S pneumoniae AI proteins of the invention may be used in immunogenic compositions for prophylactic or therapeutic immunization against 5 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 5 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 (ι e , at least 3, 4, 5, or more) 5 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 (ι 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 {ι 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 oligomeπc or hyperoligomeπc 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 oligomeπc or hyperoligomeπc 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 oligomeπc 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 oligomeπc 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

In certain preferred aspects, the invention comprises three antigens wherein each antigen is selected from a different adhesin island AI-I (PI-I), AI2 subgroup 1 (PI-2a), and AI2 subgroup 2 (PI-2b) In preferred embodiments, the antigen from AI-I is the backbone pihn antigen (GBS80 or variants thereof) In preferred embodiments, the antigen from AI 2 subgroup 1 is the ancillary pilin 1 antigen (GBS67 or variants thereof) In preferred embodiments, the antigen from AI-2 subgroup 2 is the backbone pilin antigen In preferred embodiments, the three antigens are in a vaccine composition that may be used to provoke an antibody response in a mammal or for providing broad range protection against GBS infection in a mammal (in each case preferably a human) The antigens may be in any form as disclosed throughout this specification (e g full length, fragments that are antigenic, immunogenic or otherwise can be bound by an antibody that binds the naturally occurring full length antigen from which they are derived) The three antigens may also be used in the preparation of medicaments as disclosed throughout this specification As discussed more fully below, the vaccine and medicaments may further comprise an adjuvant The various compositions including these three antigens may be used in the methods and for the uses as disclosed further below (e g , methods of administration) BRIEF DESCRIPTION OF THE FIGURES FIG 1 presents a schematic depiction of GBS Adhesin Island 1 ("AI-I") comprising open reading frames for GBS 80 GBS 52, SAG0647, SAG0648 and GBS 104 FIG 2 illustrates the identification of Al-I sequences in several GBS serotypes and strain isolates (GBS serotype V, strain isolate 2603, GBS serotype III, strain isolate nem316, GBS serotype II, strain isolate 18RS2 I GBS serotype V, strain isolate CJB l I l , GBS serotype III, strain isolate COHl and GBS serotype Ia, strain isolate A909) (An AI-I was not identified in GBS serotype Ib, strain isolate H36B or GBS serotype Ia, strain isolate 515) FIG 3 presents a schematic depiction of the correlation between AI-I and the Adhesin Island 2 ("AI-2") within the GBS serotype V, strain isolate 2603 genome (This AI-2 comprises open reading frames for GBS 67 GBS 59, SAG1406, SAG1405 and GBS 150)

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 III, strain isolate NEM316, GBS serotype Ib, strain isolate H36B, GBS serotype V, strain isolate CJBl Il, GBS serotype H, strain isolate 18RS21, and GBS serotype Ia, 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 (spbl), 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-I are co-transcπbed as an operon

FIG 7 presents schematic depictions of in-frame deletion mutations within Al-I

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 pih structures in Streptococcus agalactiae containing GBS 80 FIG 12 PHD predicted secondary structure of GBS 067

FIG 13, 14 and 15 Electron micrographs of surface exposed pill structures of strain isolate COHl of Streptococcus agalactiae containing a plasmid insert encoding GBS 80

FIG 16 and 17 Electron micrographs of surface exposed pill structure of wild type strain isolate COHl of Streptococcus agalactiae FIG 18 Alignment of polynucleotide sequences of AI-I from serotype V, strain isolates 2603 and

CJBl I l, serotype II, strain isolate 18RS21, serotype III, strain isolates COHl and NEM316, and serotype Ia, strain isolate A909

FIG 19 Alignment of polynucleotide sequences of AI-2 from serotype V, strain isolates 2603 and CJBl I l, serotype II, strain isolate 18RS21, serotype Ib, strain isolate H36B, and serotype Ia, 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 COHl and serotype Ia, strain isolate A909

FIG 22 Alignment of amino acid sequences of AI-I surface protein GBS 80 from serotype V, strain isolates 2603 and CJB 111 , serotype Ia strain isolate A909, serotype III, strain isolates COHl and NEM316

FIG 23 Alignment of amino acid sequences of AI-I surface protein GBS 104 from serotype V, strain isolates 2603 and CJB 111 , serotype III, strain isolates COHl and NEM316, and serotype II, strain isolate 18RS21 FIG 24 Alignment of amino acid sequences of AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJBl I l, serotype Ia 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 MEl 80 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 MEl 80 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 COHl, reduces the capacity of GBS to adhere to ME 180 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 COHl, containing a plasmid insert to over-express GBS 80

FIG 35 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles)

FIG 36 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 10 nm gold particles)

FIG 37 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 n m gold particles) FIG 38 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 104 antibodies or preimmune sera (visualized with 10 nm gold particles)

FIG 39 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over-express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles) and anti-GBS 104 antibodies (visualized with 10 nm gold particles)

FIG 40 Electron micrographs of surface exposed pili structures on GBS serotype III, strain isolate COHl, containing a plasmid insert to over express GBS 80, stained with anti-GBS 80 antibodies (visualized with 20 nm gold particles) and anti-GBS 104 antibodies (visualized with 10 nm gold particles)

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 Cohl , one of which is the GBS 80 pilin FlG 44 Illustrates pilin assembly

FIG 45 Illustrates that GBS 52 is a minor component of the GBS pilus

FlG 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 Cohl, only the GBS 80 protein and one sortase (sagO647 or sagO648) is required for polymerization

FIG 49 IEM image of GBS 80 staining of a GBS serotype VIII strain JM9030013 that express pill

FIG 50 IEM image of GBS 104 staining of a GBS serotype VIII strain JM9O3OO13 that express pih

FIG 51A Schematic depiction of open reading frames comprising a GAS AI 2 serotype Ml isolate, GAS AI-3 serotype M3, M5, M18, and M49 isolates, a GAS AI-4 serotype M12 isolate, and an GAS AI-I serotype M6 isolate

FIG 51B Amino acid alignment of SrtCl-type sortase of a GAS AI 2 serotype Ml isolate, SrtC2 type sortases of serotype M3, M5, M18, and M49 isolates, and a SrtC2-type sortase of a GAS AI-4 serotype M12 isolate

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-I serotype M3, S pyogenes strain MGAS8232 serotype M3, and GAS AI-2 serotype Ml

FIG 53 Amino acid alignment of F-like fibronectin-binding proteins of GAS AI-4 serotype M12 (A735) and S pyogenes strain MGAS 10394 serotype M6

FIG 54 Amino acid alignment of F2-hke fibronectin-binding proteins of GAS AI-4 serotype M12 (A735), 5 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 fimbπal proteins of GAS AI-4 serotype M12 (A735), GAS AI-3 serotype M5 (Manfredo), 5 pyogenes strain MGAS315 serotype M3, S pyogenes strain SSI serotype M3, S pyogenes strain MGAS8232 serotype M3, and S pyogenes Ml GAS serotype Ml FIG 56 Amino acid alignment of hypothetical proteins of GAS AI-4 serotype M12 (A735), 5 pyogenes strain MGAS315 serotype M3, S pyogenes strain SSI-I serotype M3, GAS AI-3 serotype M5 (Manfredo), and S pyogenes strain MGAS8232 serotype M3

FIG 57 Results of FASTA homology search for amino acid sequences that align with the collagen adhesion protein of GAS AI-I serotype M6 (MGAS 10394) FIG 58 Results of FASTA homology search for amino acid sequences that align with the fimbπal structural subunit of GAS AM serotype M6 (MGAS 10394)

FIG 59 Results of FASTA homology search for amino acid sequences that align with the hypothetical protein of GAS AI-2 serotype Ml (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 pill structures Surface expression of GBS 80 was determined by FACS analysis using an antibody that cross-hybridizes with GBS 80 Formation of pill 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 CJB l 1 1 , 7357B, 515 using GBS 59 antiserum

FIG 67 Illustrates that anti-GBS 59 antibodies are opsonic for CJB 111 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 spyM6_0159 surface expression

FIG 74 FACS analysis of GAS serotype M6 for spyM6_0160 surface expression

FIG 75 FACS analysis of GAS serotype Ml for GAS 15 surface expression

FIG 76 FACS analysis of GAS serotype Ml for GAS 16 surface expression using a first anti-GAS 16 antiserum

FIG 77 FACS analysis of GAS serotype Ml for GAS 18 surface expression using a first anti-GAS 18 antiserum

FIG 78 FACS analysis of GAS serotype Ml for GAS 18 surface expression using a second anti-GAS 18 antiserum FIG 79 FACS analysis of GAS serotype Ml for GAS 16 surface expression using a second anti-GAS 16 antisera

FIG 80 FACS analysis of GAS serotype M3 for spyM3_0098 surface expression

FIG 81 FACS analysis of GAS serotype M3 for spyM3_0100 surface expression

FIG 82 FACS analysis of GAS serotype M3 for spyM3_0102 surface expression FIG 83 FACS analysis of GAS serotype M3 for spyM3_0104 surface expression

FIG 84 FACS analysis of GAS serotype M3 for spyM3_0106 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 Ml 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 Ml 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 Ml 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 5 pneumoniae strain 670 AI FlG 102 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M I strain 2580 FlG 103 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M l strain 2913 FIG 104 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 3280 FlG 105 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS Ml strain 3348

FIG 106 Western blot analysis of GAS 15, GAS 16, and GAS 18 in GAS M l strain 2719 FIG 107 Western blot analysis of GAS 15 , GAS 16, and GAS 18 in GAS M 1 strain SF37O HG 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 SpyM3_0102

FlG 110 Western blot analysis of M6_SpyO159 and M6_SpyO16O in GAS M6 strain 2724 FIG 111 Western blot analysis of M6_Spy0159 and M6_Spy0160 in GAS M6 strain SF370 FIG 112 Western blot analysis of M6_Spyl60 in GAS M6 strain 2724 FIGS 113-115 Electron micrographs of surface exposed GAS 15 on GAS Ml strain SF370 FIGS 116 121 Electron micrographs of surface exposed GAS 16 on GAS Ml strain SF37O

FIGS 122-125 Electron micrographs of surface exposed GAS 18 on GAS Ml strain SF37O detected using anti-GAS 18 antisera

FIG 126 IEM image of a hyperoligomer on GAS Ml strain SF370 detected using anti-GAS 18 antisera FIGS 127-132 IEM images of ohgomeπc and hyperoligomeπc structures containing M6_Spy0160 extending from the surface of GAS serotype M6 3650

FIG 133 A and B Western blot analysis of L lactis transformed to express GBS 80 with anti-GBS 80 antiserum

FIGS 134 Western blot analyses of L lactis transformed to express GBS AI-I 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-I 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 5 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 5 pneumoniae strain TIGR4 AI locus

FIG 139B Base pair lengths of amplicons produced from FIG 139 A primers in S pneumoniae strain

TIGR4 FIG 140 CGH analysis of 5 pneumoniae strains for the AI locus

FIG 141 Amino acid sequence alignment of polypeptides encoded by AI orf 2 in 5 pneumoniae AI- positive strains

FIG 142 Amino acid sequence alignment of polypeptides encoded by AI orf 3 in 5 pneumoniae AI positive strains FIG 143 Amino acid sequence alignment of polypeptides encoded by AI orf 4 in 5 pneumoniae AI- positive strains FlG 144 Amino acid sequence alignment of polypeptides encoded by AI orf 5 in S pneumoniae Al- positive strains

FlG 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 SpO462 ammo acid sequence

FIG 150B Primers used to produce a clone encoding the SpO462 polypeptide

FIG 15 IA Schematic depiction of recombinant SpO462 polypeptide

FIG 151B Schematic depiction of full-length SpO462 polypeptide FIG 152A Western blot probed with serum obtained from 5 pneumoniae-mfecied patients for SpO462

FIG 152B Western blot probed with GBS 80 serum for SpO462

FIG 153A SpO463 amino acid sequence

FIG 153B Primers used to produce a clone encoding the SpO463 polypeptide

FIG 154A Schematic depiction of recombinant SpO463 polypeptide FIG 154B Schematic depiction of full-length SpO463 polypeptide

FIG 155 Western blot detection of recombinant SpO463 polypeptide

FIG 156 Western blot detection of high molecular weight SpO463 polymers

FIG 157A SpO464 amino acid sequence

FIG 157B Primers used to produce a clone encoding the SpO464 polypeptide FIG 158A Schematic depiction of recombinant SpO464 polypeptide

FIG 158B Schematic depiction of full-length SpO464 polypeptide

FIG 159 Western blot detection of recombinant SpO464 polypeptide

FIG 160 Amplification products prepared for production of SpO462, SpO463, and SpO464 clones

FIG 161 Opsonic killing by anti-sera raised against L lactis expressing GBS AI FIG 162 Schematic depicting GAS adhesm islands GAS AI-I₁ 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), Ml_SF370 (B), M5JSS4883 (C) and M12_20010296 (D)

FIGS 163 E-H Immunoblots of cell-wall fractions of deletion mutants Ml_SF370Δ128 (E) M1.SF370Δ130 (F) Ml_SF370ΔSrtCl (G) and the Ml_128 deletion strain complemented with plasmid pAM 128 which contains the Ml_128 gene (H) with antisera specific for the pilin components of M1_SF37O

FIGS 163 I-N Immunogold labelling and transmission electron microscopy of T6 (I) and Cpa (J) in M6JSS3650, Ml_128 in Ml_SF370 (K) and deletion strain Ml_SF370Δ128 (N), M5_orf80 in M5JSS4883 (L), M12_EftLSL A in M12_20010296 (M) The strains used are indicated below the panels Bars=200nm

FlG 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_SF37O stained with anti-Mi (C) or antι-M l_128 (D), M5JSS4883 stained with anti-PrtF (E) or anti-M5_orf80 (F) and M12J20010296 with antι-M 12 (G) or aπti 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

FIG 166 H and I Flow cytometry analysis of strain M1_SF37O (H) and the deletion strain Ml_SF370Δ128 (I) with T-typmg 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-I using anti-GBS 80 antibodies

FIG 168 B and C Immunogold-electronmicroscopy detects GBS 80 in oligomeπc (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-I

FIG 170 Phase contrast microscopy and immuno-electronmicroscopy shows that expression of GBS AI-I in L lactis induces L lactis aggregation

FIG 171 Purification of GBS pih from L lactis transformed to express GBS AI-I

FIG 172 Schematic depiction of GAS M6 (AI-I), Ml (AI-2), and M12 (AI-4) adhesin islands and portions of the adhesin islands inserted in the pAM401 construct for expression in L lactis

FIG 173 A-C Western blot analysis showing assembly of GAS pill in L lactis expressing GAS AI-2 (Ml) (A), GAS AI-4 (M12) (B), and GAS AI-I (M6) (C)

FIG 174 FACS analysis of GAS serotype M6 for M6_SpyO 157 surface expression

FIG 175 FACS analysis of GAS serotype M 12 for 19224139 surface expression FIG 176 A-E Immunogold electron microscopy using antibodies against M6_Spy0160 detects pih on the surface of M6 strain 2724

FIG 176 F Immunogold electron microscopy using antibodies against M6_SpyO159 detects M6_Spy0159 surface expression on M6 strain 2724

FIG 177 A-C Western blot analysis of Ml strain SF370 GAS bacteria individually deleted for Ml_130, SrtCl, or Ml_128 using anti-Ml_130 serum (A), anti Ml_128 serum (B), and anti-Ml_126 serum (C)

FIG 178 A-C Immunogold electron microscopy using antibodies against Ml_128 to detect surface expression on wildtype strain SF370 bacteria (A), Ml_128 deleted SF370 bacteria (B), and SrtCl deleted SF370 bacteria (C)

FIG 179 A-C FACS analysis to detect expression of Ml_126 (A), Ml_128 (B), and Ml_130 (C) on the surface of wildtype SF370 GAS bacteria

FIG 179 D-F FACS analysis to detect expression of Ml_126 (D), Ml_128 (E), and Ml_130 (F) on the surface of Ml_128 deleted SF37O GAS bacteria

FIG 179 G-I FACS analysis to detect expression of Ml_126 (G), M1.128 (H), and Ml_130 (I) on the surface of SrtCl deleted SF370 GAS bacteria FIG 180 A and B FACS analysis of wildtype (A) and LepA deletion mutant (B) strains of SF37O bacteria for Ml surface expression FIG 181 Western blot analysis detects high molecular weight polymers in S pneumoniae TIGR4 using aπti-RrgB aπtisera

FlG 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 5 pneumoniae to adhere to A549 alveolar cells

FIG 185 Negative staining of 5 pneumoniae strain TIGR4 showing abundant pill 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

FIG 187 Negative staining of the TIGR4 mgrA mutant showing abundant pili on the 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 (5nm) and α-RrgC (IOnm) Bar represents 200nm

FIG 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 pi Ii

FIG 194 Purification of 5 pneumoniae TIGR 4 pili by a sucrose gradient centπfugation 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-I 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 Ml (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, M 18, 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-I) adhesin island- relative to other GAS serotypes harbouring an adhesin island FIG 202 GBS 80 recombinant protein does not bind to epithelial cells Epithelial cells were incubated in the presence or absence of GBS80 protein and then a mouse a-GBS80 polyclonal antibody added The cell were then stained with FITC conjugated a-mouse IgG antibody The violet area indicates cells treated with FITC conjugated antibody alone GBS80 binding, expressed as D_cmejn channel values, was measured by FACScan cytometer as difference in fluorescence intensity between cell incubated with or without GBS80 The same protocol was used for GBS104 protein binding to epithelial cells

FIG 203 Deletion of GBS 80 protein does not affect the ability of GBS to adhere and invade ME180 cervical epithelial tells ME180 cervical carcinoma epithelial cells were infected with GBS 2603 wild type or 2603

D80 isogenic mutant After 2h infection, non-adherent bacteria were washed off and infection prolonged for further

2h and 4h In invasion experiments, after each time point followed a 2h antibiotic treatment Cells were then lysed with 1% saponin and lysates plated on TSA plates

FIG 204 GBS 80 binds to extracellular matrix proteins ELISA with purified ECM components and native GBS80 protein

FIG 205 Deletion of GBS 104 protein, but not GBS 80, reduces the capacity of GBS to invade J774 macrophage-like cells J774 cells were infected with GBS COHl wild type or COH1DGBS104/ COH1DGBS80 isogenic mutants After Ih infection, non-adherent bacteria were washed off and intracellular bacteria recovered at

2h, 4h and 6h post-antibiotic treatment At each time point cells were lysed with 025% Triton X-100 and lysates plated on TSA plates

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 Epithelial cells monolayers were inoculated with each bacterium in the apical chamber of a transwell system for 2h and then non-adherent bacteria washed off Infection was prolonged for further 2h and 4h Samples were taken from the media of the basolateral side and the number of colony forming units measured Transepithelial electrical resistance measured prior and after infection gave comparable values, indicating the maintenance of the integrity of the monolayer

FIG 208 GBS adherence to HUVEC endothelial cells HUVEC cells were infected with GBS COHl wild type or COH1DGBS104/ COH1DGBS80 isogenic mutants After Ih infection, non-adherent bacteria were washed off and cells lysed with 1% saponin and lysates plated on TSA plates

FIG 209 Strain growth rate of wildtype, GBS 80-deleted, or GBS 104 deleted COHl GBS

FIG 210 Binding of recombinant GBS 104 protein to epithelial cells by FACS analysis

FIG 21 1 Deletion of GBS 104 protein in the GBS strain COHl reduces the ability of GBS to adhere to ME180 cervical epithelial cells ME180 cervical carcinoma epithelial cells were infected with GBS COHl wild type or COHlDGBS 104/ COH1DGBS80 isogenic mutants After Ih infection, non-adherent bacteria were washed off and cells lysed with 1% saponin and lysates plated on TSA plates

FIG 212 COHl 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 COHl strain

GBS enhances the capacity of the COHl bacteria to form microcolonies on epithelial cells

FIG 214 Confocal imaging shows that overexpression of GBS 80 proteins on COHl strain GBS enhances the capacity of the COHl bacteria to form microcolonies on epithelial cells

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 HG 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

FIG 225 GBS 59 is opsonic only against a strain of GBS expressing a homologous GBS 59

FIG 226 A and B Results of FACS analysis for surface expression of GBS 59 using antibodies specific for different GBS 59isoforms

FIG 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 immunogenic^ 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

FIG 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 pill double labeled with α-RrgB (5nm) and α-RrgC (lOnm) Bar represents lOOnm

FIG 236 Immuno-gold labeling of the S pneumoniae TIGR4 rrgA-srtD deletion mutant with no visible pih on the surface detectable by α-RrgB- and α-RrgC Bar represents 200nm

FIG 237 Variability in GBS 67 amino acid sequences between strains 2603 and H36B

FIG 238 Strain variability in GBS 67 ammo acid sequences of allele I (2603) FIG 239 Strain variability in GBS 67 amino acid sequence of allele II (H36B)

FIG 240 sequence identity dendrogram showing six GBS59 polypeptide allelic families

FIG 241 Immunogenicity of pilus subunits in humans FIG 241A₁ FACS analysis of ability of human sera to recognize whole S pneumoniae TIGR4 cells P = sera from patients with diagnosed pneumococcal diseases, H = serum from a healthy donor FIG 241B, Western blot detection of 5 pneumoniae TIGR4 mutanolysin preparation by human sera Representative results obtained with three sera are shown The typical ladder constituted by polymers of pilus subunits, shown by silver staining (SS), is recognized by the patient's sera (P) but not by the healthy donor control (H) Electrophoretic migrations of relevant molecular mass markers are indicated on the left FIG 241C, ELISA quantification of specific IgG against recombinant RrgA, RrgB or RrgC in human sera from patients (P) with diagnosed pneumococcal diseases (N = 9) or from a healthy donor (H) as indicated Sera were diluted 1 500 For the sera from the patients columns = mean of the 9 sera, bars = standard deviation

FIG 242 Immunogenicity of pilus subunits in mice ELISA quantification of specific IgG titers against recombinant RrgA , RrgB or RrgC in sera of mice immunized as indicated N = 8 for each group with the exception of control group in which N = 16 Specific IgG were undetectable in control group (adjuvant plus saline) Columns represent the mean of the group, with the exception of the S pneumoniae TIGR4 vaccination group, in which sera were pooled A+B+C = combination of RrgA+B+C , bars = standard deviation

FIG 243 Protective efficacy of pilus subunits in mice Protective efficacy against S pneumoniae TIGR4 or 6B challenge of active vaccination with either recombinant pilus antigens or heat-inactivated 5 pneumoniae

TIGR4 with Freund's adjuvant or Al(0H)₃ as indicated, or passive transfer of antisera raised against the same antigens with Freund's adjuvant N = 8 for each group with the exception of control groups in the Freund's adjuvant and in the passive immunization panels, in which N = 16 FIG 243 A, Bacteremia at 24 h (for S pneumoniae TIGR4 challenge) or 5 h (for 6B challenge) post-challenge Circles = values of CFU per ml of blood of single animals, horizontal bars = geometric mean of each group, dashed line = detection limit (i e no CFU were detected in blood samples below dashed line) FIG 243B, Mortality course Diamonds = survival days of single animals, horizontal bars = median of survival days of each group, dashed line = endpoint of observation ( i e animals above the dashed line survived at the endpoint) Ctrl = mice receiving only the corresponding adjuvant plus saline, A+B+C = combination of RrgA+B+C, * = P < 005 and ** = P < 001, in comparison with the corresponding control group FIG 244 Schematic of M2 (Adhesin-Island 5) andM4 (Adhesin-Island 6)

FIG 245 Schematic ofGAS M2 AI-5

FIG 246 Schematic of GAS M4 AI-6

FIG 247 A-D Schematic of sequence identity of AI proteins (see SEQ ID NOS 318-466)

FIG 248 Immunoblots on cell-wall fractions and immuno-electron microscopy images of GAS strain SF370 wild type, ΔspyO128, ΔspyO129, Δspy0128/pAM 128, Δspy0129/pAM 129, and L lactis strains

MG1363/pAM, MG1363/pAM pilMl For immunoblots, sera against Cpa (GAS15), Backbone (GAS16/spyO128) and SpyO13O (GAS18) were used on cell-wall fractions of each strain For immuno-electron microscopy bacteria were labeled with serum against Backbone As a negative control pre-immune sera were used Bars 200 nm

FIG 249 FIGS 249A-E, confocal microscopy images of GAS SF370 wild type, ΔspyO128, ΔspyO129, Δspy0128/pAM 128 and ΔspyO129/pAM 129 grown to late exponential growth phase on polylysine-coated covershps and stained with anti-GAS (blue) and anti-spyO128 (red) sera FIGS 249F-G, light microscopy analysis of L lactis MG1363 strains transformed with pAM vector alone or with pAM pilMl

FIG 250 Confocal microscopy images of bacterial aggregation on human pharynx cell line Detroit-562 Cell monolayers were incubated with bacteria grown to OD 04 at 37°C in a 5%CO2 atmosphere, after 15 minutes of incubation wells were extensively washed 3 times with PBS to remove the unattached bacteria, and infection was let continue to 30, 60 and 120 minutes, then wells were washed again and stained with anti-phalloidin (blue) for eukaryotic cell staining and a polyclonal anti-GAS serum (green) for bacteria staining Panels A-D SF370 wild type, panels E-H ΔspyO128, panels I L ΔpyO129

FIG 251 FIGS 251 A-B, adherence assay with SF370 wild type, Δspyl28 and ΔspyO129 on Detroιt-562 pharynx cell line Confluent cell monolayers were infected with bacteria (MOI 100 1) for 5, 15, 30 and 120 minutes

The percentage of adherent bacteria was calculated as follows (n bacteria recovered after infection/ n inoculated bacteria) xlOO and reported as real percentage (A) or as normalized percentage considering the wild type as 100% (B) FIG 251C Adherence assay with L lactis transformed with pAM401 vector alone and pAM401 containing pilus region of M 1 SF370 on Detroit-562 pharynx cell line A MOl 10 1 was used to infect cells for 15 and 120 mm Each experiment was performed in triplicate and repeated three times Means and standard deviations of three experiments are shown FIG 252 Bio-film assay with SF370 wt, Δspyl28, ΔspyO129, Δspyl28/pAM 128, ΔspyO129/pAM 129 strains Bacteria were incubated in C-medium at room temperature on 24-multi well plates and at the indicated time points supernatant were removed and adherent bacteria were stained with crystal violet 02% Photograph (A) and quantification by measurement of OD at 540 nm (B) of bacterial adhesion after 24 h incubation Bacterial growth in the same conditions for 24 h was also checked as a control Experiments were performed in triplicate and repeated at least three times Means and standard deviations of one representative experiment are shown

FIG 253 X-Z and X-Y panels and three dimensional views of 72h bio-films formed by SF370 wild type (A), ΔspyO128 (B), Δspyl28/pAM 128 (C), ΔspyO129 (D), Δspy0129/pAM 129 (E) Bacteria were grown in C- medium for 72 hours at room temperature on polylysine-coated covershps placed at the bottom of 50 ml tubes Medium was changed every 24 hours Covershps were then recovered, fixed and stained with anti-GAS (blue) and anti-spyl28 (red) sera, and with FITC conjugated-ConA (green) Thickness of bio-films was also measured in different points of each field, and the average of at least 6 measures with standard deviations are reported in the table

FIG 254 Graphs showing that passive transfer of antisera to S pneumoniae TIGR4 native pilus protects against 5 pneumoniae TIGR4 challenge FIG 255 Correlation between Pilus Islands distribution and GBS capsular serotype The different colors represent the pilus islands combination found in the clinical isolates, as shown in the legend The number of isolates containing the different types of pilus islands is indicated inside each column

FIG 256 Schematic representation of sequence variability of pilus-coding genes among GBS clinical isolates (A), (B) and (C) indicate gene conservation in isolates containing PI-I, PI-2a and PI-2b, respectively The total number of strains containing the same PI allele and their serotype distribution are boxed at the right side of each allele Grey arrows represent the sortases genes present in each island Sequences with 100% identity are shown in the same color, while variants showing less than 90% sequence identity are indicated in different colors Single mutations are represented with vertical bars and the number above each bar indicates the position/substitution of the mutated residue (D) Phylogenetic trees inferred from the protein alignments by the neighbour-joining-distance- based method of variants of BP-2a and of APl-2a Numbers at the nodes indicate bootstrap values

FIG 257 Correlation between the presence of pilus islands (PCR positive, red columns) and surface exposure of pill structural components measured by flow cytometry as the difference in fluorescence between cells stained with immune sera versus pre-immune sera Numbers inside each column represent the number of strains that belong to each group, whereas the numbers shown at the top of columns indicate the average values of fold increase in fluorescence and the corresponding standard deviation Blue color columns represent the number of strains showing a greater than 2-fold increase in fluorescence Yellow columns indicate the number of strains showing a greater than 5-fold increase in fluorescence 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 Enzynwlσgv (S Colowick and N Kaplan, eds , Academic Press, Inc ) and Handbook of Experimental Immunology, VoIs 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, NY

All publications, patents and patent applications cited herein, are hereby incorporated by reference in their entireties

As used herein, an "Adhesin Island" or "AI" refers to a series of open reading frames within a bacterial genome, such as the genome for Group A or Group B Streptococcus or other gram positive bacteria, that encodes for a collection of surface proteins and sortases An Adhesin Island may 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 (e g , 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 (ι e , the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction) The transcriptional regulator may regulate the expression of the AI operon

GBS Adhesin Island 1

As discussed above, Applicants have identified a new adhesin island, "Adhesin Island 1, "AI-I or "GBS

AI-I, " within the genomes of several Group B Streptococcus serotypes and isolates Al-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("AI-I proteins") Specifically, AI-I includes open reading frames encoding for two or more

0 e , 2, 3, 4 or 5) of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648 One or more of the AM open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced

ORF Alternatively, one or more of the AI-I open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

A schematic of AI-I is presented in FIG 1 AI-I 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-I surface protein sequences typically include an LPXTG motif (e g , 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 Al-I sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins AI- I may encode for at least one surface protein Alternatively, Al-I 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 Al-I protein preferably includes GBS 80 or a fragment thereof or a sequence having sequence identity thereto

As used herein, an LPXTG motif represents an amino acid sequence comprising at least Five amino acid residues Preferably, the motif includes a leucine (L) in the first amino acid position, a proline (P) in the second amino acid position, a threonine (T) in the fourth amino acid position and a glycine (G) in the fifth amino acid position The third position, represented by X, may be occupied by any amino acid residue Preferably, the X is occupied by lysine (K), Glutamate (E), Asparagine (N), Glutamine (Q) or Alanine (A) Preferably, the X position is occupied by lysine (K) In some embodiments, one of the assigned LPXTG amino acid positions is replaced with another amino acid Preferably, such replacements comprise conservative amino acid replacements, meaning that the replaced amino acid residue has similar physiological properties to the removed amino acid residue Genetically encoded amino acids may be divided into four families based on physiological properties (1) acidic (aspartate and glutamate), (2) basic (lysine, argimne, histidine), (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) 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 ammo acid residue (leucine) is replaced with an alanine (A), valine (V), isoleucine (I), proline (P), phenylalanine (F), methionine (M), glutamic acid (E), glutamine (Q), or tryptophan (Y) residue In one preferred embodiment, the first amino acid residue is replaced with an isoleucine (I) The second amino acid residue of the LPXTG motif may be replaced with another amino acid residue

Preferably, the second amino acid residue praline (P) is replaced with a valine (V) residue

The fourth amino acid residue of the LPXTG motif may be replaced with another amino acid residue Preferably, the fourth amino acid residue (threonine) is replaced with a serine (S) or an alanine (A)

In general, an LPXTG motif may be represented by the amino acid sequence XXXXG, in which X at amino acid position 1 is an L, a V, an E, an I, an F, or a Q, X at amino acid position 2 is a P if X at amino acid position 1 is an L, an I, or an F, X at amino acid position 2 is a V if X at amino acid position 1 is a E or a Q, X at amino acid position 2 is a V or a P if X at amino acid position 1 is a V, X at amino acid position 3 is any amino acid residue, X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, I, F, or Q, and X at amino acid position 4 is a T, S, or A if X at amino acid position 1 is an L Generally, the LPXTG motif of a GBS AI protein may be represented by the amino acid sequence XPXTG, in which X at amino acid position 1 is L, I, or F, and X at amino acid position 3 is any amino acid residue Specific examples of LPXTG motifs in GBS AI proteins may include LPXTG (SEQ ID NO 122) or IPXTG (SEQ ID NO 133)

As discussed further below, the threonine in the fourth amino acid position of the LPXTG motif may be involved in the formation of a bond between the LPXTG containing protein and a cell wall precursor Accordingly, in preferred LPXTG motifs, the threonine in the fourth amino acid position is not replaced with another amino acid or if the threonine is replaced the replacement ammo 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-hnked acyl intermediate of sortase Many AI surface proteins include a pihn motif amino acid sequence which interacts with the sortase and LPXTG ammo 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 pill For example, the pilin motif can make a nucleophilic attack on the acyl enzyme providing a covalent linkage between AI subunits to form pill and regenerate the sortase enzyme Examples of pilin motifs may include ((YPKN(Xi₀)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 (ME 180) 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-I surface protein GBS 104 can bind epithelial cells such as ME180 human cervical cells, A549 human lung cells and Caco2 human intestinal cells (See FIGS 29 and 210) Further, deletion of the GBS 104 sequence in a GBS strain reduces the capacity 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-hke 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 COHl cells diminishes adherence to HUVEC endothelial cells See FIG 208 Deletion of GBS 80 or GBS 104 in COHl does not however, affect growth of COHl either with ME180 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-I surface proteins, can bind to the extracellular matrix binding proteins fibronectin and fibrinogen While GBS 80 protein apparently does not bind to certain epithelial cells or affect the capacity of a GBS bacteria to adhere to or invade cervical epithelial cells (See 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 COHl bacteria overexpressing GBS 80 protein have an impaired ability to translocate through an epithelial monolayer See FIG 212 These COHl bacteria overexpressing GBS 80 form microcolomes 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-I, exhibits some functional homology to FimA, a major fimbπal 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 Bacterid 66 1482-1491, Yeung et al (1988) J Bacteπol 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 pih formation in the Gram positive bacteria Corynebactenum diphthenae (SpaA, SpaD, and SpaH) See, Ton-That et al (2003) Molecular Microbiology 50(4) 1429-1438 and Ton-That et al (2004) Molecular Microbiology 53(1) 251- 261 The C diphthenae proteins all included a pilin motif of WxxxVx VYPK (SEQ ID NO 151, where x indicates a varying amino acid residue) The lysine (K) residue is particularly conserved in the C diphthenae pilus proteins and is thought to be involved in sortase catalyzed oligomeπzation of the subunits involved in the C diphthenae pilus structure (The C diphthenae pilin subunit SpaA is thought to occur by sortase-catalyzed amide bond cross-linking of adjacent pilin subunits As the thioester-hnked acyl intermediate of sortase requires nucleophilic attack for release, the conserved lysine within the SpaA pilin motif might function as an ammo 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-I polypeptides may include the amino acid sequences YxLxExxxxxGY (SEQ ID NO 153), YxLxExxxPxGY (SEQ ID NO 154), or YxLxETxAPxGY (SEQ ID NO 152) Specifically, the E box motif of the polypeptides may comprise the amino acid sequencer YKLKETKAPEGY (SEQ ID NO 155), YVLKEIETQSGY (SEQ ID NO 156), or YKL YEISSPDGY (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-hke 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 COHl with a plasmid insert to facilitate the overexpression of GBS 80. This EM photo was produced with a standard negative stain - no pilus structures are distinguishable In addition, the use of such AI surface proteins in immunogenic compositions for the treatment or prevention of infection against a Gram positive bacteria has not been previously described.

Surprisingly, Applicants have now identified the presence of GBS 80 in surface exposed pilus formations visible in electron micrographs These structures are only visible when the electron micrographs are specifically stained against an AI surface protein such as GBS 80. Examples of these electron micrographs are shown in FIGS. 11, 16 and 17, which reveal the presence of pilus structures in wild type COHl Streptococcus agalactiae. Other examples of these electron micrographs are shown in FIG 49, which reveals that GBS 80 is associated with pili in a wild type clinical isolate of S. agalactiae, JM9O3OO13. (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, oligomeπc 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 COHl and JM9130013 using an anti-GBS 80 antisera. Immunogold electron microscopy of the COHl and JM9130013 bacteria using anti-GBS 80 antisera demonstrates that JM913OO13 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 Cohl bacterial clones that overexpress GBS 80, but lack GBS 104, or one of the AI-I sortases sagO647 or sagO648. However, pili are not formed in Cohl bacterial clones that overexpress GBS 80 and lack both sagO647 and sagO648. Thus, for example, it appears that at least GBS 80 and a sortase, sagO647 or sagO648, may be necessary for pili formation. (See FIG 48.) Overexpression of GBS 80 in GBS strain 515, which lacks an AI-I, also assembles GBS 80 into pili. GBS strain 515 contains an AI-2, and thus AI-2 sortases. The AI-2 sortases in GBS strain 515 apparently polymerize GBS 80 into pili (See 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-I pill formation, GBS 104 and sortase SAG0648 appears to be important for efficient AI-I pili assembly. For example, high-molecular structures are not assembled in isogenic COHl strains which lack expression of GBS 80 due to gene disruption and are less efficiently assembled in isogenic COHl strains which lack the expression of GBS 104 (see FIG 41) This GBS strain comprises high molecular weight pih structures composed of covalently linked GBS 80 and GBS 104 subunits In addition, deleting SAG0648 in COHl bacteria interferes with assembly of some of the high molecular weight pill 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 hyperoligomeπc 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 )

GBS 52 also appears to be a component of the GBS pill Immunoblots using an anti-GBS 80 antisera on total cell extracts of Cohl and a GBS 52 null mutant Cohl reveal a shift in detected proteins in the Cohl wild type strain relative to the GBS 52 null mutant Cohl strain The shifted proteins were also detected in the wild type Cohl bacteria with an anti-GBS 52 antisera, indicating that the GBS 52 may be present in the pilus (See FIG 45 )

In one embodiment, the invention includes a composition comprising oligomeπc, pilus-hke structures comprising an AI surface protein such as GBS 80 The oligomeπc, pilus-hke structure may comprise numerous units of AI surface protein Preferably, the ohgomeπc, pilus-hke structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus like structure comprises a hyper-oligomeπc pilus-hke 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) oligomeπc subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc subunits may be covalently associated via a conserved lysine within a pilin motif

The oligomeπc 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 oligomeπc, pilus-hke 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 oligomeπc, pilus-like structures of the invention For example, GBS 80 and GBS 104 may be incorporated into an oligomeπc structure Alternatively, GBS 80 and GBS 52 may be incorporated into an oligomeric structure, or GBS 80, GBS 104 and GBS 52 may be incorporated into an oligomeric structure

In another embodiment, the invention includes compositions comprising two or more AI surface proteins The composition may include surface proteins from the same adhesin island For example, the composition may include two or more GBS AI-I surface proteins, such as GBS 80, GBS 104 and GBS 52 The surface proteins may be isolated from Gram positive bacteria or they may be produced recombinantly

The oligomeπc, 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 m oligomeric form, preferably in a hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more GBS Adhesin Island 1 ("AI-I") proteins and one or more GBS Adhesin Island 2 ("AI-2") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form

The oligomeric, pilus-hke 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 forth in WO 02/35771 as SEQ ID 8539 and SEQ ID 8540 and in the present specification as SEQ ID NOS 38 and 39 A particularly preferred GBS 322 polypeptide lacks the N- terminal signal peptide, amino acid residues 1-24 An example of a preferred GBS 322 polypeptide is a 407 ammo acid fragment and is shown in SEQ ID NO 40 Examples of preferred GBS 322 polypeptides are further described in WO 2005/028618

Additional GBS proteins which may be combined with the GBS AI surface proteins of the invention are also described in WO 2005/028618 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 m WO 02/34771 These GBS proteins include but are not limited to GBS293, GBS65, GBS97, GBS84, GBS147, and GBS325 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 ohgomeπc, 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 oligomeπc AI surface antigen comprising culturing a GBS bacterium that expresses the oligomeπc AI protein and isolating the expressed oligomeπc 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 hyperoligomeπc form Macromolecular structures associated with oligomeπc pill are observed in the supernatant of cultured GBS strain Cohl (See FIG 46 ) These pill are found in the supernatant at all growth phases of the cultured Cohl bacteria (See FIG 47 )

The ohgomeπc, 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 oligomeπc Adhesin Island surface antigen comprising culturing a GBS bacterium adapted for increased AI protein expression and isolation of the expressed ohgomeπc 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 AI protein Optionally, the sequence encoding the AI protein within the GBS bacterial genome may be deleted Alternatively, or in addition, the promoter regulating the GBS Adhesin Island may be modified to increase expression

GBS bacteria harbouring a GBS AI-I may also be adapted to increase AI protein expression by altering the number adenosine nucleotides present at two sites in the intergenic region between AraC and GBS 80 See FIG 197 A, which is a schematic showing the organization of GBS AI-I 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 foi results obtained from the FACS analysis Therefore, manipuldting 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 oligomeπc or hyperoligomeπc 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 pill on their surface The GBS bacteria may be adapted to have increased exposure of oligomeπc 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 bacterid (such us LepA in GAS) are expected to result in increased exposure of pih 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 pih protein Alternatively, the AI proteins of the invention may be expressed on the surface of a non-pathogenic Gram positive bacteria, such as Streptococcus gordonu (See, e g , Byrd et al , "Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonu 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 Pharyngeal 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 nonpathogenic bacteria are modified to express the AI surface protein in oligomeπc, or hyper-oligomeπc form Sequences encoding for an AI surface protein and, optionally, an AI sortase, may be integrated into the non pathogenic Gram positive bacterial genome or inserted into a plasmid The non-pathogenic Gram positive bacteria may be inactivated or attenuated to facilitate in vivo delivery of the whole bacteria, with the AI surface protein exposed on its surface Alternatively, the AI surface protein may be isolated or purified from a bacterial culture of the non pathogenic Gram positive bacteria For example, the AI surface protein may be isolated from cell extracts or culture supernatants Alternatively, the AI surface protein may be isolated or purified from the surface of the nonpathogenic Gram positive bacteria The non-pathogenic Gram positive bacteria may be used to express any of the Gram positive bacterial

Adhesin Island proteins described herein, including proteins from a GBS Adhesin Island, a GAS Adhesin Island, or a S pneumo Adhesin Island The non-pathogenic Gram positive bacteria are transformed to express an Adhesin Island surface protein Preferably, the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase The Al 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-I polypeptides GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences These L lactis expressed high molecular weight structures that were immunoreactive with anti-GBS 80 in immunoblots See 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 FIG 136A and B See also FIG 171, which shows purification of GBS pih from whole extracts of L lactis expressing the GBS AI-I following sonication and gel filtration on a Sephacryl HR 400 column

Furthermore, the L. lactis transformed with the construct encoding GBS AI-I polypeptides GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences expressed the GBS AI-I polypeptides on its surface FACS analysis of these transformed L lactis detected cell surface expression of both GBS 80 and GBS 104 The surface expression levels of GBS 80 and GBS 104 on the transformed L lactis were similar to the surface expression levels of GBS 80 and GBS 104 on GBS strains COHl and JM9130013, which naturally express GBS AI-I See FIG 169 for FACS analysis data for L lactis transformed with GBS AI-I and wildtype JM913OO13 bacteria using anti-GBS 80 and GBS 104 antisera Table 40 provides the results of FACS analysis of transformed L lactis, COHl, and JM9130013 bacteria using anti-GBS 80 and anti-GBS 104 antisera The numbers provided represent the mean fluorescence value difference calculated for immune versus pre-immune sera obtained for each bacterial strain Table 40: FACS analysis of L lactis and GBS bacteria strains expressing GBS AM

Immunogold electronmicroscopy performed with anti GBS 80 primary antibodies detected the presence of pilus structures on the surface of the L lactis bacteria expressing GBS AI-I, confirming the results of the FACS analysis See FIG 168 B and C Interestingly, this expression of GBS pih 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-I was protective in a subsequent challenge with GBS Female mice were immunized with L lactis transformed with GBS AI-I The immunized female mice were bred and their pups were challenged with a dose of GBS sufficient to kill 90% of non-immunized pups Detailed protocols for intranasal and subcutaneous immunization of mice with transformed L lactis can be found in Examples 18 and 19, respectively Table 43 provides data showing that immunization of the female mice with L locus expressing GBS AI-I (LL-AI 1) greatly increased survival rate of challenged pups relative to both a negative PBS control (PBS) and a negative L lactis control (LL 10 E9, which is wild type L lactis not transformed to express GBS AI-I) Table 43: Protection of Mice Immunized with L. lactis expressing GBS Al-I

Table 51 provides further evidence that immunization of mice with L lactis transformed with GBS AI-I is protective against GBS Table 51: Further Protection of Mice Immunized with L. lactis expressing GBS AI-I

Protection of immunized mice with L lactis expressing the GBS AI-I is at least partly due to a newly raised antibody response Table 46 provides anti-GBS 80 antibody titers detected in serum of the mice immunized with L lactis expressing the GBS AI 1 as described above Mice immunized with L lactis expressing the GBS AI-I have anti-GBS 80 antibody titres, which are not observed in mice immunized with L lactis not transformed to express the GBS AI-I Further, as expected from the survival data, mice subcutaneously immunized with L lactis transformed to express the GBS AI-I have significantly higher serum anti-GBS 80 antibody titers than mice intranasally immunized with L lactis transformed to express the GBS AI-I.

Table 46: Antibody Responses against GBS 80 in Serum of Mice Immunized with L. lactis Expressin GBS AM

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-I Table 47: Anti-CBS 80 IgA Antibodies Detected in Mouse Tissues Following Immunization with L lactis Ex ressin GBS AI-I

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-I and AI-2, i e , a hybrid GBS AI By way of example, a hybrid GBS AI may be a GBS Al 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-I 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-I with a substitution of a GBS 59 polypeptide for the GBS 52 gene and a substitution of the GBS 104 gene for genes encoding GBS 59 and the two GBS AI-2 sortases Another example of a hybrid GBS AI is a GBS AI-I with the substitution of a GBS 59 gene for the GBS 52 gene and a GBS 67 for the GBS 104 gene See the schematic at HG 232 A further example of a hybrid GBS AI is a GBS AI-I having a GBS 59 gene and genes encoding the GBS AI-2 sortases in place of the GBS 52 gene Yet another example of a hybrid GBS AI is a GBS AI-I with a substitution of either GBS 52 or GBS 104 with a fusion protein comprising GBS 322 and one of GBS 59, GBS 67, or GBS 150 Some of these hybrid GBS AIs may be prepared as briefly outlined in FIG 234 A-F

Applicants have prepared a hybrid GBS AI having a GBS AI-I sequence with a substitution of a GBS 67 coding sequence for the GBS 104 gene as depicted in FIG 231 A Transformation of L lactis with the hybrid GBS AI-I resulted in L lactis expression of high molecular weight polymers containing the GBS 80 and GBS 67 proteins See 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 oligomeπc, 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

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

As shown in FIG 1, AI 1 includes the surface exposed proteins of GBS 80, GBS 52 and GBS 104 and the sortases S AG0647 and SAG0648 AI- 1 typically appears as an insertion into the 3' end of the irmA gene In addition to the open reading frames encoding the AI-I proteins, Al-I may also include a divergently transcribed transcriptional regulator such as araC (t e the transcriptional regulator is located near or adjacent to the

Al 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 £ toll)

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-I sequences were identified in GBS serotype V, strain isolate 2603, GBS serotype III, strain isolate NEM316, GBS serotype II, strain isolate 18RS21, GBS serotype V, strain isolate CJBl I l, GBS serotype III, strain isolate COHl and GBS serotype

Ia, strain isolate A909 (Percentages shown are amino acid identity to the 2603 sequence) (An AI-I was not identified in GBS serotype Ib, strain isolate H36B or GBS serotype Ia, strain isolate 515)

An alignment of AI-I polynucleotide sequences from serotype V, strain isolates 2603 and CJBlIl, serotype II, strain isolate 18RS21, serotype III, strain isolates COHl and NEM316, and serotype Ia, strain isolate A909 is presented in FIG 18 An alignment of amino acid sequences of AI-I surface protein GBS 80 from serotype V, strain isolates 2603 and CJBl I l, serotype Ia, strain isolate A909, serotype III, strain isolates COHl and NEM316 is presented in FIG 22 An alignment of amino acid sequences of AI-I surface protein GBS 104 from serotype V, strain isolates 2603 and CJBl I l, serotype III, strain isolates COHl and NEM316, and serotype II, strain isolate 18RS21 is presented in FIG 23 Preferred AI-I polynucleotide and amino acid sequences are conserved among two or more GBS serotypes or strain isolates

The full length of surface protein GBS 80 is particularly conserved among GBS serotypes V (strain isolates 2603 and CJBIII), III (strain isolates NEM316 and COHl), and Ia (strain isolate A909) The GBS 80 surface protein is missing or fragmented in serotypes II (strain isolate 18RS21), Ib (strain isolate H36B) and Ia (strain isolate 515) Polynucleotide and amino acid sequences for AraC are set forth in FIG 30

GBS Adhesin Island 2

A second adhesin island, "Adhesin Island 2" or "AI-2" or "GBS AI-2" has also been identified in numerous GBS serotypes A schematic depicting the correlation between AI-I and AI-2 within the GBS serotype V, strain isolate 2603 is shown in FIG 3 (Homology percentages in FIG 3 represent amino acid identity of the AI-2 proteins to the AI-I proteins) Alignments of AI-2 polynucleotide sequences are presented in FIGS 19, 20, and 21 FIG 19 includes sequences from serotype V, strain isolates 2603 and CJBl I l, serotype II, strain isolate 18RS21, serotype Ib, strain isolate H36B, and serotype Ia, strain isolate 515 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 COHl and serotype Ia, strain isolate A909 An alignment of amino acid sequences of AI-2 surface protein GBS 067 from serotype V, strain isolates 2603 and CJBl I l, serotype Ia, 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 (e g , 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 Al 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 pilm 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-hnked 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 pill 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 m the GBS AI-2 proteins include ((YPKN(X₈)K, SEQ ID NO 158), (PK(X_S)K, SEQ ID NO 159), (YPK(X₉)K₁SEQ 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), YxxxExxAxxGY (SEQ ID NO 164), YxLxExxxPxDY (SEQ ID NO 165), or YxLxETx APxGY (SEQ ID NO 152)

As shown in HG 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-I, rogB is thought to regulate the expression of the AI-2 operon

A schematic depiction of AI-2 within several GBS serotypes is depicted in 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-I 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-I 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 Al-I GBS 80 surface protein appears to be missing in GBS serotype Ib, strain isolate H36B and GBS serotype Ia, strain isolate 515 Within AI-2 for these sequences, however, the GBS 67 surface protein has 97 - 99 % amino acid sequence homology with the corresponding sequence in strain isolate 2603 GBS 67 appears to have two allelic variants, which can be divided according to percent homology with strains 2603 (GBS67²⁶⁰³) and H36B (GBS67^H36B) See HGS 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% ammo acid residue homology with GBS strain 515 and GBS strain CJB l I l, 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 CJBI l 1, NEM316, and 515 (GBS59^CJB1", GBS59^Nhvnl6 and GBS59⁵¹⁵ respectively) The second isoform appears to include the GBS 59 protein of GBS strains 18RS21 2603, and H36B GBS59^18RS21, GBS59²⁶⁰³ and GBS59^H36B respectively See FIGS 63 and 224 All newly sequenced strains were deposited at American Type Culture Collection under the following accession numbers A909, BAA-1 138, CJBl I l, BAA-23, H36b, BAA-1174, 18RS21, BAA- 1 175, COH l BAA-1176, and 515, BAA- 1177 References for the eight strains are as follows NEM316 (Glaser et al , MoI Microbiol 45, 1499-1513, 2002), 2603V/R (Tettehn et al , Proc Natl Acad Sci USA 99, 12391-96, 2002), A909, H36B, and 18RS21 (Lancefield et al , J Exp Med 142, 165-79, 1975), 515 (Wessels et al , Infect Immun 61, 4760-66, 1993), COHl (Wilson & Weaver, J Infect Dis 152, 323-29, 1985), and CJB l I l (Carol Baker Collection, Division of Infectious Diseases, Baylor College of Medicine, Houston) From Tettehn et al , Proc Natl Acad Sci USA 102, 13950-55, 2005

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-cjbl 11) or GBS 59 isoform 2 (α -2603) Only one of the two antibodies detected GBS 59 surface expression on each GBS strain As a negative control, GBS strains in which a GBS 59 gene was not detectable by PCR did not have significant GBS 59 surface expression levels FIG 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 CJBl 11, NEM316, or 515 (i e , GBS59^CJBI", _{GB S59} ^NEM3I6 _and GBS59⁵¹⁵) _{The second ]soform may be the GBS 59 protein} of GBS stra_in 18RS21, 2603, or

H36B (i e , GBS59^I8RS21, GBS59²⁶⁰³ and GBS59^H36B)To further investigate GBS59 distribution, presence of GBS59 gene in 80 different GBS isolates was assessed by PCR and the resulting amplicons were sequenced Table 53 summarizes the sequence analysis results for the 65 positive strains (81%) The various GBS59 sequences thus obtained suggest that GBS59 isoforms can be further grouped in 6 mam allelic families, as schematized in FIG 240 Each sequence member of an allelic family has been compared to the first representative strain in the list of different isolates (i e , GBS59^CJB1", GBS59^DK21, GBS59⁵¹⁵, GBS59^CJB110, GBS59²⁶⁰³ and GBS59^H36B)

Members of the same allelic family will typically have 75% sequence identity or more (e g 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 5%) More preferably members of the same allelic family will have 97% or more sequence identity or more Even more preteiably, members of the same allelic family will exhibit immunological cross reactivity As used herein, the term "cross reactivity" refers to the characteristic of an antigen to elicit an immune response effective against more than one strain of GBS [e g , heterologous GBS strains) According to this classification, two new variants of GBS59 are distinguishable one represented by GBS59 from strain CJBI lO (i e , GBS59^αB"°) and the second represented by GBS59, encoded from strain DK21 (i e , GBS59^Diαι) As shown in FIG 240, GBS59^CJB"° amino acid sequence is 72% identical to that of GBS59²⁶⁰¹ and GBS59^DK21 shares 71% sequence identity with GBS59^CJBm

Accordingly, immunogenic composition of the invention for the treatment or prophylaxis of GBS infections may be further improved by combining GBS59 polypeptides from different allelic families or fragments thereof, in order to increase strain coverage In some embodiments, when no cross reactivity is detected between two or more allelic families, the combination will preferably include representative polypeptides from each allelic family In further embodiments, when GBS59 polypeptides from different allelic families cross-react, the immunogenic composition of the invention may include only one representative polypeptide In other embodiments, when an allelic family contains GBS59 polypeptides from specifically virulent strains, the immunogenic composition of the invention will preferably contain representative antigens from that allelic family

Immunizations with GBS59 polypeptides of the invention are discussed further in the Examples

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 CJB l I l, 7357B, COH31, D1363C, 5408, 1999, 5364, 5518, and 515 using antiserum raised against GBS 59 of GBS strain CJB 111 FIG 65 also shows detection of these high molecular weight GBS 59 polymers in whole extracts of GBS strains D136C, 515, and CJBl I l 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 lmmunoreactivity 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 CJB 111, DKl , DK8, Davis, 515, 2986, 5551, 1169, and 7357B by FACS analysis using mouse antiserum raised against GBS 59 of GBS CJBl 11 FACS analysis did not detect surface expression of GBS 59 in GBS strains SMU071, JM9130013, and COHl, which do not contain a GBS 59 gene (See 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 pill 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 FIG 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 GBS 59 in GBS strain 515 GBS 59 of GBS stain 515 is of a different isotype than GBS 59 of GBS stain 2603 See FIG 63, which illustrates that the homology of these two GBS 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 pill 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 extracts immunoreact with anti-GBS 67 and anti-GBS 150 antiserum In a GBS strain 515 lacking GBS 67 expression, anti-GBS 67 antiserum no longer immunoreacts with polypeptides in total extracts, while anti-GBS 150 antiserum is still able to cross-hybridize with high molecular weight structures

Likewise, formation of pi Ii 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. 221 B, 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 Srtl, Srt2, or both Srtl and Srt2. Only deletion of both Srtl and Srt2 abolishes pilus assembly as detected by antibodies that cross-hybridize with each of GBS 59, GBS 67 and GBS 150 The results of the Western blot analysis were verified by FACS, which provided similar results. See 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 COHl protein). Notwithstanding the difference in the surface protein lengths for these two strains, AI-2 within these sequences still contains two sortase proteins and three LPXTG containing surface proteins, as well as a signal peptidase sequence leading into the first surface protein One of the surface proteins in this variant of Al-2, spbl, has previously been identified as a potential adhesion protein. (See Adderson et al., Infection and Immunity (2003) 71(12):6857 - 6863). Alternatively, because of the lack of GBS 59 and GBS 67 sequences, this variant of Al-2 may be a third type of AI (Adhesin Island-3, AI-3, or GBS AI-3).

More than one AI surface protein may be present in the oligomeπc, 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 oligomeπc 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

Applicants have identified at least six 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 hyperoligomeπc 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 inappropriate 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 Pathogenesis, 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 (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 Bacteπol (1990) 172(6) 3310 - 3317 This island is known as the Fibronectin-bmding, Collagen-binding T-antigen (FCT) region because it contains, in addition to the T6 coding gene (teeό), members of a family of genes coding for Extra Cellular Matrix (ECM) binding proteins Bessen et al , Infection & Immunity (2002) 70(3) 1159-1167 Several of the protein products of this gene family have been shown to directly bind either fibronectin and/or collagen See Hanski et al , Infection & Immunity (1992) 60(12) 5119-5125, Talay et al , Infection & Immunity (1992( 60(9) 3837-3844, Jaffe et al (1996) 21(2) 373-384, Rocha et al , Adv Exp Med Biol (1997) 418 737-739, Kreikemeyer et al , J Biol Chem (2004) 279(16) 15850-15859, Podbielski et al , MoI Microbiol ( 1999) 31(4) 1051-64, and Kreikemeyer et al , Int J Med Microbiol (2004) 294(2-3) 177-88 In some cases direct evidence for a rale of these proteins in adhesion and invasion has been obtained

Applicants raised antiserum against a recombinant product of the teeό gene and used it to explore the expression of T6 in M6 strain 2724 In immunoblot of mutanolysin extracts of this strain, the antiserum recognized in addition to a band corresponding to the predicted molecular mass of the product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3 8% gradient gels used

This pattern of high molecular weight products is similar to that observed in immunoblots of the protein components of the pill identified in Streptococcus agalactiae (described above) and previously in Corynebactenum diphtheriae Electron microscopy of strain M6_2724 with antisera specific for the product of teeό revealed abundant surface staining and long pilus like structures extending up to 700 nanometers from the bacterial surface, revealing that the T6 protein, one of the antigens recognized in the original Lancefield serotyping system, is located within a GAS Adhesin Island (GAS AI-I) and forms long covalently linked pilus structures

Applicants have identified at least six 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-oligomeπc 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 pill structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysacchaπde 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 oligomeπc, or hyperoligomeπc (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 51 A and FIG 162 In all strains identified so far, the adhesin island region is flanked by highly conserved open reading frames Ml_123 and Ml_136 Between three and five genes in each GAS adhesin island code for ECM binding adhesin proteins containing LPXTG motifs

GAS Adhesin Island 1

As discussed above, Applicants have identified adhesin islands, "GAS Adhesin Island 1" or "GAS Al-I " within the genome Group A Streptococcus serotypes and isolates GAS AI-I comprises a series of approximately five open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-I proteins") GAS AI 1 preferably comprises surface proteins, a srtB sortase, and a rofA divergently transcribed transcriptional regulator GAS AI-I surface proteins may include a fibronectin binding protein, a collagen adhesion protein and a fimbria] structural subunit Preferably each of these GAS Al-I surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO 122) or LPXSG (SEQ ID NO 134) (conservative replacement of threonine with serine) Specifically, GAS AI-I includes open reading frames encoding for two or more (i e , 2, 3 4 or 5) of M6_Spy0157, M6_SpyO158, M6_SpyO159, M6_Spy0160, M6_SpyO161

Applicants have also identified open reading frames encoding fimbπal structural subunits in other GAS bacteria harbouring an AI-I These open reading frames encode fimbπal structural subunits CDC SS 410_fimbπal, ISS3650_fimbπal, and DSM2071_fιmbnal A GAS AI-I may comprise d polynucleotide encoding any one of CDC SS 410_fimbπal, ISS365O_fimbπal, and DSM2071_fimbπal

As discussed above, the hyper-oligomeπc pilus structure of GAS AI-I appears to be responsible for the T- antigen type 6 classification, and GAS AI-I corresponds to the FCT region previously identified for teeό As in GAS AI- 1, the teeό FCT region includes open reading frames encoding for a collagen adhesion protein (cpa, capsular polysaccharide adhesion) and a fibronectin binding protein (prtFl) Immunoblots of feed, a GAS AI-I fimbπal structural subunit corresponding to M6_Spyl60, reveal high molecular weight structures indicative of the hyper-oligomeπc pilus structures Immunoblots with antiserum specific for Cpa also recognize a high molecular weight ladder structure, indicating Cpa involvement in the GAS AI-I pilus structure or formation In EM photos of GAS bacteria, Cpa antiserum reveals abundant staining on the surface of the bacteria and occasional gold particles extended from the surface of the bacteria In contrast, immunoblots with antiserum specific for PrtFl recognize only a single molecular species with electrophoretic mobility corresponding to its predicted molecular mass, indicating that PrtFl may not be associated with the oligomeπc pilus structure A preferred immunogenic composition of the invention comprises a GAS AI 1 surface protein which may be formulated or purified in an oligomeπc (pilus) form In a preferred embodiment, the oligomeπc form is a hyperoligomer Another preferred immunogenic composition of the invention comprises a GAS AI-I surface protein which has been isolated in an ohgomeπc (pilus) form The oligomer or hyperohgomeric pilus structures comprising the GAS AI-I surface proteins may be purified or otherwise formulate for use in immunogenic compositions

One or more of the GAS AI-I open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the GAS AI 1 open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

One or more of the GAS AI-I surface protein sequences typically include an LPXTG motif (e g , 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 ammo 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 ammo acid position 2 is a V or a P if X at amino acid position 1 is a V, wherein X at amino acid position 3 is any amino acid residue, wherein X at amino acid position 4 is a T if X at amino acid position 1 is a V, E, or Q, and wherein X at ammo acid position 4 is a T, S, or A if X at amino acid position 1 is an L Some examples of LPXTG motifs present in GAS AI surface proteins include LPSXG (SEQ ID NO 134), VVXTG (SEQ ID NO 135), EVXTG (SEQ ID NO 136), VPXTG (SEQ ID NO 137), QVXTG (SEQ ID NO 138), LPXAG (SEQ ID NO 139), QVPTG (SEQ ID NO 140), and FPXTG (SEQ ID NO 141)

The GAS AI surface proteins of the invention may affect the ability of the GAS bacteria to adhere to and invade epithelial cells AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer Preferably, one or more GAS AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface GAS AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen

The GAS AI-I sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins GAS AI-I may encode for at least one surface protein Alternatively, GAS AI-I may encode for at least two surface exposed proteins and at least one sortase Preferably, GAS Al-I encodes for at least three surface exposed proteins and at least two sortases

The AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis See Comfort et al , Infection & Immunity (2004) 72(5) 2710 - 2722

GAS AI-I preferably includes a srtB sortase. GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO 166), particularly where the motif is followed by a serine

In one embodiment, the invention includes a composition comprising oligomeπc, pilus-like structures comprising a GAS AI-I surface protein such as M6_SpyO157, M6_SpyO159, M6_Spy0160, CDC SS 410_fimbπal, ISS3650_fimbπal, or DSM2071_fimbπaI The oligomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-like structure comprises a hyper-oligomeπc 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) oligomeπc subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc 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 oligomeπc, 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 AM") proteins and one or more GAS Adhesin Island 2 ("GAS AI-2"), GAS Adhesm Island 3 ("GAS AI-3"), GAS Adhesin Island 4 ("GAS AI-4"), GAS Adhesin Island 5 ("GAS AI-5"), or GAS Adhesin Island 6 ("GAS AI-6") proteins, wherein one or more of the GAS Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeric form

In addition to the open reading frames encoding the GAS AI-I proteins, GAS AI-I may also include a divergently transcribed transcriptional regulator such as RofA (i.e , the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction)

GAS Adhesin Island 2

A second adhesin island, "GAS Adhesin Island 2" or "GAS AI-2" has also been identified in Group A Streptococcus serotypes and isolates GAS AI-2 comprises a series of approximately eight open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-2 proteins") Specifically, GAS AI-2 includes open reading frames encoding for two or more (i e , 2, 3, 4, 5, 6, 7, or 8) of GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SpyO133, 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 (pilus) form In a preferred embodiment, the oligomeπc form is a hyperohgomer Another preferred immunogenic composition of the invention comprises a GAS AI-2 surface protein which has been isolated in an oligomeric (pilus) 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 ot the GAS Al-2 open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding tor 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 (e g 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-2 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins GAS AI-2 may encode for at least one surface protein Alternatively, GAS AI-2 may encode for at least two surface exposed proteins and at least one sortase Preferably, GAS AI-2 encodes for at least three surface exposed proteins and at least two sortases

The AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis See Comfort et al , Infection & Immunity (2004) 72(5) 2710 - 2722

In one embodiment, the invention includes a composition comprising ohgomeπc, pilus-hke structures comprising an AI surface protein such as GAS15, GAS16, or GAS18 The ohgomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus-hke structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-hke structure comprises a hyper-ohgomeπc pilus-hke 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-hke structures of the invention will preferably include a pilin motif

The oligomeric, pilus like structures may be used alone or in the combinations of the invention In one embodiment, the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 2 ("GAS Al-2") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 3 ("GAS AI-3"), GAS Adhesin Island 4 ("GAS AI-4") proteins, GAS Adhesin Island 5 ("GAS AI-5"), or GAS Adhesin Island 6 ("GAS AI-6") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeπc form

In addition to the open reading frames encoding the GAS AI-2 proteins, GAS Al-2 may also include a divergently transcribed transcriptional regulator such as rofA (ι e , the transcriptional regulator is located near or adjacent to the Al 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 seven open reading frames encoding for a collection of ammo acid sequences comprising surface proteins and sortases ("GAS Al-3 proteins") Specifically, GAS AI-3 includes open reading frames encoding for two or more (ι e , 2, 3, 4, 5, 6, or 7) of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101 , SpyM3_0102, SpyM3_0103, SpyM3_0104, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf78, orf79, orf80, orfδl, orf82, orf83, orf84, spyM18_0126, spyM 18_O127, spyM18_O128, spyM18_0129, spyM18_0130, spyM18_0131, spyM18_O132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoMO 1000151, SpyoM01000150, and SpyoM0J000149 In one embodiment, GAS AI-3 includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, or 7) of SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104 In another embodiment, GAS AI-3 includes open reading frames encoding for two or more {ι e., 2, 3, 4, 5, 6, or 7) of SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, and SPsOlOβ. In a further embodiment, GAS AI-3 includes open reading frames encoding for two or more 0 e , 2, 3, 4, 5, 6, or 7) of orf78, orf79, orf80, orfδl, orf82, orf83, and orf84 In yet another embodiment, GAS AI-3 includes open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_0130, spyM18_O131, and spyM18_0132 In yet another embodiment, GAS AI-3 includes open reading frames encoding for two or more {i.e., 2, 3, 4, 5, 6, or 7) of SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoMO 1000151, SpyoM01000150, and SpyoMO 1000149

Applicants have also identified open reading frames encoding fimbπal structural subunits in other GAS bacteria harbouring an AI-3. These open reading frames encode fimbrial structural subunits ISS3040_fimbrial, ISS3776_fimbπal, and ISS4959_fimbrial A GAS AI-3 may comprise a polynucleotide encoding any one of ISS3040_fimbπal, ISS3776_fimbπal, and ISS4959_fimbπal.

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 (pilus) 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 (pilus) form. The oligomer or hyperoligomeπc 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 {e g., 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 oligomeπc, pilus-Iike structures comprising an AI surface protein such as SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPsOlOo, orf78 orfSO, orf82, orf84, spyM18_0126, spyM18_0128, spyM18_O13O, spyM18_0132, SpyoM01000155, SpyoM01000153 SpyoM01000151, SpyoM01000149, ISS3040_fimbπaI, ISS3776_fimbπal, and ISS4959_fimbπal In one embodiment the invention includes a composition comprising oligomeπc, pilus-Iike structures comprising an AI surface protein such as SpyM3_0098, SpyM3_0100, SpyM3_0102, and SpyM3_0104 In another embodiment, the invention includes a composition comprising oligomeπc, pilus-Iike structures comprising an AI surface protein such as SPsOlOO, SPs0102, SPs0104, and SPsOlOo In another embodiment, the invention includes a composition comprising oligomeπc, pilus-Iike structures comprising an AI surface protein such as orf78, orfSO, orf82, and orf84 In yet another embodiment, the invention includes a composition comprising ohgomeric, pilus-hke structures comprising an AI surface protein such as spyM18_0126, spyM18_0128, spyM18_O13O, and spyM18_0132 In a further embodiment, the invention includes a composition comprising oligomeπc, pilus-hke structures comprising an Al surface protein such as SpyoM01000155, SpyoM01000153, SpyoM01000151, and SpyoM01000149 In yet a further embodiment, the invention includes a composition comprising oligomeπc, pilus-hke structures comprising an AI surface protein such as ISS3040_fimbπal, ISS3776_fimbπal, and ISS4959_fimbrial The ohgomeric, pilus-hke structure may comprise numerous units of AI surface protein Preferably, the ohgomeric, pilus-Iike structures compπse two or more AI surface proteins Still more preferably, the oligomeπc, pilus-hke structure comprises a hyper-oligomeπc pilus-hke 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) ohgomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The ohgomeric subunits may be covalently associated via a conserved lysine within a pilin motif The oligomeπc 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 ohgomeric, pilus-Iike structures of the invention will preferably include a pilin motif

The ohgomeric, 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 ohgomeric form, preferably in a hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 3 ("GAS AI 3") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 2 ("GAS AI-2"), GAS Adhesin Island 4 ("GAS AI-4") proteins, GAS Adhesin Island 5 ("GAS AI-5"), or GAS Adhesin Island 6 ("GAS AI-6") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperohgomeπc 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 Nm

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 ammo acid sequences comprising surface proteins and sortases ("GAS AI-4 proteins") Specifically, GAS AI-4 includes open reading frames encoding for two or more (J 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 fimbπal structural subunits in other GAS bacteria harbouring an AI-4 These open reading frames encode fimbπal structural subunits 20010296_fimbπal, 20020069_fϊmbπal, CDC SS 635_fimbnal, ISS4883_fimbrial, and ISS4538_fimbπal A GAS AI-4 may comprise a polynucleotide encoding any one of 20010296_fιmbπal, 20020069_fϊmbπal, CDC SS 635_fimbπal, ISS4883_fimbπal, and lSS4538_fimbπal

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 Al-4 surface protein which may be formulated or purified in an oligomeric (pilus) form In a preferred embodiment, the oligomeric form is a hyperohgomer Another preferred immunogenic composition of the invention comprises a GAS AI-4 surface protein which has been isolated in an oligomeric (pilus) 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 (e g , 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 AI surface proteins may also affect the ability of GAS to translocate through an epithelial cell layer Preferably, one or more AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen

The GAS AI-4 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins GAS AI 4 may encode for at least one surface protein Alternatively, GAS AI-4 may encode for at least two surface exposed proteins and at least one sortase Preferably, GAS AI-4 encodes for at least three surface exposed proteins and at least two sortases

The AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid 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_fϊmbπal, 20020069_fimbπal, CDC SS 635_fimbπal, ISS4883_fimbπal, and ISS4538_fϊmbπal 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-oligomeπc 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 subumts, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeric subumts may be covalently associated via a conserved lysine within a pilin motif The oligomeric subumts 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 pihn motif

The oligomeric, pilus like structures may be used alone or in the combinations of the invention In one embodiment, the invention comprises a GAS Adhesin Island protein in oligomeric form, preferably in a hyperoligomeric form In one embodiment, the invention comprises a composition comprising one or more GAS Adhesin Island 4 ("GAS AI-4") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 2 ("GAS AI-2 '). GAS Adhesin Island 3 ( 'GAS AI-3") proteins, GAS Adhesin Island 5 ("GAS AI 5"), or GAS Adhesin Island 6 ("GAS AI 6") proteins, wherein one or more of the Adhesin Island proteins is in the form of an oligomer, preferably in a hyperoligomeπc form

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 (ι 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 5

A fifth adhesin island, "GAS Adhesin Island 5" or "GAS AI-5" has also been identified in Group A Streptococcus serotypes and isolates GAS AI-5 comprises a series of approximately 10 open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS AI-5 proteins") Specifically, GAS AI 5 includes open reading frames encoding for two or more (; e , 2, 3, 4, 5, 6, 7, 8, 9, or 10) of MGAS10270_Spy0108, MGAS10270_Spy0109, MGAS 10270_Spy0110, MGAS10270_Spy0111,

MGAS10270_Spy01 12, MGAS10270_Spy0113, MGAS10270_Spy0114, MGAS 10270_Spy0115,

MGAS10270_Spy0116, and MGAS10270_Spy0117.

One or more of the GAS AI-5 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-5 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-5 surface protein which may be formulated or purified in an oligomeπc (pilus) form In a preferred embodiment, the oligomeric form is a hyperoligomer Another preferred immunogenic composition of the invention comprises a GAS AI-5 surface protein which has been isolated in an oligomeric (pilus) form The oligomer or hyperoligomeπc pilus structures comprising the GAS AI-5 surface proteins may be purified or otherwise formulate for use in immunogenic compositions

One or more of the GAS AI-5 surface protein sequences typically include an LPXTG motif (such as IPxTG (SEQ ID NO 133) or FPxTG (SEQ ID NO 141) 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 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-5 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins GAS AI-5 may encode for at least one surface protein Alternatively, GAS AI-5 may encode for at least two surface exposed proteins and at least one sortase Preferably, GAS AI 5 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-hke structures comprising an AI surface protein such as MGAS10270_Spy0108, MGAS10270_Spy0109, MGAS10270_Spy0110, MGAS10270_Spy01 11 MGAS10270_Spy0112, MGAS10270_Spy01 13, MGAS10270_Spy01 14, MGAS10270_Spy0115, MGAS10270_Spy01 16, and MGAS10270_Spy0117. 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, 1 1, 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 5 ("GAS AI-5") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 2 ("GAS AI-2"), GAS Adhesin Island 3 ("GAS AI-3"), GAS Adhesin Island 4 ("GAS AI-4"), or GAS Adhesin Island 6 ("GAS AI-6") 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-5 proteins, GAS AI-5 may also include a divergently transcribed transcriptional regulator such as rofA (Le., the transcriptional regulator is located near or adjacent to the AI protein open reading frames, but it transcribed in the opposite direction).

Table 54. AI-5 proteins in M2 (10270)

GAS Adhesin Island 6 A sixth adhesin island, "GAS Adhesin Island 6" or "GAS AI-6" has also been identified in Group A Streptococcus serotypes and isolates GAS Al-6 comprises a series of approximately 10 open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases ("GAS Al-6 proteins") Specifically, GAS AI-6 includes open reading frames encoding for two or more (i e , 2, 3, 4, 5, 6, 7, or 8) of MGAS10750_Spy01 13, MGAS10750_Spy0U4, MGAS10750_Spy01 15, MGAS10750_Spy0116, MGAS 10750_Spy0117,

MGAS10750_Spy0118, MGAS10750_Spy01 19, and MGAS10750_Spy0120.

One or more of the GAS AI-6 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-6 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-6 surface protein which may be formulated or purified in an oligomeπc (pilus) form In a preferred embodiment, the oligomeπc form is a hyperohgomer Another preferred immunogenic composition of the invention comprises a GAS AI-6 surface protein which has been isolated in an oligomeπc (pilus) form The oligomer or hyperoligomeπc pilus structures comprising the GAS AI-6 surface proteins may be purified or otherwise formulate for use in immunogenic compositions

One or more of the GAS AI-6 surface protein sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO 122), IPxTG (SEQ ID NO 133) or FPxTG (SEQ ID NO 141) or other sortase substrate motif The AI surface proteins of the invention may effect the ability of the GAS bacteπa 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-6 sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins GAS AI-6 may encode for at least one surface protein Alternatively, GAS AI-6 may encode for at least two surface exposed proteins and at least one sortase Preferably, GAS AI-6 encodes for at least three surface exposed proteins and at least two sortases

The Al 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 oligomeπc, pilus-like structures comprising an AI surface protein such as MGASlO75O_SpyO113, MGAS10750_Spy01 14, MGAS10750_Spy0115, MGAS10750_Spy0116, MGAS 10750_Spy0117, MGAS10750_Spy0118, MGAS10750_Spy0119, and MGAS10750_Spy0120 The oligomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the ohgomeπc, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, 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) oligomers subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc 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 6 ("GAS AI-6") proteins and one or more GAS Adhesin Island 1 ("GAS AI-I"), GAS Adhesin Island 2 ("GAS AI-2"), GAS Adhesin Island 3 ("GAS AI-3"), GAS Adhesin Island 4 ("GAS AI-4"), or GAS Adhesin Island 5 ("GAS AI-5") 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-6 proteins, GAS AI-6 may also include a divergently transcribed transcriptional regulator such as rofA (i.e., the transcriptional regulator is located near or adjacent to the A] protein open reading frames, but it transcribed in the opposite direction). Table 55. AI-6 proteins in M4 (10750)

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 il 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 hyperoligomeπc 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, ^ 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 oligomeπc 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 pih on their surface The GAS bacteria may be adapted to have increased exposure of oligomeπc or hyperoligomeric AI proteins on its surface by increasing expression levels of LepA polypeptide, or an equivalent signal peptidase, in the GAS bacteria Applicants have shown that deletion of LepA in strain SF37O bacteria, which harbour a GAS AI-2, abolishes surface exposure of M and pih proteins on the GAS Increased levels of LepA expression in GAS are expected to result in increased exposure of M and pih 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 pih 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 gordontt 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 Pharyngeal 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 oligomeπc, or hyper-oligomeπc form Sequences encoding for an Al 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 Al polypeptides L lactis was transformed with pAM401 constructs encoding entire pill gene clusters of AI- I Al-2, and Al-4 adhesin islands Briefly, the pAM401 is a promoterless high-copy plasmid The entire pill gene clusters of an M6 (AI-I), Ml (AI-2), and M12 (AI-4) bacteria were inserted into the pAM401 construct The gene clusters were transcribed under the control their own (M6, Ml, or M12) promoter or the GBS promoter that successfully initiated expression of the GBS AI- 1 adhesin islands in L lactis, described above FIG 172 provides a schematic depiction of GAS M6 (AI-I), Ml (AI-2), and M12 (AI-4) adhesin islands and indicates the portions of the adhesin island sequences inserted in the pAM401 construct

Each of the L lactis transformed with one of the M6, Ml, 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 (FlG 173 A), Ml (FIG 173 B), or M12 (FIG 173 C) adhesin island gene clusters using antibodies that bind to the fimbπal 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 Ml AI-2 using an antibody that binds to fimbπal structural subunit SpyO128 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 fimbπal 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-I using an antibody that binds to fϊmbπal structural subunit M6_Spy0160 In FIGS 173 A-C, "pi" immediately following the notation of AI subtype indicates that the promoter present in the Adhesin Island is used to drive transcription of the adhesin island gene cluster and "p2" indicates that the promoter was the GBS promoter described above Thus, it appears that L lactis is capable of expressing the fimbπal structural subunits encoded by GAS adhesin islands in an ohgomeπc form

Alternatively, the oligomeπc, 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 oligomeπc or hyperoligomeπc 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 (/ 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 oligomeπc (pilus) form In a preferred embodiment, the oligomeπc form is a hyperohgomer Another preferred immunogenic composition of the invention comprises a 5 pneumoniae from TIGR4 AI surface protein which has been isolated in an oligomeric (pilus) form The oligomer or hyperohgomer pilus structures comprising S pneumoniae surface proteins may be purified or otherwise formulated for use in immunogenic compositions

One or more of the S pneumoniae from TIGR4 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the S pneumoniae from TIGR4 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF One or more of the 5 pneumoniae from TIGR4 AI surface protein sequences typically include an LPXTG motif (e g , SEQ ID NO 122) or other sortase substrate motit

The 5 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 5 pneumoniae to translocate through an epithelial cell layer Preferably, one or more 5 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 5 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 Al 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 oligomeπc, pilus-hke structures comprising a S pneumoniae from TIGR4 AI surface protein such as SP0462, SP0463, SP0464, or SP0465 The oligomeπc, pilus-hke structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus like structures comprise two or more AI surface proteins Still more preferably, the ohgomeπc, pilus-hke structure comprises a hyper-oligomeπc pilus-hke 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) ohgomeπc subunits, wherein each subumt 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-hke structures of the invention will preferably include a pilin motif

The oligomeric pilus like structures may be used alone or in the combinations of the invention In one embodiment, the invention comprises a S pneumoniae from TIGR4 AI protein in oligomeric form, preferably in a hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more S pneumoniae from TIGR4 AI proteins and one or more 5 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 (ι e , 2, 3, 4, 5, 6, or 7) of orfl_670, orf3_670, orf4_670 orf5_670, orfό 670, orf7_670, orf8_670 A preferred immunogenic composition of the invention comprises a 5 pneumoniae strain 670 AI surface protein which may be formulated or purified in an oligomeπc (pilus) form Another preferred immunogenic composition of the invention comprises a S pneumoniae strain 670 AI surtace protein which has been isolated in an oligomeπc (pilus) 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 ot 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 (e g , 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 5 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 5 pneumoniae strain 670 AI sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins S pneumoniae strain 670 AI may encode for at least one surface protein Alternatively, S pneumoniae strain 670 AI may encode for at least two surface exposed proteins and at least one sortase Preferably, S pneumoniae strain 670 AI encodes for at least three surface exposed proteins and at least two sortases

The AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase The sortase may function to cleave the surface protein, 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 oligomeπc, pilus-hke structures comprising a S pneumoniae strain 670 AI surface protein such as orf3_670, orf4_670, or orf5_670 The oligomeπc, pilus-hke structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus-hke structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-hke structure comprises a hyper- ohgomeπc pilus-hke 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) oligomers subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc subunits may be covalently associated via a conserved lysine within a pilin motif The oligomeπc subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively

AI surface proteins or fragments thereof to be incorporated into the oligomeπc, pilus-hke structures of the invention will preferably include a pilin motif

The oligomeπc, 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 ohgomeπc form, preferably in a hyperohgomeric 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 hyperohgomeric 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 5 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 ORF2_14CSR, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF6_14CSR, ORF7_14CSR, ORF8_14CSR

A preferred immunogenic composition of the invention comprises a 5 pneumoniae strain 14 CSR 10 AI surface protein which may be formulated or purified in an oligomeπc (pilus) 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 oligomeπc (pilus) form

One or more of the 5 pneumoniae strain 14 CSR 10 AI open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF Alternatively, one or more of the S pneumoniae strain 14 CSR 10 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

One or more of the S pneumoniae strain 14 CSR 10 AI surface protein sequences typically include an LPXTG motif (e g , 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, 5 pneumoniae strain 14 CSR 10 AI may encode for at least two surface exposed proteins and at least one sortase Preferably, S pneumoniae strain 14 CSR 10 AI encodes for at least three surface exposed proteins and at least two sortases

The AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis See Comfort et al , Infection & Immunity (2004) 72(5) 2710 - 2722

In one embodiment, the invention includes a composition comprising oligomeπc, pilus-like structures comprising a S pneumoniae strain 14 CSR 10 AI surface protein such as orO_CSR, orf4_CSR, or orf5_CSR The oligomeπc, 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-oligomeπc 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 AJ surface proteins or fragments thereof to be incorporated into the oligomeπc pilus-like structures of the invention will preferably include a pilin motif

The oligomeπc pilus like structures may be used alone or in the combinations of the invention In one embodiment, the invention comprises a 5 pneumoniae strain 14 CSR 10 AI protein in oligomers form, preferably in a hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more 5 pneumoniae strain 14 CSR 10 AI proteins, and one or more Al proteins of any of 5 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 5 pneumoniae AI proteins is in the form of an oligomer, preferably in a hyperoligomeπc 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 5 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 (ι e , 2, 3, 4, 5, 6, or 7) of ORF2_19AH, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF6_19AH, ORF7_19AH, ORF8_19AH

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 19A Hungary 6 AI surface protein which may be formulated or purified in an oligomeπc (pilus) 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 ohgomeric (pilus) 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 5 pneumoniae strain 19A Hungary 6 AI surface protein sequences typically include an LPXTG motif (e g , 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 5 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 pneumonuie strain 19A Hungary 6 AI may encode for at least two surface exposed proteins and at least one sortase Preferably, S pneumoniae strain 19A Hungary 6 AI encodes for at least three surface exposed proteins and at least two sortases

The AI surface proteins may be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as an AI sortase The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis See Comfort et al , Infection & Immunity (2004) 72(5) 2710 - 2722 In one embodiment, the invention includes a composition comprising oligomeπc pilus-like structures comprising a 5 pneumoniae strain 19A Hungary 6 AI surface protein such as orf3_19AH, orf4_19AH, or orf5_19AH The oligomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the ohgomeric, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-like structure comprises a hyper-oligomeπc 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) ohgomeric subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc subunits may be covalently associated via a conserved lysine within a pilin motif The ohgomeric 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 ohgomeric, pilus-like structures of the invention will preferably include a pilin motif

The ohgomeric, 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 ohgomeric form, preferably in a hyperoligomeπc 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 Spam 2, 9V Spam 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 hyperohgomeric form

In addition to the open reading frames encoding the S pneumoniae strain 19A Hungary 6 AI proteins, 5 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 ammo 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 (ι e , 2, 3, 4, 5, 6, or 7) of ORF2_19FTW, ORF3_19FTW, ORF4_19FTW, ORF5_19FTW, ORF6_19FTW, ORF7_19FTW, ORF8_19FTW

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 19F Taiwan 14 AI surface protein which may be formulated or purified in an ohgomeric (pilus) 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 oligomeπc (pilus) 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 (e g , SEQ ID NO 122) or other sortase substrate motif

The 5 pneumoniae strain 19F Taiwan 14 Al 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 5 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 Al 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, 5 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 oligomeπc, pilus-hke structures comprising a S pneumoniae strain 19F Taiwan 14 AI surface protein such as orO_19FTW, orf4_19FTW, or orf5_19FTW The oligomeπc, pilus-hke structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus- like structure comprises a hyper-oligomeπc pilus-hke 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) oligomeπc 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 pihn 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 pihn 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 hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more S pneumoniae strain 19F Taiwan 14 Al proteins and one or more AI proteins of any one or more of 5 pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spam 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 hyperohgomeric form

In addition to the open reading frames encoding the S pneumoniae strain 19F Taiwan 14 AI proteins, S pneumoniae strain 19F Taiwan 14 Al may also include a transcriptional regulator S pneumoniae strain 23F Poland 16 Adhesin Island

As discussed above, Applicants have identified adhesin islands within the genome of S pneumoniae strain 23F Poland 16 The S pneumoniae strain 23F Poland 16 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases Specifically, the S pneumoniae strain 23F Poland 16 AI proteins includes open reading frames encoding for two or more (i e , 2, 3, 4, 5, 6, or 7) of ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP, and ORF8_23FP

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 23F Poland 16 AI surface protein which may be formulated or purified in an oligomeric (pilus) 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 (pilus) form One or more of the 5 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 5 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 (e g , 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 also affect the ability of S pneumoniae to translocate through an epithelial cell layer Preferably, one or more S pneumoniae strain 23F Poland 16 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface S pneumoniae strain 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, 5 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 oligomeπc, pilus-like structures comprising a S pneumoniae strain 23F Poland 16 AI surface protein such as orf3_23FP, orf4_23FP, or orf5_23FP The oligomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-like structure comprises a hyper-oligomeπc 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) oligomers subumts, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeric subumts may be covalently associated via a conserved lysine within a pihn motif The oligomeric subumts may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively

AI surface proteins or fragments thereof to be incorporated into the oligomeπc, pilus-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 5 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 Poland 16 AI proteins, S pneumoniae strain 23F Poland 16 AI may also include a transcriptional regulator S pneumoniae strain 23F Taiwan 15 Adhesin Island

As discussed above Applicants have identified adhesin islands within the genome of 5 pneumoniae strain 23F Taiwan 15 The S pneumoniae strain 23F Taiwan 15 Adhesin Island comprises a series of approximately seven open reading frames encoding for a collection of amino acid sequences comprising surface proteins and sortases Specifically, the S pneumoniae strain 23F Taiwan 15 AI proteins includes open reading frames encoding for two or more (i e , 2, 3, 4, 5, 6, or 7) of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, ORF8_23FTW

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 23F Taiwan 15 AI surface protein which may be formulated or purified in an oligomeric (pilus) 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 oligomeπc (pilus) form

One or more of the 5 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 (<? g , 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 5 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 5 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 5 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, 5 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 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 Taiwan 15 AI surface protein such as orO_23FTW, orf4_23FTW, or orf5_23FTW The oligomeric, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeric, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeric, pilus- like structure comprises a hyper-oligomeπc 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 tragments thereof to be incorporated into the oligomeric, pilus-like structures of the invention will preferably include a pilin motif

The oligomeπc, 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 hyperoligomeπc form In one embodiment, the invention comprises a composition comprising one or more 5 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 hyperoligomeπc 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 (r e , 2, 3, 4, 5, 6, or 7) of ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF, ORF8_6BF

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 6B Finland 12 AI surface protein which may be formulated or purified in an oligomeric (pilus) 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 oligomeπc (pilus) 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 Alternatively, one or more of the S pneumoniae strain 6B Finland 12 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

One or more of the S pneumoniae strain 6B Finland 12 AI surface protein sequences typically include an LPXTG motif (e g , 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 transgl ycoslylation and transpeptidation reactions of bacterial wall synthesis See Comfort et a! , Infection & Immunity (2004) 72(5) 2710 - 2722

In one embodiment, the invention includes a composition comprising oligomeπc, pilus-like structures comprising a S pneumoniae strain 6B Finland 12 AI surface protein such as orf3_6BF, orf4_6BF, or orf5_6BF The oligomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-like structure comprises a hyper-oligomeπc 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) oligomeπc subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc subunits may be covalently associated via a conserved lysine within a pilin motif The oligomeπc subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or serine amino acid residue, respectively

AI surface proteins or fragments thereof to be incorporated into the oligomeπc, pilus-like structures of the invention will preferably include a pilin motif

The oligomeπc, 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 Finland 12 AI protein in oligomeπc 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 Spam 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 Spam 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 Spam 2 AI proteins includes open reading frames encoding for two or more (i e , 2, 3, 4, 5, 6, or 7) of ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP, and ORF8_6BSP

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 6B Spain 2 AI surface protein which may be formulated or purified in an oligomeric (pilus) 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 (pilus) 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 Spam 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 {e g , 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 5 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 5 pneumoniae strain 6B Spain 2 AI surface proteins are capable of binding to or otherwise associating with an epithelial cell surface 5 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, 5 pneumoniae strain 6B Spain 2 AI may encode for at least two surface exposed proteins and at least one sortase Preferably, S pneumoniae strain 6B Spain 2 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 oligomeπc, pilus-like structures comprising a S pneumoniae strain 6B Spain 2 AI surface protein such as orf3_6BSP, orf4_6BSP, or orf5_6BSP The oligomeπc, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeπc, pilus-like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-like structure comprises a hyper-oligomeπc 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) oligomeπc subunits, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc subunits may be covalently associated via a conserved lysine within a pihn motif The oligomeπc subunits may be covalently associated via an LPXTG motif, preferably, via the threonine or seπne amino acid residue, respectively

AI surface proteins or fragments thereof to be incorporated into the ohgomeπc, pilus-like structures of the invention will preferably include a pihn motif

The oligomeπc, pilus like structures may be used alone or in the combinations of the invention In one embodiment, the invention comprises a 5 pneumoniae strain 6B Spain 2 AI protein in oligomeπc form, preferably in a hyperoligomeric form In one embodiment, the invention comprises a composition comprising one or more S pneumoniae strain 6B Spam 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 Al proteins includes open reading frames encoding for two or more (/ e , 2, 3, 4, 5, 6, or 7) of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP ORF7_9VSP, and ORF8_9VSP

A preferred immunogenic composition of the invention comprises a S pneumoniae strain 9V Spain 3 AI surface protein which may be formulated or purified in an oligomeπc (pilus) form Another preferred immunogenic composition of the invention comprises a 5 pneumoniae strain 9V Spain 3 AI surface protein which has been isolated in an oligomeric (pilus) 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 Spam 3 AI open reading frames may be replaced by a sequence having sequence homology to the replaced ORF

One or more of the 5 pneumoniae strain 9V Spain 3 AI surface protein sequences typically include an LPXTG motif (e g , SEQ ID NO 122) or other sortase substrate motif

The 5 pneumoniae strain 9V Spain 3 AI surface proteins of the invention may affect the ability of the 5 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 Spam 3 AI surface proteins may also be able to bind to or associate with fibrinogen, fibronectin, or collagen

The 5 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 AJ may encode for at least one surface protein Alternatively, 5 pneumoniae strain 9V Spam 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 mto 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 orO_9VSP, orf4_9VSP, or orf5_9VSP The oligomeric, pilus-like structure may comprise numerous units of AI surface protein Preferably, the oligomeric, pilus like structures comprise two or more AI surface proteins Still more preferably, the oligomeric, pilus-like structure comprises a hyper-oligomeπc 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 hyperoligomeπc 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 5 pneumoniae from TIGR4, 670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 14 CSR 10, 19F Ta.wan 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 hyperoligomeπc 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 oligomeπc, pilus-like structures may be isolated or purified from bacterial cultures in which the bacteria express an 5 pneumoniae AI surface protein The invention therefore includes a method for manufacturing an oligomeπc AI surface antigen comprising cultuπng 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 AJ 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 5 pneumoniae oligomeric Adhesin Island surface antigen comprising culturing a S pneumoniae bacterium adapted for increased AI protein expression and isolation of the expressed oligomeπc 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 5 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 pill on their surface The S pneumoniae bacteria may be adapted to have increased exposure of oligomeric or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pill 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 pill protein

Alternatively, the AI proteins of the invention may be expressed on the surface of a non-pathogenic Gram positive bacteria, such as Streptococcus gordonu (See, e g , Byrd et al , "Biological consequences of antigen and cytokine co expression by recombinant Streptococcus gordonu vaccine vectors, ' Vaccine (2002) 202197-2205) or Lactococcus lactis (See, e g , Mannam et al , "Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis Protects Mice against Pharyngeal 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 oligomeπc or hyper oligomeπc 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 isoloted 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 Preferably, the non-pathogenic Gram positive bacteria also express at least one Adhesin Island sortase The AI transformed non-pathogenic Gram positive bacteria of the invention may be used to prevent or treat infection with pathogenic S pneumoniae

FIGS 190 A and B, and 193-195 provide examples of three methods successfully practiced by applicants to purify pill 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 5 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 ammo acid sequence that is at least 50% (ι 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 Al 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 (ι 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% (/ 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 oligomeπc pilus structures may be formulated or purified for use in immunization Isolated AI surface protein oligomeπc 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 may be a surface protein Such surface proteins may contain an LPXTG motif or other sortase substrate motif The first and second AI proteins may be from the same or different genus or species of Gram positive bacteria If within the same species, the First and second AI proteins may be from the same or different AI subtypes If two AIs are of the same subtype, the AIs have the same numerical designation For example, all AIs designated as AI 1 are of the same AI subtype If two AIs are of a different subtype, the AIs have different numerical designations For example, AI- I is of a different AI subtype from AI-2, AI-3, AI-4, etc Likewise, AI-2 is of a different AI subtype from AI-I, AI-3, and AI-4, etc

For example, the invention includes an immunogenic composition comprising one or more GBS AI-I proteins and one or more GBS AI-2 proteins One or more of the Al proteins may be a surface protein Such surface proteins may contain an LPXTG motif (e g , SEQ ID NO 122) and may bind fibrinogen, fibronectin, or collagen One or more of the AI proteins may be a sortase The GBS AI-I proteins may be selected from the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648 Preferably, the GBS AI-I proteins include GBS 80 or GBS 104

The GBS AI-2 proteins may be selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523, 01523, 01524 and 01525 In one embodiment, the GBS AI-2 proteins are selected from the group consisting of GBS 67, GBS 59, GBS 150, SAG1405, and SAG1406 In another embodiment, the GBS AI-2 proteins may be selected from the group consisting of 01520, 01521, 01522, 01523, 01523, 01524 and 01525 Preferably, the GBS AI-2 protein includes GBS 59 or GBS 67

As another example, the invention includes an immunogenic composition comprising one or more of any combination of GAS AI-I, GAS AI-2, GAS AI-3, or GAS Al-4 proteins One or more of the GAS AI proteins may be a sortase The GAS AJ-I proteins may be selected from the group consisting of M6_SpyO156, M6_SpyO157, M6_Spy0158, M6_SpyO159, M6_Spy0160, M6_SpyO161, CDC SS 410_fimbπal, ISS3650_fϊmbπal, and DSM2071_fimbπal Preferably, the GAS AI-I proteins are selected from the group consisting of M6_SpyO157, M6_SpyO159, M6_Spy0160, CDC SS 410_fimbπal, ISS3650_fimbπal, and DSM2071_fimbπal

The GAS AI-2 proteins may be selected from the group consisting of SpyO124, GAS15, SpyO127, GAS16, GAS17, GAS18, SpyO131, SpyO133, 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 SpyM3_0097, SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, SpyM3_0104, SPs0099, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf77, orf78, orf79, orf80, orfδl, orf82, orf83, orf84, spyM18_0125, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_O13O, spyM18_O131, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoMO 1000153, SpyoM01000152, SpyoMOlOOOlSl, SpyoM01000150, SpyoMO 1000149, ISS3040_fimbπal, ISS3776_fimbπal, and ISS4959_fimbπal In one embodiment the GAS AI-3 proteins are selected from the group consisting of SpyM3_0097, SpyM3_0098, SpyM3_0099, SpyM3_0100, SpyM3_0101, SpyM3_0102, SpyM3_0103, and SpyM3_0104 In another embodiment, the GAS AI-3 proteins are selected from the group consisting of SPs0099, SPsOlOO, SPsOlOl, SPs0102, SPs0103, SPs0104, SPs0105, and SPsOlOδ 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 spyM18_0125, spyM18_0126, spyM18_0127, spyM18_0128, spyM18_0129, spyM18_O13O, spyM18_0131, and spyM18_0132 In yet another embodiment the GAS AI-3 proteins are selected from the group consisting of SpyoM01000156, SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoMO 1000152, 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_fimbπal, 20020069_fimbnal, CDC SS 635_fimbπal, ISS4883_fimbπal, and lSS4538_fimbπal. Preferably, the GAS-AJ4 proteins are selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fιmbπal, 20020069_fϊmbπal, CDC SS 635_fimbπal, ISS4883_fimbπal, and ISS4538_fimbπal.

As yet another example, the invention includes an immunogenic composition comprising one or more of any combination of 5. pneumoniae from TIGR4, S. pneumoniae strain 670, S pneumoniae from 19A Hungary 6, S pneumoniae from 6B Finland 12, S pneumoniae from 6B Spain 2, S. pneumoniae from 9V Spain 3, S. pneumoniae from 14 CSR 10, S. pneumoniae from 19F Taiwan 14, S. pneumoniae from 23F Taiwan 15, or S. pneumoniae 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 (e.g., SEQ ID NO: 122) and may bind fibrinogen, fibronectin, or collagen One or more of the AI proteins may be a sortase

The S. pneumoniae from TIGR4 AI proteins may be selected from the group consisting of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, SP0468 Preferably, the 5. pneumoniae from TIGR4 AI proteins include SP0462, SP0463, or SP0464.

The S. pneumoniae strain 670 Al proteins may be selected from the group consisting of Orfl_670, Orf3_670, Orf4_670, Orf5_670, Orf6_670, Orf7_670, and Orf8_670. Preferably, the S pneumoniae strain 670 AI proteins include OrO_670, Orf4_670, or Orf5_670

The S. pneumoniae from 19A Hungary 6 AI proteins may be selected from the group consisting of ORF2_19AH, ORF3.19AH, ORF4_19AH, ORF5.19AH, ORF6_19AH, ORFT.^AH, or ORF8_19AH.

The S. pneumoniae from 6B Finland 12 AI proteins may be selected from the group consisting of ORF2_6BF, ORF3_6BF, ORF4_6BF, ORF5_6BF, ORF6_6BF, ORF7_6BF , or ORF8_6BF.

The S. pneumoniae from 6B Spain 2 AI proteins may be selected from the group consisting of ORF2_6BSP, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF6_6BSP, ORF7_6BSP , or ORF8_6BSP.

The 5. pneumoniae from 9V Spam 3 AI proteins may be selected from the group consisting of ORF2_9VSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, ORF6_9VSP, ORF7_9VSP _t or ORF8_9VSP.

The S. pneumoniae from 14 CSR 10 AI proteins may be selected from the group consisting of ORF2_14CSR, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF6_14CSR, ORF7J4CSR , or ORF8_14CSR.

The S. pneumoniae from 19F Taiwan 14 AI proteins may be selected from the group consisting of ORF2_19FTW, ORF3_19FTW, ORF4_19FTW, ORF5_19FTW, ORF6_19FTW, ORF7_19FTW „ or ORF8_19FTW

The S. pneumoniae from 23F Taiwan 15 AI proteins may be selected from the group consisting of ORF2_23FTW, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF6_23FTW, ORF7_23FTW, or ORF8_23FTW.

The S. pneumoniae from 23F Poland 16 Al proteins may be selected from the group consisting of ORF2_23FP, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF6_23FP, ORF7_23FP , or ORF8_23FP.

Preferably, the Gram positive bacteria AI proteins included in the immunogenic compositions of the invention can provide protection across more than one serotype or strain isolate. For example, the immunogenic composition may comprise a first AI protein, wherein the amino acid sequence of said AI protein is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 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 Al 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% (; 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 AJ 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 Al 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-I protein or an AI-2 protein For example, the first AI protein may be a GBS AI-I surface protein such as GBS 80 The amino acid sequence of GBS 80 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 80 amino acid sequence from GBS serotype III, strain isolates NEM316 and COHl and the GBS 80 amino acid sequence from GBS serotype Ia, strain isolate A909

As another example, the first AI protein may be GBS 104 The amino acid sequence of GBS 104 from GBS serotype V, strain isolate 2603 is greater than 90% homologous to the GBS 104 amino acid sequence from GBS serotype III, strain isolates NEM316 and COHl, the GBS 104 amino acid sequence from GBS serotype Ia, strain isolate A909, and the GBS 104 amino acid sequence serotype II, strain isolate 18RS21

Table 12 provides the amino acid sequence identity of GBS 80 and GBS 104 across GBS serotypes Ia, Ib, II, III, V, and VIII The GBS strains in which genes encoding GBS 80 and GBS 104 were identified share, on average, 99 88 and 9996 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 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 ammo acid sequence from GBS serotype III, strain isolate NEM316, the GBS 67 amino acid sequence from GBS serotype Ib, strain isolate H36B, and the GBS 67 amino acid sequence from GBS serotype II, strain isolate 17RS21

As another example, the first AI protein may be an AI-2 protein such as spbl The amino acid sequence of spbl from GBS serotype III, strain isolate COHl is greater than 90% homologous to the spbl amino acid sequence from GBS serotype Ia, strain isolate A909

As yet another example, the first AI protein may be an AI-2 protein such as GBS 59 The amino acid sequence of GBS 59 from GBS serotype II, strain isolate 18RS21 is 100% homologous to the GBS 59 amino acid sequence from GBS serotype V, strain isolate 2603 The amino acid sequence of GBS 59 from GBS serotype V, strain isolate CJB 111 is 98% homologous to the GBS 59 amino acid sequence from GBS serotype III, strain isolate NEM316

The compositions of the invention may also be designed to include Gram positive AI proteins from divergent serotypes or strain isolates, i e , to include a first AI protein which is present in one collection of serotypes or strain isolates of a Gram positive bacteria and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein

For example, the invention may include an immunogenic composition comprising a first and second Gram positive bacteria AI protein, wherein a polynucleotide sequence encoding for the full length sequence of the first AI protein is not present in a similar Gram positive bacterial genome comprising a polynucleotide sequence encoding for the second AI protein The compositions of the invention may also be designed to include AI proteins from divergent GBS serotypes or strain isolates, i.e., to include a first AI protein which is present in one collection of GBS serotypes or strain isolates and a second AI protein which is present in those serotypes or strain isolates not represented by the first AI protein.

For example, the invention may include an immunogenic composition comprising a first and second GBS AI protein, wherein a polynucleotide sequence encoding for the full length sequence of the first GBS Al protein is not present in a genome comprising a polynucleotide sequence encoding for the second GBS AI protein. For example, the first AI protein could be GBS 80 (such as the GBS 80 sequence from GBS serotype V, strain isolate 2603). As previously discussed (and depicted in 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 12. Conservation of GBS 80 and GBS 104 amino acid sequences

Table 15: Antigen surface exposure of GBS 80, GBS 322, GBS 104, and GBS 67

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 % (ι β , 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 % (ι e , less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second GBS AI protein For example, the first GBS AI protein could be GBS 67 (such as the GBS 67 sequence from GBS serotype Ib, strain isolate H36B) As shown in 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 FACS analysis of different GBS strains demonstrates that at least one of these five proteins is always found to be expressed on the surface of GBS bacteria An initial FACS analysis of 70 strains of GBS bacteria, obtained from the CDC in the United States (33 strains), ISS in Italy (17 strains), and Houston/Harvard (20 strains), detected surface exposure of at least one of GBS 80, GBS 104, GBS 322, GBS 67, or GBS 59 on the surface of the GBS bacteria 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 Ml 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, CBS 104, GBS 67, GBS 322, and GBS 59 on GBS Strains

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

The invention may include an immunogenic composition comprising a first and second GBS59 polypeptide, wherein the amino acidic sequence encoding the sequence of the first GBS59 polypeptide is less than 90 % (ι 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 encoded in the genome of the second GBS59 polypeptide As previously shown, 6 different allelic families of GBS59 polypeptides have been identified (see FIG 240) which have less than 90% sequence identity Accordingly, the first and the second GBS59 polypeptides of the invention include but are not limited to any one of the 6 allelic families ancestors (i e , GBS59^CJB1", GBS59^DK21, GBS59⁵¹⁵, GBS59^CJBπo, GBS59²⁶⁰³ and GBS59^H36B), leading tol5 possible combinations of two For example, GBS59^DK21 and GBS59^CJB"° Other possible such combinations are GBS59^CJB1" and G_BS59 ^DK2I GBS₅₉ ^{CJBi I i} _and QB₅₅₉ ^SiS _{1 GBS59} ^{CJBi i}ι _{and G}BS59^CJB"°, GBS59^CJB1" and GBS59²⁶⁰³ GBS59^αB1" and

GBS59^H36B, GBS59^DK21 and GBS59⁵¹⁵, GBS59^DK21 and GBS59²⁶⁰³ GBS59^DK21 and GBS59^H36B, GBS59⁵¹⁵ and GBS59^αB"°, GBS59⁵¹⁵ and GBS59²⁶⁰³ GBS59⁵¹⁵ and GBS59^H36B, GBS59^CJB"° and GBS59²⁶⁰³ GBS59^CJB"° and GBS59^H16B or GBS59²⁶⁰³ and GBS59^H36B GBS By way ot another example, preferably, the GAS A] 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 ot said AI protein is at least 90% (ι 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, / 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_SpyO159 (from M6 isolate (MGAS 10394), which comprises an AI-I) or GAS15 (from Ml isolate (SF370), which comprises an AI-2)

Further, the invention may include an immunogenic composition comprising a first and second GAS AI protein, wherein the first GAS AI protein has detectable surface exposure on a first GAS strain or serotype but not a second GAS strain or serotype and the second GAS AI protein has detectable surface exposure on a second GAS strain or serotype but not a first GAS strain or serotype

The invention may include an immunogenic composition comprising a first and second GAS AI protein, wherein the polynucleotide sequence encoding the sequence of the first GAS AI protein is less than 90 % (i e , less than 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent) homologous than the corresponding sequence in the genome of the second GAS AI protein Preferably the first and second GAS AI proteins are subunits of the pilus More preferably the first and second GAS AI proteins are selected from the major pilus forming proteins (i e , M6_SpyO16O from M6 strain 10394, SPyO128 from Ml strain SF370, SpyM3_0100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM18_0128 from M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from M12 strain A735, fimbria! structural subunit from M77 strain ISS4959, fimbπal structural subunit from M44 strain ISS3776, fimbπal structural subunit from M50 strain ISS3776 ISS 4538, fimbπal structural subunit from M12strain CDC SS635, fimbπal structural subunit from M23 strain DSM2071, fimbπal structural subunit from M6 strain CDC SS410) Table 45 provides the percent identity between the amino acidic sequences of each of the main pilus forming subunits from GAS AI-I, AI-2, AI 3, and AI-4 representative strains (i e , M6_Spy0160 from M6 strain 10394, SPyO128 from Ml strain SF370, SpyM3_0100 from M3 strain 315, SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo, spyM18_0128 from M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from M12 strain A735, Fimbria! structural subunit from M77 strain ISS4959, fimbπal structural subunit from M44 strain ISS3776, fimbria! structural subunit from M50 strain ISS3776 ISS 4538, fimbπal structural subunit from M12strain CDC SS635, fimbria! structural subunit from M23 strain DSM2071, fimbπal structural subunit from M6 strain CDC SS410)

Table 45 Comparison of Amino Acid Sequences of Major Pilus Proteins in the Four GAS AIs

For example, the first main pilus subunit may be selected from bacteria of GAS serotype M6 strain 10394 and the second main pilus subunit may be selected from bacteria of GAS serotype Ml strain 370. As can be seen from Table 45, the main pilus subunits encoded by these strains of bacteria share only 23% nucleotide identity. An immunogenic composition comprising pilus main subunits from each of these strains of bacteria is expected to provide protection across a wider group of GAS strains and serotypes. Other examples of main pilus subunits that can be used in combination to provide increased protection across a wider range of GAS strains and serotypes include proteins encoded by GAS serotype M5 Manfredo isolate and serotype M6 strain 10394, which share 23% sequence identity, GAS serotype M18 strain 8232 and serotype Ml strain 370, which share 38% sequence identity, GAS serotype M3 strain 315 and serotype M12 strain A735, which share 61% sequence identity, and GAS serotype M3 strain 315 and serotype M6 strain 10394 which share 25% sequence identity.

As also can be seen from Table 45, the amino acid sequences of the four types of main pilus subunits present in GAS are relatively divergent. 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-I, GAS AI-2, GAS AI-3, and GAS AI-4). See also further discussion below.

Immunizations with the Adhesin Island proteins of the invention are discussed further in the Examples. Co-expression of GBS Adhesin Island proteins and role of GBS AI proteins in surface presentation

In addition to the use of the GBS adhesin island proteins for cross strain and cross serotype protection, Applicants have identified interactions between adhesin island proteins which appear to affect the delivery or presentation of the surface proteins on the surface of the bacteria.

In particular, Applicants have discovered that surface exposure of GBS 104 is dependent on the concurrent expression of GBS 80. As discussed further in Example 2, reverse transcriptase PCR analysis of AI-I shows that all of the AI genes are co-transcribed as an operon. Applicants constructed a series of mutant GBS containing in frame deletions ot various AI-I 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 GBS 104 operon did not affect surface exposure of GBS 80 While not being limited to a specific theory, it is thought that GBS 80 is involved in the transport or localization of GBS 104 to the surface of the bauena The two proteins may be oligomeπzed 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

Pill structures that comprise GBS 104 appear to be of a lower molecular weight than pill 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 α-GBS 80 POLIC )

In addition, Applicants have shown that removal of GBS 80 can cause attenuation, further suggesting the protein contributes to virulence As described in more detail in Example 3, the LD₅₀'s for the Δ80 mutant and the Δ80, Δ104 double mutant were reduced by an order of magnitude compared to wildtype and Δ104 mutant

The sortases within the adhesin island also appear to play a role in localization and presentation of the surface proteins As discussed further in Example 4, FACS analysis of various sortase deletion mutants showed that removal of sortase SAG0648 prevented GBS 104 from reaching the surface and slightly reduced the surface exposure of GBS 80 When sortase SAG0647 and sortase SAG0648 were both knocked out, neither GBS 80 nor GBS 104 were surface exposed Expression of either sortase alone was sufficient for GBS 80 to arrive at the bacterial surface Expression of SAG0648, however, was required for GBS 104 surface localization

Accordingly, the compositions of the invention may include two or more AI proteins, wherein the AI proteins are physically or chemically associated For example the two AI proteins may form an oligomer In one embodiment, the associated proteins are two AI surface proteins, such as GBS 80 and GBS 104 The associated proteins may be AI surface proteins from different adhesin islands, including host cell adhesin island proteins if the AI surface proteins are expressed in a recombinant system For example, the associated proteins may be GBS 80 and GBS 67 Adhesin Island proteins from other Gram positive bacteria

Applicants' identification and analysis of the GBS adhesin islands and the immunological and biological functions of these AI proteins and their pilus structures provides insight into similar structures in other Gram positive bacteria

As discussed above, "Adhesin Island" or "AI ' refers to a series of open reading frames within a bacterial genome that encode for a collection of surface proteins and sortases An Adhesin Island may encode for amino acid sequences comprising at least one surface protein The Adhesin Island may encode at least one surface protein Alternatively, an Adhesin Island may encode for at least two surface proteins and at least one sortase Preferably, an Adhesin Island encodes for at least three surface proteins and at least two sortases One or more of the surface proteins may include an LPXTG motif (e g , 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

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 hyperohgomeπc 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 pneumoniae, 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 AJ 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 oligomeπc, pilus-like structures comprising a Gram positive bacteria AI surface protein The oligomeπc, pilus-like structure may comprise numerous units of the AI surface protein Preferably, the oligomeπc, pilus like structures comprise two or more AI surface proteins Still more preferably, the oligomeπc, pilus-like structure comprises a hyper-oligomeπc pilus-like structure compπsing 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) oligomeπc subumts, wherein each subunit comprises an AI surface protein or a fragment thereof The oligomeπc subumts may be covalently associated via a conserved lysine within a pilin motif The oligomeric subumts 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 oligomeπc, 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 oligomeπc form, preferably in a hyperoligomeric form

The oligomeπc 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 oligomeπc, piius-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 oligomeπc Adhesm Island surface antigen comprising cultuπng a Gram positive bacteria adapted for increased AI protein expression and isolation of the expressed oligomeπc 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 oligomeπc 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 pih on their surface The Gram positive bacteria may be adapted to have increased exposure of ohgomeπc or hyperoligomeric AI proteins on its surface by increasing expression levels of a signal peptidase polypeptide Increased levels of a local signal peptidase expression in Gram positive bacteria (such us LepA in GAS) are expected to result in increased exposure of pih 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 pih 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 Pharyngeal 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 oligomeπc form and on its surface

Alternatively, the ohgomeπc, 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 oligomeπc or hyperoligomeric formation of the AI surface protein subunits Gram positive Al 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

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 ohgomeπc 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 (GASI

As discussed above, Applicants have identified at least six 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, poststreptococcal autoimmune responses are still a major cause of cardiac pathology in children

Group A Streptococcal infection of its human host can generally occur in three phases The first phase involves attachment and/or invasion of the bacteria into host tissue and multiplication of the bacteria within the extracellular spaces Generally this attachment phase begins in the throat or the skin The deeper the tissue level infected, the more severe the damage that can be caused In the second stage of infection, the bacteria 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 inappropriate 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 Pathogenesis, 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 (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 Bacteπol (1990) 172(6) 3310 - 3317 This island is known as the Fibronectin-binding, Collagen-binding T-antigen (FCT) region because it contains, in addition to the T6 coding gene (teeό), members of a family of genes coding for Extra Cellular Matrix (ECM) binding proteins Bessen et al , Infection & Immunity (2002) 70(3) 1159-1167 Several of the protein products of this gene family have been shown to directly bind either fibronectin and/or collagen See Hanski et al , Infection & Immunity (1992) 60(12) 5119-5125, Talay et al , Infection & Immunity (1992( 60(9) 3837-3844, Jaffe et al (1996) 21(2) 373-384, Rocha et al , Adv Exp Med Biol (1997) 418 737-739, Kreikemeyer et al , J Biol Chem (2004) 279(16) 15850 15859, Podbielski et al , MoI Microbiol (1999) 31(4) 1051-64, and Kreikemeyer et al , Int J Med Microbiol (2004) 294(2-3) 177-88 In some cases direct evidence for a role of these proteins in adhesion and invasion has been obtained

Applicants raised antiserum against a recombinant product of the teeό gene and used it to explore the expression of T6 in M6 strain ISS3650 In immunoblot of mutanolysin extracts of this strain, the antiserum recognized, in addition to a band corresponding to the predicted molecular mass of the teeό gene product, very high molecular weight ladders ranging in mobility from about 100 kDa to beyond the resolution of the 3-8% gradient gels used See FIG 163 A, last lane labeled "M6_Tee6 "

This pattern of high molecular weight products is similar to that observed in immunoblots of the protein components of the pill identified in Streptococcus agalactiae (described above) and previously in Corynebactenum diphtheriae Electron microscopy of strain M6 ISS3650 with antisera specific for the product of teeό revealed abundant surface staining and long pilus like structures extending up to 700 nanometers from the bacterial surface, revealing that the T6 protein, one of the antigens recognized in the original Lancefield serotypmg system, is located within a GAS Adhesm Island (GAS AI-I) and forms long covalently linked pilus structures See FIG 1631

In addition to the teeό gene, the FCT region in M6_ISS3650 (GAS AI-I) contains two other genes (prtFl and cpa) predicted to code for surface exposed proteins, these proteins are characterized as containing the cell wall attachment motif LPXTG Western blot analysis using antiserum specific for PrtFl detected a single molecular species with electrophoretic mobility corresponding to the predicted molecular mass of the protein and one smaller band of unknown origin Western blot analysis using antisera specific for Cpa recognized a high molecular weight covalently linked ladder (Fig 163 A, second lane) Immunogold labelling of Cpa with specific antiserum followed by transmission electron microscopy detected an abundance of Cpa at the cell surface and only occasional structures extending from the cell surface (HG 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, Ml and M12 differ See FIG 164 As discussed below, these four FCT regions correlate to four GAS Adhesin Island types (AI-I, AI- 2, AI-3 and AI-4)

Applicants discovery of genes coding for pill 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_SF37O, M5JSS4883 and M12_20010296) representing the other three FCT variants Each gene present in the FCT region of each bacteria was cloned and expressed Antisera specific for each recombinant protein was then used to probe mutanolysin extracts of the respective strains (6) In Ml strain SF370, there are three predicted surface proteins (Cpa (also referred to as M l_126 and GAS 15), Ml_128 (a fϊmbπal protein also referred to as SpyO128 and GAS 16), and Ml_130 (also referred to as Spy0130 and GAS 18)) (GAS Al-2) Antisera specific for each surface protein reacted with a ladder of high molecular weight material (FIG 163B) Immunogold staining of Ml strain SF370 with antiserum specific for Ml_128 revealed pih structures similar to those seen when M6 strain ISS3650 was immunogold stained with antiserum specific for teeό (See Fig 1163K) Antisera specific for surface proteins Cpa and Ml_130 revealed abundant surface staining and occasional structures extending from the surface of Ml strain SF37O bacteria (FIG 163S)

The Ml_128 protein appears to be necessary for polymerization of Cpa and Ml_130 proteins If the Ml_128 gene in Ml_SF370 was deleted, Western blot analysis using antibodies that hybridize to Cpa and Ml_130 no longer detected high molecular weight ladders comprising the Cpa and Ml_130 proteins (FIG 163 E) See also FIGS 177 A-C which provide the results of Western blot analysis of the Ml_128 (Δ128) deleted bacteria using anti-Ml_130 antiserum (FIG 177 A), anti-Ml_128 antiserum (FIG 177 B), and anti-M l_126 antiserum (FIG 177 C) High molecular weight ladders, indicative of pilus formation on the surface of Ml strain 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 Ml_128, Western blot analysis using antisera specific for Cpa and Ml_130 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 Ml_128 antisera (FIG 178 B) Although Δ128 SF37O bacteria were unable to form pih, Ml_126 (cpa) and Ml_130, which contain sortase substrate motifs, were present on the bacteria's surface FACS analysis of the Ml_128 deleted (Δ128) strain SF370 bacteria also detected both Ml_126 and Ml_130 on the surface of the Δ128 strain SF370 bacteria See FIG 179 D and F, which show a shift in fluorescence when antibodies immunoreactive to Ml_126 and Ml_130 are used on Δ128 bacteria As expected, virtually no shift in fluorescence is observed when antibodies immunoreactive to Ml_128 are used with the Δ128 bacteria (FIG 179 E)

By contrast, deletion of the Ml_130 gene did not effect polymerization of Ml_128 (FIG 163 F) See also FIGS 177 A-C, which provide Western blot analysis results of the Ml_130 deleted (Δ130) strain SF37O bacteria using anti- Ml_130 (FIG 177 A), anti-Ml_128 (FIG 177 B), and anti-Ml_l 26 antiserum (FIG 177 C) The anti-Ml_128 and anti- Ml_126 antiserum both detected the presence of high molecular weight ladders in the Δ130 strain SF370 bacteria, indicating that the Δ130 bacteria form pih that comprise Ml_126 and Ml_128 polypeptides in the absence of Ml_130 As expected, the Western blot probed with antiserum immunoreactive with Ml_130 did not detect any proteins for the Δ130 bacteπa(FIG 177A)

Hence, the composition of the pih in GAS resembles that previously described for both C diphtheria (7, S) and S agalactiae (described above) (9) in that each pilus is formed by a backbone component which abundantly stains the pih in EM and is essential for the incorporation of the other components

Also similar to C diphtheria, elimination of the srtCl gene from the FCT region of M1_SF37O abolished polymerization of all three proteins and assembly of pill (FIG 163 G) See also FIGS 177 A-C, which provide Western blot analysis of the SrtCl deleted (ΔSrtCl) strain SF370 bacteria using anti-Ml_130 (FIG 177 A), anti-Ml_128 (FlG 177 B), and anti-Ml_126 antiserum (FIG 177 C) None of the three antisera immunoreacted with high molecular weight structures (pill) in the ΔSrtCl bacteria Confirming that deletion of the SrtCl gene abrogates pilus assembly in strain SF370, immunoelectron microscopy using antisera against Ml_128 failed to detect pilus formation on the bacteria surface See FIG 178 C Although no assembled pill were detected on ΔSrtCl SF37O, Ml_128 proteins could be detected on the surface of SF370 Thus, it appeared that SrtCl deletion prevented pilus assembly on the surface of the SF370 bacteria, but not anchoring of the proteins that comprise pill to the bacterial cell wall FACS analysis of the ΔSrtCl strain SF37O confirmed that deletion of SrtCl does not eliminate cell surface expression of M 1_126, Ml_128 or Ml_130 See FIG 179 G I, which show a shift in fluorescence when antibodies immunoreactive to M 1_126 (FIG 179 G), Ml_128 (FIG 179 H), and M l_130 (FIG 179 1) are used to detect cell surface protein expression on ΔSrtCl bacteria Thus, SrtCl deletion prevents pilus formation, but not surface anchoring of proteins involved in pilus formation on the surface of bacteria Another sortase is possibly involved in anchoring of the proteins to the bacteria surface Pilus polymerization in C diphthenae is also dependent on particular sortase enzyme whose gene resides at the same genetic locus as the pilus components (7 S)

The LepA signal peptidase, SpyO127, also appears to be essential for pilus assembly in strain SF370 LepA deletion mutants (ΔLepA) of strain SF370 fail to assemble pih on the cell surface Not only are the ΔLepA mutants unable to assemble pili, they are also deficient at cell surface Ml expression See FIG 180, which provides a FACS analysis of the wildtype (A) and ΔLepA mutant (B) SF370 bacteria using Ml antisera No shift in fluorescence is observed for the ΔLepA mutant bacteria in the presence of Ml immune serum It is possible that these deletion mutants of LepA will be useful for detecting non-M, non-pih, surface exposed antigens on the surface of GAS, or any Gram positive bacteria These antigens may also be useful in immunogenic compositions

Pih 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 M5JSS4883 (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 M 12 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 pih were visible when antisera against EftLSL A were used (FIG 163 M)

The major pilus forming proteins identified in the four strains studied by applicants (T6, Ml_128, M5_orf80 and EftLSL A) share between 23% and 65% amino acid identity in any pairwise comparison, indicating that each pilus may represent a different 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 pilus proteins was assayed by indirect immunofluorescence and flow cytometry using antibodies specific for the pilus proteins, the bacteria's respective M proteins, or surface proteins not associated with the pili (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 pilus components (T6, Ml_128, M5_orf80 and EftLSL A) identified in the strains of bacteria discussed above Pool U, which contains the T6 serum, recognized the T6 protein specifically (a surface exposed pilus protein from GAS AI-I)(FIG 166 B) Pool T specifically recognized M l_128 (a surface exposed pilus protein from GAS AI 2) (FlG 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- I)(FIG 166 E) and identified M l_128 as antigen Tl (corresponding to a surface exposed pilus protein from GAS AI-2) (FIG 166 D), EftLSL A as antigen T12 (corresponding to a surface exposed pilus protein from GAS AI-4) (FIG 166 G) and M5_orf80 as a common antigen recognized by the related sera T5 T27 and T44 (corresponding to a surface exposed pilus protein from GAS AI-3)

Confirming applicants observations, discussed above, that deleting the Ml_128 gene from Ml_SF370 abolishes pilus formation, the pool T sera stained whole Ml_SF370 bacteria (FIG 166 H) but failed to stain M l_SF370 bacteria lacking the Ml_128 gene (FIG 1661)

As discussed above, Applicants have identified at least six different Group A Streptococcus Adhesm 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-oligomeπc 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 pih may be involved in formation of biofilms Applicants have discovered that the GBS pih structures appear to be implicated in the formation of biofilms (populations of bacteria growing on a surface, often enclosed in an exopolysacchaπde 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 btofilm 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 oligomeπc, 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 AM and AI-2, GAS AI-I and GAS AI-3, GAS AI-I and GAS AI-4, GAS AI-2 and GAS AI-3, GAS AI-2 and GAS AI-4, and GAS AI-3 and GAS AI-4 Preferably the two GAS AI proteins are derived from different T-types

A schematic arrangement of GAS Adhesin Island sequences is set forth in FIG 162 In all strains, the AI region is flanked by the highly conserved open reading frames Ml_123 and Ml-136 Between three and five genes in each locus code for surface proteins containing LPXTG motifs These surface proteins also all belong to the family of genes coding for ECM binding adhesins

Adhesin island sequences can be identified in numerous M types of Group A Streptococcus Examples of AI sequences within Ml , M6, M3, M5, M 12, M 18, 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 Adhesm 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 (e g , 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 fimbπal 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 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 FIG , all GAS strains and serotypes thus far characterized as an AI-I have a SrtB type sortase, all GAS strains and serotypes thus far characterized as an AI-2 have SrtB and SrtCl type sortases, all GAS strains and serotypes thus far characterized as an AI- 3 have a SrtC2 type sortase, and all GAS strains and serotypes thus far characterized as an AI-4 have SrtB and SrtC2 type sortases A comparison of the percentage identity of sequences within the adhesin islands was presented in Table 45, see above

(1) Adhesin Island sequence within M6 GAS Adhesin Island 1 ("GAS AM")

A GAS Adhesin Island within M6 serotype (MGAS10394) is outlined in Table 4 below This GAS adhesin island 1 ("GAS AI-I") comprises surface proteins, a srtB sortase and a rofA divergently transcribed transcriptional regulator.

GAS AI-I surface proteins include SpyO157 (a fibronectin binding protein), SpyO159 (a collagen adhesion protein) and SpyOlόO (a fimbπal structural subunit) Preferably, each of these GAS AI-I surface proteins includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO 122) or LPXSG (SEQ ID NO 134) (conservative replacement of threonine with serine)

GAS AI-I includes a srtB type sortase GAS srtB sortases may preferably anchor surface proteins with an LPSTG motif (SEQ ID NO 166), particularly where the motif is followed by a serine Table 4: GAS AI-I sequences from M6 isolate (MGAS10394)

M6_Spy0160 appears to be present on the surface of GAS as part of oligomeπc (pilus) structures FIGS 127- 132 present electron micrographs of GAS serotype M6 strain 3650 immunogold stained for M6_Spy0160 using anti M6_Spy0160 antiserum Oligomeπc or hyperoligomeπc structures labelled with gold particles can be seen extending from the surface of the GAS in each of these FIGS , indicating the presence of multiple M6_Spy0160 polypeptides in the oligomeπc or hyperoligomeπc 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_SpyO159 antiserum (FIG 176 F) Oligomeπc or hyperoligomeπc structures labelled with gold particles can again be seen extending from the surface of the M6 strain 2724 GAS bacteria immunogold stained for M6_Spy0160 M6_SpyO159 is also detected on the surface of the M6 strain 2724 GAS

FACS analysis has confirmed that the GAS AI-I surface proteins spyM6_0159 and spyM6_0160 are indeed expressed on the surface of GAS FIG 73 provides the results of FACS analysis for surface expression of spyM6_0159 on each of GAS serotypes M6 2724, M6 3650, and M6 2894 A shift in fluorescence is observed for each GAS serotype when anti-spyM6_0159 antiserum is present, demonstrating cell surface expression Table 18, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-spyM6_0159 antiserum, and the difference in fluorescence value between the pre-immune and anti-spyM6_0159 antiserum Table 18 Summary of FACS values for surface expression of spyM6_0159

FIG 74 provides the results of FACS analysis for surface expression of spyM6_0160 on each of GAS serotypes M6 2724, M6 3650, and M62894 In the presence of anti-spyM6_0160 antiserum, a shift in fluorescence is observed for each GAS serotype, which demonstrates its cell surface expression Table 19, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-spyM6_0160 antiserum, and the change in fluorescence value between the pre-immune and anti-spyM6_0160 antiserum Table 19 Summary of FACS values for surface expression of spyM6_0160

Surface expression of M6_SpyO159 and M6_Spy0160 on M6 serotype GAS has also been confirmed by Western blot analysis FIG 98 shows that while pre-immune sera (P cc-0159) does not detect expression of M6_Spy0159 in GAS serotype M6, antι-M6_SpyO159 immune sera (I α 0159) is able to detect M6_SpyO159 protein in both total GAS M6 extracts (M6 tot) and GAS M6 tractions enriched for cell surface proteins (M6 surf prot) The M6_Spy0159 proteins detected in the total GAS M6 extracts or the GAS M6 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that M6_SpyO159 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 veπfy the presence of M6_SpyO159 and M6_Spy0160 in higher molecular weight structures on the surface of GAS FIG 110 provides a Western blot performed to detect M6_SpyO159 and M6_Spy0160 in GAS M6 strain 2724 extracts enriched for surface proteins Antiserum raised against either M6_SpyO159 (Anti-159) or M6_Spy0160 (Anti 160) cross-hybridizes with high molecular weight structures (pill) m these extracts FIG 111 provides a similar Western blot that verifies the presence of M6_ SpyO159 and M6_SpyO16O in high molecular weight structures in GAS M6 strain 3650 extracts enriched for surface proteins

SpyM6_0157 (a fibronectin-bindmg 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 spyM6_0157 on M6 strain 3650 A slight shift in fluorescence is observed, which demonstrates that some spyM6_0157 may be expressed on the GAS cell surface Adhesin Island sequence within M6 GAS Adhesin Island 2 ("GAS AI-2")

A GAS Adhesin Island within Ml serotype (SF370) is outlined in Table 5 below This GAS adhesin island 2 ("GAS AI-2") comprises surface proteins, a SrtB sortase, a SrtCl sortase and a RofA divergently transcribed transcriptional regulator

GAS AI-2 surface proteins include GAS 15 (Cpa), SpyO128 (thought to be a fimbπal protein) and SpyODO (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 srtCl sortase As discussed above, GAS SrtB sortases may preferably anchor surface proteins with an LPSTG (SEQ ID NO 166) motif, particularly where the motif is followed by a serine GAS SrtCl sortase may preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO 167) motif GAS SrtCl may be differentially regulated by RofA

GAS AI-2 may also include a LepA putative signal peptidase I protein Table S : GAS AI-2 sequence from Ml isolate (SF37Q)

GAS 15, GAS 16, and GAS 18 appear to be present on the surface of GAS as part of oligomeπc (pilus) structures FIGS 1 13-1 15 present electron micrographs of GAS serotype M l strain SF370 immunogold stained for GAS 15 using anti-GAS 15 antiserum FIGS 116-121 provide electron micrographs of GAS serotype Ml strain SF370 immunogold stained for GAS 16 using anti-GAS 16 antiserum FIGS 122-125 present electron micrograph of GAS serotype Ml strain SF370 immunogold stained for GAS 18 using anti-GAS 18 antiserum Oligomers of these proteins can be seen on the surface of SF370 bacteria in the immuno-gold stained micrographs

FIG 126 reveals a hyperohgomer on the surface of a GAS serotype Ml strain SF370 bacterium immunogold stained for GAS 18 This long hyperoligomeπc 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 Ml 2719, Ml 2580, Ml 3280, Ml SF37O, Ml 2913, and Ml 3348 A shift in fluorescence is observed for each GAS serotype when anti-GAS 15 antiserum is present, demonstrating cell surface expression Table 20, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immuπe 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

FIGS 76 and 79 provide the results of FACS analysis for surface expression of GAS 16 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348 The FACS data in HG 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, demonstrating its cell surface expression Table 21, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-GAS 16 antiserum, and the change in fluorescence value between the pre-immune and anti-GAS 16 antiserum Table 21 Summary of FACS values for surface expression of GAS 16

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: GAACAAGAGACATCTACTGATAAAGATATGACCATTACTTTTACAAATAAAAAAGATTT

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

FIGS. 77 and 78 provide the results of FACS analysis for surface expression of GAS 18 on each of GAS serotypes Ml 2719, Ml 2580, Ml 3280, Ml SF370, Ml 2913, and Ml 3348 The antiserum 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

Surface expression of GAS 15, GAS 16, and GAS 18 on Ml serotype GAS has also been confirmed by Western blot analysis FIG 89 shows that while pre-immune sera does not detect GAS Ml expression of GAS 15, anti GAS 15 immune sera is able to detect GAS 15 protein in both total GAS Ml extracts and GAS Ml proteins enriched for cell surface proteins The GAS 15 proteins detected in the Ml extracts enriched for surface proteins are also present as high molecular weight structures, indicating that GAS 15 may be in an oligomeπc (pilus) form FIG 90 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 15 antisera Again, the lanes that contain GAS M l extracts enriched for surface proteins (Ml 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 pre- immune 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 Ml expression of GAS 16, anti-GAS 16 immune sera is able to detect GAS 16 protein in GAS Ml extracts enriched for cell surface proteins The GAS 16 proteins detected in the Ml extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 16 may be in an oligomeric (pilus) form FIG 93 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 16 antisera The lanes that contain total GAS Ml protein (Ml tot new and Ml tot old) and the lane that contains GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 16 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 Ml expression of GAS 18, anti-GAS 18 immune sera is able to detect GAS 18 protein in GAS Ml extracts enriched for cell surface proteins The GAS 18 proteins detected in the Ml extracts enriched for surface proteins are present as high molecular weight structures, indicating that GAS 18 may be in an oligomeric (pilus) form FIG 96 also shows the results of Western blot analysis of Ml serotype GAS using anti-GAS 18 antisera The lane that contains GAS Ml extracts enriched for surface proteins (Ml prot sup) show the presence of high molecular weight structures that may be oligomers of GAS 18 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

HGS 102-106 provide additional Western blots to verify the presence of GAS 15, GAS 16, and GAS 18 in high molecular weight structures in GAS Each Western blot was performed using proteins from a different GAS Ml strain, 2580, 2913, 3280, 3348, and 2719 Each Western blot was probed with antisera raised against each of GAS 15, GAS 16, and GAS 18 As can be seen in 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 Ml

FIG 107 provides a similar Western blot performed to detect GAS 15, GAS 16, and GAS 18 proteins in a GAS serotype M 1 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 Ml 8 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 fimbπal protein, a F2 like fibronectin binding protein GAS AI-3 surface proteins may also include a hypothetical surface protein Preferably, each of these GAS AI-3 surface proteins include an LPXTG sortase substrate motit, 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)

Table 7: GAS AI-3 se uence from M3 isolate SSI-I

Table 10: GAS AI-3 sequences from M5 isolate (Manfredo)

Table 8: GAS AI-3 se uences from M18 isolate (MGAS8232

Table 44: GAS AI-3 sequences from M49 isolate (591)

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-hke fibronectin-binding protein of each these type 3 adhesin islands contains a pihn motif and an E-box FIG 60 indicates the amino acid sequence of the pihn motif and E-box of each of GAS AI-3 serotype M3 MGAS315 (SpyM3_0104/21909640), GAS AI-3 serotype M3 SSI (SpsOlOό/28895018), GAS AI-3 serotype M18 (SpyM18_0132/19745307), and GASAI-3 serotype M5 (orf84)

FACS analysis has confirmed that the GAS AI-3 surface proteins SpyM3_0098, SpyM3_0100, SpyM3_0102, and SpyM3_0104 are expressed on the surface of GAS FIG 80 provides the results of FACS analysis for surface expression of SpyM3_0098 on each of GAS serotypes M3 2721 and M3 3135 A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0098 antiserum is present, demonstrating cell surface expression Table 25, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre- immune antiserum, anti-SpyM3_0098 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0098 antiserum Table 25 Summary of FACS values for surface expression of SpyM3_0098

FIG 81 provides the results of FACS analysis for surface expression of SpyM3_0100 on each of GAS serotypes M3 2721 and M3 3135 A shift in fluorescence is observed for each GAS serotype when antι-SpyM3_0100 antiserum is present, demonstrating cell surface expression Table 26, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0100 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0100 antiserum. Table 26: Summary of FACS values for surface expression of SpyM3_0100

FIG 82 provides the results of FACS analysis for surface expression of SρyM3_0102 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0102 antiserum is present, demonstrating cell surface expression. Table 27, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0102 antiserum. Table 27: Summary of FACS values for surface expression of SpyM3_0102 m M3 serotypes

FIG 82 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM3_0102 identified in a different GAS serotype, M6. FACS analysis conducted with the SpyM3_0102 antisera was able to detect surface expression of the homologous SpyM3_0102 antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894. Table 28, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0102 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0102 antiserum. Table 28- Summary of FACS values for surface expression of SpyM3_0102 in M6 serotypes

SpyM3_0102 is also homologous to pilin antigen 19224139 of GAS serotype M12 Antisera raised against SpyM3_0102 is able to detect high molecular weight structures in GAS serotype M12 strain 2728 protein fractions enriched for surface proteins, which would contain the 19224139 antigen. See FIG. 109 at the lane labelled M12 2728 surf prot.

FIG. 83 provides the results of FACS analysis for surface expression of SpyM3_0104 on each of GAS serotypes M3 2721 and M3 3135. A shift in fluorescence is observed for each GAS serotype when anti-SpyM3_0104 antiserum is present, demonstrating cell surface expression. Table 29, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0104 antiserum, and the difference in fluorescence value between the pre-immune and anti-SpyM3_0104 antiserum.

Table 29: Summary of FACS values for surface expression of SpyM3_0104 in M3 serotypes

FIG 83 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SpyM3_0104 identified in a different GAS serotype, M12 FACS analysis conducted with the SpyM3_0104 antisera was able to detect surface expression of the homologous SpyM3_0104 antigen on GAS serotype M12 2728 Table 30, below quantitatively summarizes the FACS fluorescence values obtained for this GAS serotype in the presence of pre-immune antiserum, anti-SpyM3_0104 antiserum, and the difference in fluorescence value between the pre-immune and anti- SpyM3_0l04 antiserum Table 30 Summary of FACS values for surface expression of SpyM3_0104 in an M 12 serotype

FlG 84 provides the results of FACS analysis for surface expression of SPs_0106 on each of GAS serotypes M3 2721 and M3 3135 A shift in fluorescence is observed for each GAS serotype when anti-SPs_0106 antiserum is present, demonstrating cell surface expression Table 31, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SPs_0106 antiserum, and the difference in fluorescence value between the pre-immune and anti-SPs_0106 antiserum Table 31 Summary of FACS values for surface expression of SPs_0106 in M3 serotypes

FIG 84 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to SPs_0106 identified in a different GAS serotype, M 12 FACS analysis conducted with the SPs_0106 antisera was able to detect surface expression of the homologous SPs_0106 antigen on GAS serotype M12 2728 Table 32, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre-immune antiserum, anti-SPs_0106 antiserum, and the difference in fluorescence value between the pre-immune and anti-SPs_0106 antiserum Table 32 Summary of FACS values for surface expression of SPs_0106 in an M 12 serotype

(4) Adhesin Island sequence within M12 GAS Adhesin Island 4 ("GAS AJ-4")

GAS Adhesin Island sequences within M 12 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 fimbπal protein, an F or F2 like fibronectin-bindmg 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)

A schematic of AI-4 serotype M12 is shown in FIG 51 A

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 above See FIG 52B for an amino acid sequence alignment for each of the SrtC2 amino acid 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 ammo acid alignment of the capsular polysaccharide adhesion protein (cpa) of AI-4 serotype M12 (19224135), GAS AI-3 serotype M5 (ORF78), S pyogenes strain MGAS315 serotype M3 (21909634), S pyogenes SSI-I serotype M3 (28810257), S pyogenes MGAS8232 serotype M3 (19745301), and GAS AI-2 serotype Ml (GAS15) The amino acid sequence of the AI-4 serotype M12 cpa shares a high degree of homology with other cpa proteins

FIG 53 shows that the F-like fibronectin-bindmg 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-hke fibronectin-bindmg 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 fimbπal protein of AI-4 serotype M12 (19224137) shares homology with the fimbπal protein of GAS AI-3 serotype M5 (ORF80), and the hypothetical protein of S pyogenes strain MGAS315 serotype M3 (21909636), S pyogenes strain SSI serotype M3 (28810259), S pyogenes strain MGAS8732 serotype M3 (19745303), and S pyogenes strain Ml GAS serotype Ml (13621428)

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 5 pyogenes strain MGAS315 serotype M3 (21909638), S pyogenes strain SSI-I serotype M3 (28810261), GAS AI-3 serotype M5 (ORF82), and S pyogenes strain MGAS8232 serotype M3 (19745305)

The protein F2-hke fibronectin-binding protein of the type 4 adhesin island also contains a highly conserved pilin motif and an E-box FIG. 60 indicates the ammo acid sequence of the pihn motif and E-box in AI-4 serotype M 12

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 M 12 serotype

FlG 85 also provides the results of FACS analysis for surface expression of a pilin antigen that has homology to 19224134 identified in a different GAS serotype, M6 FACS analysis conducted with the 19224134 antisera was able to detect surface expression of the homologous 19224134 antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894 Table 34, below, quantitatively summarizes the FACS fluorescence values obtained for each GAS serotype in the presence of pre immune antiserum, anti 19224134 antiserum, and the difference in fluorescence value between the pre- immune and antι-19224134 antiserum Table 34 Summary of FACS values for surface expression of 19224134 in M6 serotypes

FlG 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 M 12 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

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 M 12 serotype

FlG 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 M 12 serotype

19224139 (designated as orf2) may also be expressed on the surface of GAS serotype M 12 bacteria FlG 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 M 12 serotype GAS has also been confirmed by Western blot analysis FIG 99 shows that while pre-immune sera (P α-4135) does not detect GAS M12 expression of 19224135, anti-19224135 immune sera (1 cc-4135) is able to detect 19224135 protein in both total GAS M 12 extracts (M12 tot) and GAS M12 fractions enriched for cell surface proteins (M 12 surf prot) The 19224135 proteins detected in the total GAS M12 extracts or the GAS M 12 extracts enriched for surface proteins are also present as high molecular weight structures indicating that 19224135 may be in an oligomers (pilus) form See also FIG 108, which provides a further Western blot showing that anti-19224135 antiserum (Antι-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 FlG 100 shows that while pre-immune sera (P α-4137) does not detect GAS M12 expression of 19224137, anti-19224137 immune sera (I α-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 M 12 extracts enriched for surface proteins are also present as high molecular weight structures, indicating that 19224137 may be in an oligomeπc (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 ammo 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 (e g , SEQ ID NO 122) or other sortase substrate motif One or more 5 pneumoniae AI surface proteins may participate in the formation of a pilus structure on the surface of the S pneumoniae bacteria

S pneumoniae Adhesin Islands of the invention preferably include a divergently transcribed transcriptional regulator The transcriptional regulator may regulate the expression of the S pneumoniae AI operon

The S pneumoniae AI surface proteins may bind or otherwise adhere to fibrinogen, fibronectin, or collagen

A schematic of the organization of a S pneumoniae AI locus is provided in 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 5 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

The full-length nucleotide sequence of the 5 pneumoniae strain 670 AI is also shown in FIG 101, as ts 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

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 139 A 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 5 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 CSRlO) 9V Spain 3 (9VSP3), 19F Taiwan 14 ( 19FTW 14), 23F Taiwan 15 (19FTW 15) and 23F Poland 16 (23FP16) See FlG 140

The AI locus has been sequenced for each ot 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 ot 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 5 pneumoniae strain 670 and TIGR4 adhesin islands Table 39: Percent identity comparison of S. pneumoniae strains AI sequences

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 Al-b 5 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 5 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 5. pneumoniae AI locus

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 5. 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 5. pneumoniae strains that comprise the adhesin island locus depicted in FIG. 137, these strains are indicated as rlrA+. Confirming these findings, electron microscopy and negative staining detects the presence of pili extending from the surface of S. pneumoniae. See 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 SpO463 (S. pneumoniae TIGR4 rrgB) adhesion island polypeptide is expressed in oligomeric form. Whole cell extracts were analyzed by Western blot using a SpO463 antiserum. The antiserum cross-hybridized with high molecular weight SpO463 polymers. See 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 TIGR 4 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 5. 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 5. 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, SpO462 (rrgA), SpO463 (rrgB), and SpO464 (rrgC), have been cloned and expressed. See examples 15-17. A polyacrylamide gel showing successful recombinant expression of RrgA is provided in 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.

In addition to the identification of these S. pneumoniae adhesion islands, coding sequences for SrtB type sortases have been identified in several S. pneumoniae clinical isolates, demonstrating conservation of a SrtB type sortase across these isolates. Recombinantlv Produced AI polypeptides

It is also an aspect of the invention to alter a non-AI polypeptide to be expressed as an AI polypeptide The non- AI polypeptide may be genetically manipulated to additionally contain AI polypeptide sequences, e g , a sortase substrate, pilin, or E-box motif, which may cause expression of the non-AI polypeptide as an AI polypeptide Alternatively the non-AI polypeptide may be genetically manipulated to replace an amino acid sequence within the non-AI polypeptide for AI polypeptide sequences, e g , a sortase substrate, pilin, or E-box motif, which may cause expression of the non-AI polypeptide as an AI polypeptide Any number of amino acid residues may be added to the non-AI polypeptide or may be replaced within the non-AI polypeptide to cause its expression as an AI polypeptide At least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 75, 100, 150, 200, or 250 amino acid residues may be replaced or added to the non-AI polypeptide amino acid sequence GBS 322 may be one such non-AI polypeptide that may be expressed as an AI polypeptide GBS Adhesin Island Sequences

The GBS AI polypeptides of the invention can, of course, be prepared by various means (e g recombinant expression, purification from GBS, chemical synthesis etc ) and in various forms (e g native, fusions, glycosylated, non- glycosylated etc ) They are preferably prepared in substantially pure form (ι 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=l

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%, 97%, 98%, 99%, 995% 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 vary depending on the polynucleotide sequence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, (e g at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more)

The 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 ammo 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 I, 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 ammo acid residue may be deleted Other fragments omit one or more domains of the protein (e g omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain) GBS 80 Examples of preferred GBS 80 fragments are discussed below Polynucleotide and polypeptide sequences of GBS 80 from a variety of GBS serotypes and strain isolates are set forth in HGS 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

GCTTTTGCTGTTAAGGGGATGAAGCGTCGTACAAAAGATAAC

SEQ ID NO:2

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNY

NSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDY EKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATAN TDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLA SDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIE FTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIP-WrGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN

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-termmal 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

AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEA

ADAKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPK

NWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTID

EPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTP

DKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSH

TDGTFEIKGLAYAVDANAEGTAVTYKLKETK?

TGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN 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

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNY AKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYLYVEDLK

EKFEITDKF ADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATAN TDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDK^ SDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDG FTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPWTG

GBS 80 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:5 IPNTG (shown in italics in SEQ ID NO 2 above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 80 protein from the host cell. Accordingly, in one preferred fragment of GBS 80 for use in the invention, the transmembrane and/or cytoplasmic regions and the cell wall anchor motif are removed from GBS 80 An example of such a GBS 80 fragment is set forth below as SEQ ID NO:6.

SEQ ID NO:6

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNY AKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYL YVEDLK NSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDY EKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATAN TDDAAFLEIPVASTINEKAVLGKAI ENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLA SDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIE FTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS

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

AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEA AD AKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYL YVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPK

EPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTP DKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSH TDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVI PDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS

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

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMS IVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNY

AKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYLYVEDLK

EKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATAN TDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLA SDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIE FTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPWTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN SEQ ID NO:8

AEVSQERPAKTTVNI YKLQADSYKSEITSNGGIENKDGEVISNY AKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEA

NWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTI PANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTID EPTVDNQNTLKITFKPEKFKEIAELLKG

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

MTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKD

STETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLK ETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPS

Both an Active Maternal Immunization Assay and a Passive Maternal Immunization Assay were conducted on this collection of proteins

As used herein, an Active Maternal Immunization assay refers to an in vivo protection assay where female mice are immunized with the test antigen composition The female mice are then bred and their pups are challenged with a lethal dose of GBS Serum titers of the female mice during the immunization schedule are measured as well as the survival time of the pups after challenge

Specifically, the Active Maternal Immunization assays referred to herein used groups of four CD-I female mice (Charles River Laboratories, Calco Italy) These mice were immunized intrapeπtoneally with the selected proteins in Freund's adjuvant at days 1, 21 and 35, prior to breeding 6-8 weeks old mice received 20 μg protein/dose when immunized with a single antigen, 30-45 μg protein/dose (15 μg each antigen) when immunized with combination of antigens The immune response of the dams was monitored by using serum samples taken on day 0 and 49 The female mice were bred 2-7 days after the last immunization (at approximately 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 50μl of THB medium Preferably, the pup challenge takes place at 56 to 61 days after the first immunization The challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB prior to use Survival of pups was monitored for 5 days after challenge

As used herein, the Passive Maternal Immunization Assay refers to an in vivo protection assay where pregnant mice are passively immunized by injecting rabbit immune sera (or control sera) approximately 2 days before delivery The pups are then challenged with a lethal dose of GBS

Specifically, the Passive Maternal Immunization Assay referred to herein used groups of pregnant CDl mice which were passively immunized by injecting 1 ml of rabbit immune sera or control sera via I P , 2 days before delivery Newborn mice (24-48 hrs after birth) are challenged via I P with a 70 - 90% lethal dose of GBS serotype III COHl The challenge dose, obtained by diluting a frozen mid log phase culture, was administered in 50μl 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

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, pihn motifs, containing conserved lysine (K) residues have been identified in GBS 80 The pihn 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 ammo acid residues 368 and 375 The pilm sequences, in particular the conserved lysine residues, are thought to be important for the formation of ohgomeric, pilus-hke structures of GBS 80 Preferred fragments of GBS 80 include at least one conserved lysine residue Preferably, fragments include at least one pihn sequence SEQ ID NO:2

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNY AKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYLYVEDLK NSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDY

TDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVΉTGGKRFVKKDSTETQTLGGAEFDLLA SDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIE FTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN

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 oligomeπc 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

MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNY

AKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSKSNVRYLYVEDLK NSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDY EKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATAN TDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLA SDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIE FTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN

GBS 104 Similarly, the following offers examples of preferred GBS 104 fragments. Nucleotide and amino acid sequences of GBS 104 sequenced from serotype V isolated strain 2603 are set forth below as SEQ ID NOS 10 and 1 1 : SEQ ID NO. 10

ATTGGTACAATTGTCTATATATTAGTTGGTTCTACTTTTATGATACTTACCATTTGTTCTTTCCGTCGTAAACAATTG

SEQ ID NO. 11

MKKROKIWRGLSVTLLILSOIPFGILVOGETODTNOALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEAT FENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATI IEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKV GEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVWLLDNSNSMNNERANNSQ RALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTND ANEWILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQN QFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLY WRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVD

QKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKT KPWTFT I QNGEVTNLKAD PNANKNOIGYLEGNGKHLITNTPKRPPGVFPJCTGGIGTIVYILVGSTFMILTICSFRRKOL

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 amino acid sequences from the leader or signal sequence region of GBS 104 are removed. An example of such a GBS 104 fragment is set forth below as SEQ ID NO 12.

SEQ ID NO 12

GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWK

TGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVWLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVT YASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTND ANEVNILKSRIPKEAEHINGDRTLYQFG ATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQ IVKGDGESFKLFSDRKVPVTGGTTQAA YRVPQNQLSVMSNEGYAINSGYIYL YWRD YNWVYPFDPKTKKVSATKQIKTHGE PTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNV TDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKWLTYD

QIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPWTFTIQNGEVTNLKADPNANKNQIG YLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL 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 1 1 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

MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEAT FENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKV GEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVWLLDNSNSMNNERAlNlNSQ RALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTND ANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQN QFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLY WRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVD DTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVT VTYDKTSQTIKINHLNLGSGQKWLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISN QKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKT KPWTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNT

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

GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWK

VKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKI TGVNDLDKNKYKIELTVEGKTTVETKELNQPLDWVLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVT YASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQFG ATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQ

IVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGE PTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNV TDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKWLTYD VRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESLLGAKFQL QIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPWTFTIQNGEVTNLKADPNANKNQIG YLEGNGKHLITNT

GBS 104, like GBS 80, contains an ammo 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 invention, only the transmembrane and/or cytoplasmic regions and the cell wall anchor motif are removed from GBS 104 Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two pihn 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 oligomeπc, pilus-hke 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

MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSGEAT

GEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQ RALKAGEAVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTND ANEVNILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQN QFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLY

VTYDKTSQTIKINHLNLGSGQKWLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISN QKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDGYIEVKT KPWTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL 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 1 1 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 oligomeπc 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

MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVL KNDNDKSETSHETVEGSGEAT

FENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATI IEGMDADKAEKRKEVLNAOYPKSAIYEDTKENYPLVNVERSKV GEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDWVLLDNSNSMNNERANNSQ RALKAGEAVEKLIDKITSNKDNRVAL VTYASTIFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTND ANEWILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTSYQN QFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAA YRVPQNQLSVMSNEGYAINSGYIYLY WRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVD DTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVT

OKKMGEVEFIKVNKDKHSESLLGAKFOLOIEKDFSGYKOFVPEGSDVTTKNDGKIYFKALODGNYKLYEISSPDGYIEVKT KPWTFTIQNGEVTNLKAD PNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL

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

AAATCTAGTGATATGTCCATCAAAAAAGAT

SEQ ID NO:16

MRKYQKFSKILTLSLFCLSQI PLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGE ATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYP PTGIYEDTKESYKLEHVK GSVPNGKSEAKA VNPYSSEGEHIREI PEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNN DGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLT NNAEEI IKRI PTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTM KAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAIN NFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQI ISGNLQKLHYLDLNLNYPKGTIYRNGP VKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIV TSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGG PNNDGGI LKGVKLEYIGNKL YVRGLNLGEGQKVTLTYDVKLDDSFI SNKFYDTNGRTTLNPKSEDPNTLRDFPIPKI RDVREYPTITI KNEKKLGEIEFIKVD KDNNKLLLKGATFELQEFNEDYKL YLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPEDYQK ITNKPILTFEWKGSIKNI IAVNKQISEYHEEGDKHLITNTHIPPKGIJPMTGGKGILSFILIGGAMMSIAGGIYIWKRYK KSSDMSIKKD

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 transmembrane region An example of such a GBS 067 fragment is set forth below as SEQ ID NO 17 SEQ ID NO:17

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGE ATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVK GSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNN DGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLT NNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAIN

NFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGP VKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIV TSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGI LKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITI KNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPEDYQK ITNKPILTFEWKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILS

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

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGE ATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVK GSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNN DGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLT NNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAIN NFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGP VKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIV TSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGI LKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITI KNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPEDYQK ITNKPILTFEWKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGI

Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Three pilin motifs, containing conserved lysine (K) residues have been identified in GBS 67 The pilin motif sequences are underlined in SEQ ID NO 16, below Conserved lysine (K) residues are marked in bold, at amino acid residues 478 and 488 at amino acid residues 340 and 342, and at amino acid residues 703 and 717 The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, 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

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGE ATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVK GSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNN DGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLT NNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAIN NFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGP VKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIV TSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGI

KNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPEDYQK ITNKPILTFEWKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYK KSSDMSIKKD

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 oligomeπc 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

MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHPESKIEKVTAELTGE ATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYEDTKESYKLEHVK GSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVGDLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNN DGPNFQRHNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLT NNAEEIIKRIPTEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTRSYAIN NFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGP VKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEITELMRSFSSKPEYYTPIV TSADTSNNEILSKIQQQFETILTKENSIVNGTIEDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGI

LKGVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITI

KNEKKLGEIEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPEDYQK ITNKPILTFEWKGSIKNIIAVNKQISEYHEEGDKHLITNTHIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYK KSSDMSIKKD

Predicted secondary structure for the GBS 067 amino acid sequence is set forth in FIG 33 As shown in this

FIG , 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 oligomeπzation 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 (pfamO5738) 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 extracellular proteins such as integπns and it thought to mediate adhesion, including adhesion to 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

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

GATTCTTTTCAT

SEQ ID NO:126

MKRINKYFAMF SALLLTLTSLLSVAPAF ADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKGGAISDLAGYFGEGS KEIEGAFFALALKEDKSGKVQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKGLKGEFKIVEVKS

LEKAAKTADIEFTLTYSATVNGQAI IDNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNWYTLKD EPKVETHGKKFVKTNEQGDRLAGAQFWKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYNKLSATDQKGEKGITAKE NiDYVANSNQKDATRVENKKVT JPQTGGIGTILFTI IGLSIMLGAWIMKRRQSKEA

Nucleotide and amino acid sequences of GBS 59 sequenced from serotype V isolated strain CJB l 11 are set forth below as SEQ ID NOS- 127 and 128 The GBS 59 polypeptide of SEQ ID NO:128 is referred to as BO1575 SEQ ID NO:127

SEQ ID NO:128

MKKINKCLTMFSTLLLILTSLFSVAPAF ADDATTDTVTLHKIVMPQAAFDNFTEGTKGKNDSDYVGKQINDLKSYFGSTDA KEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKSNYDNNGSILADS KAVPVKITLPLVNNQGWKDAHIYPKNTETKPQVDKNFADKDLDYTDNRKDKGWSATVGDKKEYIVGTKILKGSDYKKLV WTDSMTKGLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDKDVEIKITYSATVNGSTTVEIPETN DVKLDYGNNPTEESEPQEGTPANQEIKVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEHTFTGL DNAKTYRWERVSGYTPEYVSFKNGWTIKNNKNSND PTPINPSEPKWTYGRKFVKTNQANTERLAGATFLVKKEGKYLA

SNAGGQFEITGLDKGTYGLEETQAPAGYATLSGDVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVT JPOTGGIGT ILFTIIGLSIMLGAWIMKKRQSEEA

The GBS 59 polypeptides contain an amino acid motif indicative of a cell wall anchor' SEQ ID NO:129 IPQTG (shown in italics in SEQ ID NOS: 126 and 128 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 59 protein from the host cell. Alternatively, in some recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the Final composition

Pilin motifs, containing conserved lysine (K) residues have been identified in the GBS 59 polypeptides. The pilin motif sequences are underlined in each of SEQ ID NOS: 126 and 128, below. Conserved lysine (K) residues are marked in bold. The conserved lysine (K) residues are located at amino acid residues 202 and 212 and amino acid residues 489 and 495 of SEQ ID NO: 126 and at amino acid residues 188 and 198 of SEQ ID NO: 128. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus- like structures of GBS 59 Preferred fragments of GBS 59 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence

SEQ ID NO:126

MKRINKYFAMFSALLLTLTSLLSVAPAF ADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKGGAISDLAGYFGEGS

LEKAAKTADIEFTLTYSATVNGQAIIDNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNWYTLKD

EPKVETHGKKFVKTNEQGDRLAGAQFWKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYNKLSATDQKGEKGITAKE LIKTKQADYDAAFIEARTAYEWITDKARAITYTSNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFWNQGSYITGG NIDYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAWIMKRRQSKEA

SEQ ID NO:128

KEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNT AKLKGIYQIVELKEKSNYDNNGSILADS KAVPVKITLPLVNNQGWKDAHIYPKNTETKPQVDKNFADKDLDYTDNRKDKGWSATVGDKKEYIVGTKILKGSDYKKLV WTDSMTKGLTF^πSlNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDKDVEIKITYSATVNGSTTVEIPETN DVKLDYGNNPTEESEPQEGTPANQEIKVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEHTFTGL DNAKTYRWERVSGYTPEYVSFKNGWTIKNNKNSND PTPINPSEPKWTYGRKFVKTNQANTERLAGATFLVKKEGKYLA RKAGAATAEAKAAVKTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANYSYEWVADKKADNVVKLI SNAGGQFEITGLDKGTYGLEETQAPAGYATLSGDVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGT ILFTIIGLSIMLGAWIMKKRQSEEA

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 ammo 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 oligomeπc 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

MKRINKYFAMFSALLLTLTSLLSVAPAF ADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKGGAISDLAGYFGEGS KEIEGAFFALALKEDKSGKVQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKGLKGEFKIVEVKS

LEKAAKTADIEFTLTYSATVNGQAI IDNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNWYTLKD

EPKVETHGKKFVKTNEQGDRLAGAQFWKNSAGKYLAL KADQSEGQKTLAAKKIALDEAIAA YNKLSATDQKGEKGITAKE LIKTKQADYDAAFIEARTAYEWITDKARAITYTSNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFWNQGSYITGG NIDYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAWIMKRRQSKEA

SEQ ID NO:128

MKKINKCLTMFSTLLLILTSLFSVAPAF ADDATTDTVTLHKIVMPQAAFDNFTEGTKGKNDSDYVGKQINDLKSYFGSTDA KEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKSNYDNNGSILADS KAVPVKITLPLVNNQGWKDAHIYPKNTETKPQVDKNFADKDLDYTDNRKDKGWSATVGDKKEYIVGTKILKGSDYKKLV WTDSMTKGLTFNN]WKVTLDGEDFPVLI^YKLVTDDQGFRLALNATGLAAVAAAAKDKDVEIKITYSATVNGSTTVEIPETN DVKLDYGNNPTEESEPQEGTPANQEIKVI KDWAVDGTITDANVAVKAI FTLQEKQTDGTWVNVASHEATKPSRFEHTFTGL DNAKTYRWERVSGYTPEYVSFKNGWTIKNNKNSNDPTPINPSEPKWTYGRKFVKTNQANTERLAGATFLVKKEGKYLA RKAGAATAEAKAAVKTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANYSYEWVADKKADNWKLI SNAGGQFEITGLDKGTYGLEETQAPAGYATLSGDVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGT ILFTIIGLSIMLGAWIMKKRQSEEA

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.

Opsonophagocytosis assays also demonstrated that antibodies against BO1575 are opsonic for GBS serotype V, strain CJB l I l. 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

TCAAAACATCGGAAACATCAAAATAAGGAT

SEQ ID NO: 21

MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVLYQLYQLKSTEDGDLLAH WNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKIIWSTGELDLLKVG VDGDTKKPLAGWFELYEKNGRTPIRVKNGVHSQDIDAAKHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVT IEKSKTVTVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKHRKHQNKD

GBS 52 contains an ammo acid motif indicative of a cell wall anchor: SEQ ID NO:124 JPKTG (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

MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVLYQLYQLKSTEDGDLLAH

WNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKI IWSTGELDLLKVG VDGDTKKPLAGWFELYEKNGRTPIRVKNGVHSQDIDAAKHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVT IEKSKTVTVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKHRKHQNKD 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)₁ 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

MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVL YQL YQLKSTEDGDLLAH WNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKI IWSTGELDLLKVG

IEKSKTVTVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKHRKHQNKD SAG0647

Examples of polynucleotide and amino acid sequences for SAG0647 are set forth below. SEQ ID NO:22 and 23 represent SAG0647 sequences from GBS serotype V, strain isolate 2603. SEQ ID NO:22

CAAAATAGTCACAATAATTCGAAATAA

SEQ ID NO:23

MGQKSKISLATNIRIWIFRLIFLAGFLVLAFPIVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKT NGEYPALKDPYSAEQKQAGWEYARML EVKEQIGHVIIPRINQDIPIYAGSAE ENLQRGVGHLEGTSLPVGGESTHAVLTA HRGLPT AKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDL YVIQGEDHVTLLTCTPYMINSHRLLVRGKRIP YVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNSHNNSK

SAG0648

Examples of polynucleotide and amino acid sequences for SAG0648 are set forth below. SEQ ID NO:24 and 25 represent SAG0648 sequences from GBS serotype V, strain isolate 2603. SEQ ID NO: 24

ATTGGACTCTTCATCGTGATAATGATGAGAAGATGGATGCAACATCGTCAATAA

SEQ ID NO: 25

MGSLILLFPIVSQVSYYLASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLIDPFTSKQKEGLREYARMLEVH EQIGHVAI PSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQIFYVTNIKET LAYKWSIKWD PT ALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEAEKHKEQTVQDYRLSLVLKILLVLL IGLFIVIMMRRWMQHRQ

GBS 150 Examples of polynucleotide and amino acid sequences for GBS 150 are set forth below SEQ ID NO 26 and 27 represent GBS 150 sequences from GBS serotype V, strain isolate 2603 SEQ ID NO: 26

AAAAATAGCAAATCTGAAAGAAACGATACAGTA

SEQ ID NO: 27

MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGISYRLWTVTDNLKVDLLSQMTDSELNQKY KSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLIKYTKEGKIKKRL SGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGEIEVEGLLPGKYIFREAKALTGYRISMKDAWAWANKTQEVE VENEKETPPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNSKSERNDTV

GBS 150 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:130 LPKTG (shown in italics in SEQ ID NO 27 above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GBS 150 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

As discussed above, a pihn motif, containing a conserved lysine (K) residue has been identified in GBS 150 The pihn motif sequence is underlined in SEQ ID NO 27, below Conserved lysine (K) residues are marked in bold, at amino acid residues 139 and 148 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of ohgomeπc, pilus-hke structures of GBS 150 Preferred fragments of GBS 150 include a conserved lysine residue Preferably, fragments include the pihn sequence

SEQ ID NO: 27

MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGISYRLWTVTDNLKVDLLSQMTDSELNQKY KSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLIKYTKEGKIKKRL SGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGEIEVEGLLPGKYIFREAKALTGYRISMKDAWAWANKTQEVE VENEKETPPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNSKSERNDTV

An E box containing a conserved glutamic residue has also been identified in GBS 150 The E box motif is underlined in SEQ ID NO 27 below The conserved glutamic acid (E), at amino acid residue 216, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of ohgomeπc pilus-hke structures of GBS 150 Preferred fragments of GBS 150 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO: 27

MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGISYRLWTVTDNLKVDLLSQMTDSELNQKY KSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLIKYTKEGKIKKRL SGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGEIEVEGLLPGKYIFREAKALTGYRISMKDAWAWANKTQEVE VENEKETPPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNSKSERNDTV

SAG1405

Examples of polynucleotide and amino acid sequences for SAG1405 are set forth below SEQ ID NO 28 and 29 represent SAG1405 sequences from GBS serotype V, strain isolate 2603 SEQ ID NO: 28

CTTATATTAGTCGCTTTACTATTATATTTAAAACGAAAATTTAAAGAGAGAAAGAGAAAGGGAAATC AAAAATGA

SEQ ID NO: 29

MGGKFQKNLKKSWLNRWMNVGLILLFLVGLLITSYPFISNWYYNIKANNQVTNFDNQTQKLNTKEINRRFELAKAYNRTL DPSRLSDPYTEKEKKGIAEYAHMLEIAEMIGYIDIPSIKQKLPIYAGTTSSVLEKGAGHLEGTSLPIGGKSSHTVITAHRG LPKAKLFTDLDKLKKGKIFYIHNIKEVLA YKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLL VRGHRIKYVP PVKEKNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK

SAG1406

Examples of polynucleotide and amino acid sequences for SAG 1405 are set forth below. SEQ ID NO:30 and 31 represent SAG1405 sequences from GBS serotype V, strain isolate 2603. SEQ ID NO:30

TTACTTTGGTTAATAAAACGTCAACGTCAAAAAAATCGTTTAGCAAGTGTTAGAAAAGGAATTGAATCATAA

SEQ ID NO: 31

MKTKKIIKKTKKKKKSNLPFIILFLIGLSILLYPWSRFYYTIESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNV HLEDPYEKKRIQKGVAEYARML EVSEKIGTISVPKIGQKLPIFAGSSQEVLSKGAGHLEGTSLPIGGNSTHTVITAHSGIP DKELFSNLKKLKKGDKFYIQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMINTHRLLVRGHRIPYKGPI DEKLIKDGHLNTIYRYLFYISLVIIAWLLWLIKRQRQKNRLASVRKGIES

01520

An example of an amino acid sequence for 01520 is set forth below. SEQ ID NO:32 represents a 01520 sequence from GBS serotype III, strain isolate COHl.

SEQ ID NO: 32

MIRRYSANFLAILGIILVSSGIYWGWYNINQAHQADLTSQHIVKVLDKSITHQVKGSENGELPVKKLDKTDYLGTLDIPNL KLHLPVAANYSFEQLSKTPTRYYGSYLTNNMVICAHNFPYHFDALKNVDMGTDVYFTTTTGQIYHYKISNREIIEPTAIEK VYKTATSDNDWDLSLFTCTKAGVARVLVRCQLIDVKN

01521

An example of an amino acid sequence for 01521 is set forth below. SEQ ID NO:33 represents a 01521 sequence from GBS serotype III, strain isolate COHl.

SEQ ID NO: 33

MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNK YNNQGIAGGNLLVYQVAQAKDVDGNQVFTLTTPFQGIGIKD

FEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWPIPILIMSGLLCLIIALKWRRRRD 01521 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO.132 LPFTG (shown in italics in SEQ ID NO 33 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 01521 protein from the host cell. Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two pilin motifs, containing conserved lysine (K) residues have been identified in 01521. The pilin motif sequences are underlined in SEQ ID NO:33, below. Conserved lysine (K) residues are marked in bold, at amino acid residues 154 and 165 and at amino acid residues 174 and 188. The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of 01521. Preferred fragments of 01521 include at least one conserved lysine residue. Preferably, fragments include at least one pilin sequence.

SEQ ID NO: 33

MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGNLLVYQVAQAKDVDGNQVFTLTTPFQGIGIKD DDLTQVI^DSNQAKYVNLLTKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFILSIPKDGKYDITA FEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWPIPILIMSGLLCLIIALKWRRRRD

An E box containing a conserved glutamic residue has also been identified in 01521. The E box motif is underlined in SEQ ID NO:33 below. The conserved glutamic acid (E), at ammo acid residue 177, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of 01521. Preferred fragments of 01521 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.

SEQ ID NO: 33

MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGNLLVYQVAQAKDVDGNQVFTLTTPFQGIGIKD

DDLTQVNLDSNQAKYVNLLTKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFILSIPKDGKYDITA

FEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWPIPILIMSGLLCLIIALKWRRRRD

01522

An example of an amino acid sequence for 01522 is set forth below. SEQ ID NO:34 represents a 01522 sequence from GBS serotype III, strain isolate COHl.

SEQ ID NO: 34

MAYPSLANYWNSFHQSRAIMDYQDRVTHMDENDYKKIINRAKEYNKQFKTSGMKWHMTSQERLDYNSQLAIDKTGNMGYIS IPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWTVSILNETYTYQVDQ IRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALWAEAIQIEPIYIAPFIAIFLTLILLLISL EVTRRARQRKKILKQAMRKEENNDL

01523

An example of an amino acid sequence for 01523 is set forth below. SEQ ID NO:35 represents a 01523 sequence from GBS serotype III, strain isolate COHl.

SEQ ID NO: 35

MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKDGASYLIPQGKEAEY KASTDFNSLFTTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYYVSSTVNNGAVIM VTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNYHGTEKVYQYVIKDTMPSASWDLNEGSYEVT ITDGSGNITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFYKGINTITVTYTGVLKSGAKPGSAD LPENTNIATINPNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTEANATEYTTGA

VIGAGIVLVARRRLRS

01523 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:131 LPSTG (shown in italics in SEQ ID NO:35 above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 01523 protein from the host cell. Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

An E box containing a conserved glutamic residue has also been identified in 01523 The E box motif is underlined in SEQ ID NO 35 below The conserved glutamic acid (E), at amino acid residue 423, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of 01523 Preferred fragments of 01523 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO: 35

MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKDGASYLIPQGKEAEY KASTDFNSLFTTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYYVSSTVNNGAVIM VTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNYHGTEKVYQYVIKDTMPSASVVDLNEGSYEVT ITDGSGNITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFYKGINTITVTYTGVLKSGAKPGSAD LPENTNIATINPNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTEANATEYTTGA DGIITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTNSDNLLVNPTVENNKGTELPSTGGIGTTIFYIIGAIL VIGAGIVLVARRRLRS

01524

An example of an amino acid sequence for 01524 is set forth below SEQ ID NO'36 represents a 01524 sequence from GBS serotype III, strain isolate COHl

SEQ ID NO: 36

MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEETKTDDVGITLENKNSSQVTSSTSSSQSSVEQ SKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKKALTEVAKQDSEI KNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDENLKWHFKDDGQTEVLKSKDTAETKNTSSDVAFKTDSFSIYAI VQEDNTEVPRLTYHFQNNDGTDYDFLTASGMQVHHQIIKDGESLGEVGIPTIKAGEHFNGWYTYDPTTGKYGDPVKFGEPI TVTETKEICVRPFMSKVATVTLYDDSAGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLSESEIQ ALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPASTIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTK DTTLYAHWSPAQTTYTINYWQQSATDNKNATDAQKTYEYAGQVTRSGLSLSNQTLTQQDINDKLPTGFKVNNTRTETSVMI KDDGSSWNVYYDRKLITIKFAKYGGYSLPEYYYSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGM SYLGEFILPNDTVDSDVIKLFPKGNIVQTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQ YASQTSAGVKVPISDEYYDRYGAYHKDYLNLWWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYAPDTTTVQPKP

SRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYAGWQKVTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYI QDPSGTYYYKYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPDGSIRPYNFSGAVTQDINLRAIW RKAGDYHIIYSNDAVGTDGKPALDASGQQLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSIDID

DLADTGRVEFTAGQSIGIDNNPDATNTLYAVWQPKEYTVRVSKTWGLDEDKTKDFLFNPSETLQQENFPLRDGQTKEFKV PYGTSISIDEQAYDEFKVSESITEKNLATGEADKTYDATGLQSLTVSGDVDISFTNTRIKQKVRLQKVNVENDNNFLAGAV FDIYESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPGYLLPKNDISVLVISTGVTFEQNGNNAT PIKENLVDGSTVYTFKITNSKGTELPSTGGIGTHIYILVGLALALPSGLILYYRKKI

01524 contains an amino acid motif indicative of a cell wall anchor- SEQ ID NO:131 LPSTG (shown in italics in SEQ ID NO:36 above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 01524 protein from the host cell. Alternatively, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Three pilin motifs, containing conserved lysine (K) residues have been identified in 01524. The pilin motif sequences are underlined in SEQ ID NO 36, below Conserved lysine (K) residues are marked in bold, at amino acid residues 128 and 138, amino acid residues 671 and 682, and amino acid residues 809 and 820 The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures of 01524. Preferred fragments of 01524 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO: 36 MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEETKTDDVGITLENKNSSQVTSSTSSSQSSVEQ SKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVTVTLTKEAKI PKNADLKVTELKDNSATFKDYKKKALTEVAKQDSEI

VQEDNTEVPRLTYHFQNNDGTDYDFLTASGMQVHHQI IKDGESLGEVGI PTIKAGEHFNGWYTYDPTTGKYGDPVKFGEPI TVTETKEICVRPFMSKVATVTLYDDSAGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLSESEIQ ALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPASTIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTK DTTLYAHWSPAQTTYTINYWQQSATDNKNATDAQKTYEYAGQVTRSGLSLSNQTLTQQDINDKL PTGFKVNNTRTETSVMI

KDDGSSWNVYYDRKLITIKFAKYGGYSLPEYYYSYNWSSDADTYTGL YGTTLAANGYQWKTGAWGYLANVGNNQVGTYGM SYLGEFILPNDTVDSDVIKLFPKGNIVQTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQ YASQTSAGVKVPISDEYYDRYGAYHKD YLNL WWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYAPDTTTVQPKP SRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYΆGWQKVTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYI QDPSGTYYYKYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPDGSIRPYNFSGAVTQDINL RAIW RKAGDYHIIYSNDAVGTDGKPALDASGQQLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSIDID AHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNWTQVETPRMELNSTTTIPENQYFTRTGYNLIGWHHDK DLADTGRVEFTAGQSIGIDNNPDATNTLYAVWQPKEYTVRVSKTWGLDEDKTKDFLFNPSETLQQENFPLRDGQTKEFKV PYGTSISIDEQAYDEFKVSESITEKNLATGEADKTYDATGLQSLTVSGDVDISFTNTRIKQKVRLQKVNVENDNNFLAGAV FDI YESDANGNKASHPMYSGLVTNDKGLLLVD ANNYLSLPVGKYYLTETKAPPGYLLPKNDISVLVISTGVTFEQNGNNAT PIKENLVDGSTVYTFKITNSKGTELPSTGGIGTHIYILVGLALALPSGLILYYRKKI

An E box containing a conserved glutamic residue has also been identified in 01524 The E box motif is underlined in SEQ ID NO 36 below The conserved glutamic acid (E), at ammo acid residue 1344, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-hke structures of 01524 Preferred fragments of 01524 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO: 36

MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEETKTDDVGITLENKNSSQVTSSTSSSQSSVEQ SKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVWTLTKEAKIPKNADLKVTELKDNSATFKDYKKKALTEVAKQDSEI KNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDENL KWHFKDDGQTEVLKSKDTAETKNTSSDVAFKTDSFSIYAI VQEDNTEVPRLTYHFQNNDGTDYDFLTASGMQVHHQIIKDGESLGEVGIPTIKAGEHFNGWYTYD PTTGKYGDPVKFGEPI TVTETKEICVRPFMSKVATVTL YDDSAGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLSESEIQ ALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPASTIKPND PTRPGYTFAGWYTAASGGAAFDFNQVLTK

KDDGSSWNVYYDRKLITIKF AKYGGYSLPEYYYSYNWSSDADTYTGL YGTTLAANGYQWKTGAWGYLANVGNNQVGTYGM SYLGEFILPNDTVDSDVI KLFPKGNIVQTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQ YASQTSAGVKVPISDEYYDRYGAYHKDYLNLWWYERNSYKIKYLDPLDNTELPNF PVKDVLYEQNLSSYAPDTTTVQPKP SRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYAGWQKVTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYI QDPSGTYYYKYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKL VGWYYVNPDGSIRPYNFSGAVTQDINL RAIW RKAGDYHIIYSNDAVGTDGKPALDASGQQLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSIDID AHLADANKN I T I KPVI I PVGD I KLEDT S I KYNGNGGTRVENGNWTQVETPRMELNSTTT I PENQYFTRTGYNLIGWHHDK DLADTGRVEFTAGQSIGIDNNPDATNTLYAVWQPKEYTVRVSKTWGLDEDKTKDFLFNPSETLQQENFPLRDGQTKEFKV PYGTSISIDEQAYDEFKVSESITEKNLATGEADKTYDATGLQSLTVSGDVDISFTNTRIKQKVRLQKVNVENDNNFLAGAV FDIYESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPGYLLPKNDISVLVISTGVTFEQNGNNAT PIKENL VDGSTVYTFKITNSKGTELPSTGGIGTHIYILVGLALALPSGLILYYRKKI

01525

An example of an amino acid sequence for 01525 is set forth below SEQ ID NO 37 represents a 01525 sequence from GBS serotype III, strain isolate COHl

SEQ ID NO: 37

MKRQISSDKLSQELDRVTYQKRFWSVI KNTIYILMAVASIAILIAVLWLPVLRIYGHSMNKTLSAGDWFTVKGSNFKTGD WAFYYNNKVLVKRVIAESGDWVNIDSQGDVYVNQHKLKEPYVIHKALGNSNIKYPYQVPDKKIFVLGDNRKTSIDSRSTS VGDVSEEQIVGKISFRIWPLGKISSIN

GBS 322 refers to a surface immunogenic protein, also referred to as "sip" Nucleotide and amino acid sequences of GBS 322 sequenced from serotype V isolated strain 2603 V/R are set forth in Ref 3 as SEQ ID 8539 and SEQ ID 8540 These sequences are set forth below as SEQ ID NOS 38 and 39 SEQ ID NO. 38

SEQ ID NO. 39 mKKVLLTSTMAASLLSVASVQAQETDTTWTARWSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIAD INLIYPETTLWTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSS APALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAA PRVASVKWTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQP HVAAYKEKVASTYGVNEFSTYRAGD PGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGP ANTWNAMPDRGGVTANHYDHVHVSFNK

GBS 322 contains an N-terminal leader or signal sequence region which is indicated by the underlined sequence near the beginning of SEQ ID NO 39 In one embodiment, one or more amino acids from the leader or signal sequence region of GBS 322 are removed An example of such a GBS 322 fragment is set forth below as SEQ ID NO 40

SEQ ID NO:40

DLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTTAT VDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVPAA KEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKWTPKVETGASPEHVSAPAVPVTTTSPATDSKL QATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVG TNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK

Additional preferred fragments of GBS 322 comprise the immunogenic epitopes identified in WO 03/068813, each of which are specifically incorporated by reference herein

There may be an upper limit to the number of GBS proteins which will be in the compositions of the invention Preferably, the number of GBS proteins in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3 Still more preferably, the number of GBS proteins in a composition of the invention is less than 6, less than 5, or less than 4 Still more preferably, the number of GBS proteins in a composition of the invention is 3

The GBS proteins and polynucleotides used in the invention are preferably isolated, i e , separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose Group A Streptococcus Adhesin Island Sequences

The GAS AI polypeptides of the invention can, of course, be prepared by various means (e g recombinant expression, purification from GAS, chemical synthesis etc ) and in various forms (e g native, fusions glycosylated, non- glycosylated etc ) They are preferably prepared in substantially pure form (ι e substantially free from other streptococcal or host cell proteins) or substantially isolated form The GAS AI proteins of the invention may include polypeptide sequences having sequence identity to the identified GAS proteins The degree of sequence identity may vary depending on the amino acid sequence (a) in question but is preferably greater than 50% (e g 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, 995% or more) Polypeptides having sequence identity include homologs, orthologs, allelic variants and functional mutants of the identified GBS proteins Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence Identity between proteins is preferably determined by the Smith Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affinity gap search with parameters gap open penalty- 12 and gap extension penalty=l

The GAS adhesin island polynucleotide sequences may include polynucleotide sequences having sequence identity to the identified GAS adhesin island polynucleotide sequences The degree of sequence identity may vary depending on the polynucleotide sequence in question, but is preferably greater than 50% (e g 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 995% or more)

The GAS adhesin island polynucleotide sequences of the invention may include polynucleotide fragments of the identified adhesin island sequences The length of the fragment may vary depending on the polynucleotide sequence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, (e g at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more)

The GAS adhesin island amino acid sequences of the invention may include polypeptide fragments of the identified GAS proteins The length of the fragment may vary depending on the amino acid sequence of the specific GAS antigen, but the fragment is preferably at least 7 consecutive amino acids, (e g 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more) Preferably the fragment comprises one or more epitopes from the sequence Other preferred fragments include (1) the N-terminal signal peptides of each identified GAS protein, (2) the identified GAS protein without their N-terminal signal peptides, and (3) each identified GAS protein wherein up to 10 amino acid residues (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the N-terminus and/or the C-termmus e g the N-terminal amino acid residue may be deleted Other fragments omit one or more domains of the protein (e g omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain)

GAS Al-I sequences

As discussed above, a GAS AI-I sequence is present in an M6 strain isolate (MGAS 10394) Examples of GAS AI-I sequences from M6 strain isolate MGAS 10394 are set forth below

M6_SpyO156 SpyO156 is a rofA transcriptional regulator An example of an amino acid sequence for M6_Spy0156 is set forth in SEQ ID NO 41

SEQ ID NO:41

MIEKYLESSIESKCQLVVLFFKTSYLPITEVAEKTGLTFLQLNHYCEELNAFFPDSLSMTIQKRMISCQFTHPFKETYLYQ LYASSNVLQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALIPLLRNFELKLSKNKIVGEEYRIRYLIALLYSKFGI KVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRHQFSVTIPQTRIFQQLKKLFIYDSLKKSSRDIIE TYCQLNFSAGDLDYLYLIYITANNSFASLQWTPEHIRQCCQLFEENDTFRLLLKPIITLLPNLKEQKPSLVKALMFFSKSF LFNLQHFIPETNLFVSPYYKGNQKLYTSLKLIVEEWLAKLPGKRYLNHKHFHLFCHYVEQILRNIQPPLVWFVASNFINA HLLTDSFPRYFSDKSIDFHSYIAR

M6_SpyO157 M6_Spy0157 is a fibronectin binding protein It contains a sortase substrate motif LPXTG (SEQ ID NO 122), shown in italics in the amino acid sequence SEQ ID NO 42

SEQ ID NO:42

MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACAGAIGFGQVAYAADEKTVPSHSSPNPEFPWYG YDAYGKEYPGYNIWTRYHDLRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHTTKLGKEELEQRL LSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDNSQYQFETLWESEAKEGKISRSQVTLMREALKKLIDPNLEATAV NKIPSGYRLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDGTPIPEDPKHPDDNLEPTLPPVMLDGEEVPEV

NMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETED HFDNNEPKVEENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQSNKKV

M6_SpyOI57 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:180 LPATG (shown in italics in SEQ ID NO42, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant M6_SpyO157 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pihn motif, discussed above, containing a conserved lysine (K) residue has also been identified in M6_SpyO157. The pilin motif sequence is underlined in SEQ ID NO:42, below. Conserved lysine (K) residues are also marked in bold, at amino acid residues 277, 287, and 301 The pihn sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures. Preferred fragments of M6_SpyO157 include at least one conserved lysine residue. Preferably, fragments include the pilin sequence

SEQ ID NO:42

MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACAGAIGFGQVAYAADEKTVPSHSSPNPEFPWYG YDAYGKEYPGYNIWTRYHDLRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHTTKLGKEELEQRL LSLLYNAYPNDANGYMKGLEHLNAITVTQYAWHYSDNSQYQFETLWESEAKEGKISRSQVTLMREALKKLIDPNLEATAV NKIPSGYRLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDGTPIPEDPKHPDDNLEPTLPPVMLDGEEVPEV PSESLEPALPPLMPELDGQEVPEKPSIDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSG NMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETED TKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSGFSETATWEDTRPKLVF HFDNNEPKVEENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQSNKKV

A repeated series of four E boxes containing a conserved glutamic residue have been identified in M6_SpyO157.

The E-box motifs are underlined in SEQ ID NO:42, below. The conserved glutamic acid (E) residues, at amino acid residues 415, 452, 489, and 526 are marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of M6_Spy0157. Preferred fragments of M6_SpyO157 include at least one conserved glutamic acid residue. Preferably, fragments include at least one E box motif.

SEQ ID NO:42

MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACAGAIGFGQVAYAADEKTVPSHSSPNPEFPWYG YDAYGKEYPGYNIWTRYHDLRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHTTKLGKEELEQRL LSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDNSQYQFETLWESEAKEGKISRSQVTLMREALKKLIDPNLEATAV NKIPSGYRLNIFESENEAYQNLLSAEWPDDPPKPGETSEHNPKTPELDGTPIPEDPKHPDDNLEPTLPPVMLDGEEVPEV PSESLEPALPPLMPELDGQEVPEKPSIDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSG NMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETED TKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSGFSETATWEDTRPKLVF HFDNNEPKVEENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQSNKKV

M6_Spy0158: M6_Spy0158 is a reverse transcriptase. An example of SpyO158 is shown in the amino acid sequence SEQ ID NO 43.

SEQ ID NO:43

MSLRHQNKKGIRKEGWKSRPQSRWSDHCQLVAQKSVLKQAISKTVLAERGLFSCLDDYLERHALKVN

M6_SpyO159: M6_SpyO159 is a collagen adhesion protein. It contains a sortase substrate motif LPXSG, shown in italics in the amino acid sequence SEQ ID NO:44.

SEQ ID NO: 44

MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQTKVIPQEIVTQTETQGTQWATKQKLESENS SLKVALKRESGFEHNATIDASLDTESQGDNSQRSVTQAIVTMALELRKQGLS IVDTKIVRIQSSTNQRNDITTTLTFKNGL SLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADGKVKNLVFTGRLGKQVI IVSTTRLKEEQTISLDSYGELVIDGAV GLSQKDRPPYSKPITVNILKPKLSSIESSLDSKDFEIVKTIDNL YTWDDQFYLLDFISKQYEVLKTDYQSAKDSTPQTRDI

NQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVWDKSGSMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSS YQGESFNRGQIHYRYRGIVSVSDGIRRDDAVKNSLLGVNGLLQRFVNINPENKLSVIGFQGSADYHAGKWYPDQSPRGGFY SKLSQLGISDSLSQYVDYYDKQPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKWFTPKTTSQPKGKVTLTFKSDYKV DDEYTYTLSFNVKASDEAYEKYKDNEGRYSEMGDSDTDYGTNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKT VPITFTKVDADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYETKAKLGYTLPENPWEVAVANNG DIKVKHPIEGELKSKDGSYMIKNYKIYQLPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY

M6_SpyO159 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:181 LPSSG (shown in italics in SEQ ID NO 44, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant M6_SpyO159 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in M6_SpyO159 The pihn motif sequence is underlined in SEQ ID NO 44, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 265 and 276 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of M6_Spy0159 include at least one conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO: 44

MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQTKVIPQEIVTQTETQGTQWATKQKLESENS SLKVALKRESGFEHNATIDASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRNDITTTLTFKNGL SLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADGKVKNLVFTGRLGKQVIIVSTTRLKEEQTISLDSYGELVIDGAV GLSQKDRPPYSKPITVNILKPKLSSIESSLDSKDFEIVKTIDNLYTWDDQFYLLDFISKQYEVLKTDYQSAKDSTPQTRDI LFGEYTVEPLVMNKGHNNTINIYIRSTRPLGLKPIGAAPALIQPRSFRSLTPRSTRMKRSAPVEKFEGELEHHKRIDYLGD NQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVWDKSGSMQEGIGSVQRYRYYAQRWDDYΎSQWVYHGTFDYSS YQGESFNRGQIHYRYRGIVSVSDGIRRDDAVKNSLLGVNGLLQRFVNINPENKLSVIGFQGSADYHAGKWYPDQSPRGGFY

GSSNDRNNVTRSQEGSKLAIDEFKARYPNLSIYSLGVSKDINSDTASSSPWLKYLSGEEHYYGITDTAELEKTLNKIVED SKLSQLGISDSLSQYVDYYDKQPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKWFTPKTTSQPKGKVTLTFKSDYKV DDEYTYTLSFNVKASDEAYEKYKDNEGRYSEMGDSDTDYGTNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKT VPITFTKVDADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYETKAKLGYTLPENPWEVAVANNG DIKVKHPIEGELKSKDGSYMIKNYKIYQLPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY

An E box containing a conserved glutamic residue has been identified in M6_SpyO159 The E-box motif is underlined in SEQ ID NO 44, below The conserved glutamic acid (E), at amino acid residue 950, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of M6_SpyO159 Preferred fragments of M6_Spy0159 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO: 44

MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQTKVIPQEIVTQTETQGTQWATKQKLESENS SLKVALKRESGFEHNATIDASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRNDITTTLTFKNGL SLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADGKVKNLVFTGRLGKQVIIVSTTRLKEEQTISLDSYGELVIDGAV GLSQKDRPPYSKPITVNILKPKLSSIESSLDSKDFEIVKTIDNLYTWDDQFYLLDFISKQYEVLKTDYQSAKDSTPQTRDI LFGEYTVEPLVMNKGHNNTINIYIRSTRPLGLKPIGAAPALIQPRSFRSLTPRSTRMKRSAPVEKFEGELEHHKRIDYLGD NQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILWVDKSGSMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSS YQGESFNRGQIHYRYRGIVSVSDGIRRDDAVKNSLLGVNGLLQRFVNINPENKLSVIGFQGSADYHAGKWYPDQSPRGGFY QPNLNNSRDAELLKGWSTNSLLDPNTLTALHNNGTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTFYFGEDGYRSGN GSSNDRNNVTRSQEGSKLAIDEFKARYPNLSIYSLGVSKDINSDTASSSPWLKYLSGEEHYYGITDTAELEKTLNKIVED SKLSQLGISDSLSQYVDYYDKQPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKVTLTFKSDYKV DDEYTYTLSFNVKASDEAYEKYKDNEGRYSEMGDSDTDYGTNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKT VPITFTKVDADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYETKAKLGYTLPENPWEVAVANNG DIKVKHPIEGELKSKDGSYMIKNYKIYQLPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY

M6_Spy0160 M6_Spy0160 is a fimbria! structural subunit It contains a sortase substrate motif LPXTG (SEQ ID NO 122), shown in italics in amino acid sequence SEQ ID NO 45 SEQ ID NO:45

MTNRRETVREKILITAKKLMLACLAILAWGLGMTRVSALSKDDTAQLKITNIEGGPTVTLYKIGEGVYNTNGDSFINFKY

VTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVWKKTTSLGSVLSYSLTFELPSYTKEAVNKTVYVSDNMSEGLTFN

NPTKGNTYDNLDKKPDKGNGITSKEDSKIVΎTYQIAFRKVDSVSKTPLIGAIFGVYDTSNKLIDIVTTNKNGYAISTQVSS GKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTSDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAY IESTKALTDGTTFSKSNEGSGTVLLETDIPNTKLGELPSTGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA

M6_SpyO16O contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:131 LPSTG (shown in italics in SEQ ID NO:45, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant M6_Spy0160 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.

An E box containing a conserved glutamic residue has been identified in M6_Spy0160. The E-box motif is underlined in SEQ ID NO:45, below. The conserved glutamic acid (E), at amino acid residue 412, is marked in bold. The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of M6_Spy0160. Preferred fragments of M6_Spy0160 include the conserved glutamic acid residue. Preferably, fragments include the E box motif.

SEQID NO:45

MTNRRETVREKILITAKKLMLACLAILAWGLGMTRVSALSKDDTAQLKITNIEGGPTVTLYKIGEGVYNTNGDSFINFKY AEGVSLTETGPTSQEITTIANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGATDGRTYNPILLAASYNGEGNL VTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVNKKTTSLGSVLSYSLTFELPSYTKEAVNKTVYVSDNMSEGLTFN

NPTKGNTYDNLDKKPDKGNGITSKEDSKIVYTYQIAFRKVDSVSKTPLIGAIFGVYDTSNKLIDIVTTNKNGYAISTQVSS GKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTSDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAY IESTKALTDGTTFSKSNEGSGTVLLETDIPNTKLGELPSTGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA

M6_SpyO16I is a srtB type sortase. An example of an amino acid sequence of M6_Spy-161 is shown in SEQ ID NO:46.

SEQ ID NO:46

MTERLKNLGILLLFLLGTAIFLYPTLSSQWNAYRDRQLLSTYHKQVIQKKPSEMEEVWQKAKAYNARLGIQPVPDAFSFRD

LVKKGDTFYFRVLNKVLAYKVDQILIVEPDQATSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKKYQQAKKAMK LVDKSRMWAEWCAAFGWIAIILVFMYSRVSAKKSK

As discussed above, applicants have also determined the nucleotide and encoded amino acid sequence of fϊmbrial structural subunits in several other GAS AI-I strains of bacteria. Examples of sequences of these fimbrial structural subunits are set forth below.

M6 strain isolate CDC SS 410 is a GAS AI-I strain of bacteria. CDC SS 410_fϊmbrial is thought to be a fimbrial structural subunit of M6 strain isolate CDC SS 410. An example of a nucleotide sequence encoding the CDC SS 410_fimbrial protein (SEQ ID NO:267) and a CDC SS 410_fimbrial protein amino acid sequence (SEQ ID NO:268) are set forth below. SEQ ID NO:267 AAATCGAATGAAGGTTCAGGTACAGTATTATTAGAAACTGACATCCCTAACACCAAGCTAGGTGAACTC

SEQ ID NO 268

KDDTAQLKITNI EGGPTVTLYKIGEGVYNTNGDSFINFKYAEGVSLTETGPTSQEITTIANGINTGKIKPFSTENVSISNG TATYNARGASVYIALLTGATDGRTYNPILLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVNKKT

FIYDSLESISPNISYKAWNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKEDSKIVYTYQIAFRKVD SVSKTPLIGAIFGVYDTSNKLIDIVTTNKNGYAISTQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKST TYTSDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKALTDGTTFSKSNEGSGTVLLETDIPNTKLGEL

M6 strain isolate ISS 3650 is a GAS AI-I strain of bacteria ISS3650_fimbπal is thought to be a fimbπal structural subumt of M6 strain isolate ISS 3650 An example of a nucleotide sequence encoding the ISS3650_fimbπal protein (SEQ ID NO 269) and an ISS3650_fimbπal protein amino acid sequence (SEQ ID NO 270) are set forth below. SEQ ID NO 269

TTAACTGATGGAACAACTTTCTCAAAATCGAATGAAGGTTCAGGTACAGTATTATTAGAAACTGACATCC SEQ ID NO 270

TYDNLDKKPDKGNGITSKEDSKIVYTYQIAFRKVDSVSKTPLIGAIFGVYDTSNKLIDIVTTNKNGYAISTQVSSGKYKIK ELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTSDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKA LTDGTTFSKSNEGSGTVLLETDI

M23 strain isolate DSM2071 is a GAS AI-I strain of bacteria DSM2071_fimbπal is thought to be a fimbπal structural subumt of M23 strain DSM2071 An example of a nucleotide sequence encoding the DSM207 l_fimbπal protein (SEQ ID NO 251) and a DSM2071_fimbπal protein amino acid sequence (SEQ ID NO 252) are set forth below. SEQ ID NO 251

ATTTATATTGTTAAACGTCGTAAAGCTTAA SEQ ID NO 252

MREKILIAAKKLMLACLAILA WGLGMTRVSALSKDDKAELKITNIEGKPTVTLYKIGDGKYSERGDSFIGFELKQGVELN KAKPTSQEINKIANGINKGSVKAEWNIKEHASTTYSYTTTGAGIYLAILTGATDGRAYNPILLTASYNEENPLKGGQIDA TSHYLFGEEAVAKSSQPTISKSITKSTKDGDKDTASVGEKVDYKLTVQLPSYSKDAINKTVFITDKLSQGLTFLPKSLKII WNGQTLTKVNEEFKAGDKVIAQLKVENNGFNLNFNYDNLDNHAPEVNYSALLNENAWGKGGNDNNVD YYYSNNPNKGETH KTTEKPKEGEGTGITKKTDKKTVYTYRVAFKKTGKDHAPLAGAVFGIYSDKEAKQLVDIWTNAQGYAASSEVGKGTYYIK EIKSPKGYSLNTNIYEVETSWEKATTTSTTNRLETIYTTDDNQKSPGTNTVGWLEDGVFYKENPGGDAKLAYIKQSTEETS TTIEVKENQAEGSGTVLLETEIPNTKLGELPSTGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA GAS AI-2 sequences

As discussed above, a GAS AI-2 sequence is present in an Ml strain isolate (SF370) Examples of GAS AI-2 sequences from Ml strain isolate SF370 are set forth below

SpyO124 is a rofA transcriptional regulator An example of an ammo acid sequence for SpyO124 is set forth in SEQ ID NO 47

SEQ ID NO:47

MIEKYLESSIESKCQLIVLFFKTSYLPITEVAEKTGLTFLQLNHYCEELNAFFPGSLSMTIQKRMISCQFTHPFKETYLYQ

KVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRHQFSVTIPQTRIFQQLKKLFVYDSLKKSSHDIIE TYCQLNFSAGDLDYLYLIYITANNSFASLQWTPEHIRQYCQLFEENDTFRLLLNPIITLLPNLKEQKASLVKALMFFSKSF LFNLQHFIPETNLFVSPYYKGNQKLYTSLKLIVEEWMAKLPGKRDLNHKHFHLFCHYVEQSLRNIQPPLVWFVASNFINA

HLLTDSFPRYFSDKSIDFHSYYLLQDNVYQIPDLKPDLVITHSQLIPFVHHELTKGIAVAEISFDESILSIQELMYQVKEE KFQADLTKQLT

GAS 015 is also referred to as Cpa It contains a sortase substrate motif VVXTG (SEQ ID NO 135), shown in italics in SEQ ID NO 48 SEQ ID NO:48

SYVRGHPYYKQFRVAHDLRVNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPRITGDELNQKLRA VMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAPISNPDESFKRESESNLVSTSQLSLMRQALKQLIDPNLATKMPK QVPDDFQLSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLIRKYAIGDYSKLLEGATLQLTGDNV NSFQARVFSSNDIGERIELSDGTYTLTELNSPAGYSIAEPITFKVEAGKVYTIIDGKQIENPNKEIVEPYSVEAYNDFEEF SVLTTQNYAKFYYAKNKNGSSQWYCFNADLKSPPDSEDGGKTMTPDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKH IKKVIEKGYREKGQAIEYSGLTETQLRAATQLAIYYFTDSAELDKDKLKDYHGFGDMNDSTLAVAKILVEYAQDSNPPQLT DLDFFIPNNNKYQSLIGTQWHPEDLVDIIRMEDKKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKT DKTNLEFKDGKATINLKHGESLTLQGLPEGYSYLVKETDSEGYKVKVNSQEVANATVSKTGITSDETLAFENNKEPWPTG VDQKINGYLALIVIAGISLGIWGIHTIRIRKHD

GAS 015 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:182 VVPTG (shown in italics in SEQ ID NO 48, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant GAS 015 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif discussed above, containing a conserved lysine (K) residue has also been identified in GAS 015 The pilin motif sequence is underlined in SEQ ID NO 48, below Conserved lysine (K) residues are also marked in bold, at amino acid residue 243 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of GAS 015 include the conserved lysine residue Preferably, fragments include the pilin sequence SEQ ID NO:48

LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFAL VTSMVGAKTVFGLVESSTPNAINPDSSSEYRWYGYE

SYVRGHPYYKQFRVAHDLRVNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPRITGDELNQKLRA VMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAPISNPDESFKRESESNLVSTSQLSLMRQALKQLIDPNLATKMPK QVPDDFQLSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLIRKYAIGDYSKLLEGATLQLTGDNV NSFQARVFSSNDIGERIELSDGTYTLTELNSPAGYSIAEPITFKVEAGKVYTIIDGKQIENPNKEIVEPYSVEAYNDFEEF SVLTTQNYAKFYYAKNKNGSSQWYCFNADLKSPPDSEDGGKTMTPDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKH IKKVIEKGYREKGQAIEYSGLTETQLRAATQLAIYYFTDSAELDKDKL KD YHGFGDMNDSTLAVAKILVEYAQDSNPPQLT DLDFFI PNNNKYQSLIGTQWHPEDLVDIIRMEDKKEVI PVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKT DKTNLEFKDGKATINLKHGESLTLQGLPEGYSYLVKETDSEGYKVKWSQEVANATVSKTGITSDETLAFENNKE PWPTG VDQKINGYLALIVIAGISLGIWGIHTIRIRKHD

An E box containing a conserved glutamic residue has been identified in GAS 015 The E-box motif is underlined in SEQ ID NO 48, below The conserved glutamic acid (E), at amino acid residue 352, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-like structures of GAS 015 Preferred fragments of GAS 015 include the conserved glutamic acid residue Preferably, fragments include the E box motif SEQ ID NO:48

LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFALVTSMVGAKTVFGLVESSTPNAINPDSSSEYRWYGYE SYVRGHPYYKQFRVAHDLRVNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPRITGDELNQKLRA VMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAPISNPDESFKRESESNLVSTSQLSLMRQALKQLIDPNLATKMPK QVPDDFQLSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLIRKYAIGDYSKLLEGATLQLTGDNV NSFQARVFSSNDIGERIELSDGTYTLTELNSPAGYSIAEPITFKVEAGKVYTIIDGKQIENPNKEIVEPYSVEAYNDFEEF SVLTTQNYAKFYY AKNKNGSSQWYCFNADLKSPPDSEDGGKTMTPDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKH

IKKVIEKGYREKGQAI EYSGLTETQLRAATQLAIYYFTDSAELDKDKLKDYHGFGDMNDSTLAVAKILVEYAQDSNPPQLT DLDFFIPNNNKYQSLIGTQWHPEDLVDIIRMEDKKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKT

VDQKINGYLALIVIAGISLGIWGIHTIRIRKHD

SpyO127 is a LepA putative signal peptidase An example of an ammo acid sequence for SpyO127 is set forth in SEQ ID NO 49

SEQ ID NO:49

MI IKRNDMAPSVKAGDAILFYRLSQTYKVE EAWYEDSKTSITKVGRI IAQAGDEVDLTEQGELKINGHIQNEGLTFIKSR EANYPYRIADNSYLILNDYYSQESENYLQDAIAKDAIKGTINTLIRLRNH

SpyO128 is thought to be a fimbπal protein It contains a sortase substrate motif EVXTG (SEQ ID NO 136) shown in italics in SEQ ID NO 50 SEQ ID NO:50

TYTNSDKGGSNTKTAEFDFSEVTFEKPGVYYYKVTEEKIDKVPGVSYDTTSYTVQVHVLWNEEQQKPVATYIVGYKEGSKV PIQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKANQYYKASEKVMIEKTTKGGQAPVQTEASIDQLYHFTLKDGESI KVTNLPVGVD YWTEDDYKSEKYTTNVEVSPQDGAVKNIAGNSTEQETSTDKDMTITFTNKKDF SVPTGVAMTVAPYIALG IVAVGGALYFVKKKNA

SpyO128 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:183 EVPTG (shown in italics in SEQ ID NO 50, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyO128 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two E boxes containing a conserved glutamic residue have been identified in SpyO128 The E-box motifs are underlined in SEQ ID NO 50, below The conserved glutamic acid (E) residues, at amino acid residues 271 and 290, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-hke structures of SpyO128 Preferred fragments of SpyO128 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif SEQ ID NO:50

TYTNSDKGGSNTKTAEFDFSEVTFEKPGVYYYKVTEEKIDKVPGVSYDTTSYTVQVHVLWNEEQQKPVATYIVGYKEGSKV PIQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKANQYYKASEKVMIEKTTKGGQAPVQTEASIDQLYHFTLKDGESI KVTNL PVGVD YWTEDDYKSEKYTTNVEVSPQDGAVKNIAGNSTEQETSTDKDMTITFTNKKDFEVPTGVAMTVAPYIALG IVAVGGALYFVKKKWA

SpyO129 is a srtCl type sortase An example of an amino acid sequence for SpyO129 is set forth in SEQ ID NO 51

SEQ ID NO:51

MIVRLIKLLDKLINVIVLCFFFLCLLIAALGIYDALTVYQGANATNYQQYKKKGVQFDDLLAINSDVMAWLTVKGTHIDYP IVQGENNLEYINKSVEGEYSLSGSVFLDYRNKVTFEDKYSLIYAHHMAGNVMFGELPNFRKKSFFNKHKEFSIETKTKQKL KINIFACIQTDAFDSLLFNPIDVDISSKNEFLNHIKQKSVQYREILTTNESRFVALSTCEDMTTDGRIIVIGQIE"

SpyO13O is referred to as a hypothetical protein It contains a sortase substrate motif LPXTG (SEQ ID NO 122), shown in italics in SEQ ID NO 52

SEQ ID NO:52

MKKSILRILAIGYLLMSFCLLDSVEAENLTASINIEVINQVDVATNKQSSDIDETFMFVIEALDKESPLPNSVTTSVKGNG

KTSFEQLTFSEVGQYHYKIHQLLGKNSQYHYDETVYEWIYVLYNEQSGALETNLVSNKLGETEKSELIFKQEYSEKTPEP HQPDTTEKEKPQKKRNGILPSTGEMVSYVSALGIVLVATITLYSIYKKLKTSK

SpyO13O contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:131 LPSTG (shown in italics in SEQ ID NO 52, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyO13O protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two E boxes containing conserved glutamic residues have been identified in SpyO13O The E-box motifs are underlined in SEQ ID NO 52, below The conserved glutamic acid (E) residues, at amino acid residues 118 and 148, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus like structures of SpyO13O Preferred fragments of SpyO13O include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif

SEQ ID NO:52

MKKSILRILAIGYLLMSFCLLDSVEAENLTASINIEVINQVDVATNKQSSDIDETFMFVIEALDKESPLPNSVTTSVKGNG KTSFEQLTFSEVGQYHYKIHQLLGKNSQYHYDETVYEWIYVLYNEQSGALETNLVSNKLGETEKSELIFKQEYSEKTPEP HQPDTTEKEKPQKKRNGILPSTGEMVSYVSALGIVLVATITLYSIYKKLKTSK

SpyO131 is referred to as a conserved hypothetical protein An example of an amino acid sequence of SpyO131 is set forth in SEQ ID NO 53

SEQ ID NO:53

MTRTNYQKKRMTCPVETEDITYRRKKIKGRRQAILAQFEPELVHHELIGDSCTCPDCHGTLTEIGSWQRQELVFIPAQLK RINHVQHAYKCQTCSDNSLSDKIIKAPVPKAPLAHSLGSASIIAHTVHQKFTLKVPNYRQEEDWNKLGLSISRKEIANWHI KSSQYYFEPLYDLLRDILLSQEVIHADETSYRVLESDTQLTYYWTFLSGKHEKKGITLYHHDKRRSGLVTQEVLGDYSGYV HCDMHGAYRQLEHAKLVGCWAHVRRKFFEATPKQADKTSLGRKGLVYCDKLFALEAEWCELPPQERLVKRKEILTPLMTTF FDWCREQVVLSGSKLGLAIAYSLKHERTFRTVLEDGHIVLSNNMAERAIKSLVMGRKNWLFSQSFEGAKAAAIIMSLLETA KRHGLNSEKYISYLLDRLPNEETLAKREVLEAYLPWAKKVQTNCQ

SpyO133 is referred to as a conserved hypothetical protein An example of an amino acid sequence of SpyO133 is set forth in SEQ ID NO 54

SEQ ID NO:54

MTIRLNDLGQVYLVCGKTDMRQGIDSLAYLVKSQHELDLFSGAVYLFCGGRRDRFKALYWDGQGFWLLYKRFENGKLAWPR NRDEVKCLTAVQVDWLMKGFFISPNIKISKSHDFY Spy0135 is a SrtB type sortase It is also referred to as a putative fimbria associated protein An example of an amino acid sequence of SpyOl 35 is set forth in SEQ ID NO 55

SEQ ID NO.55

MECYRDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPDAFSFRDGIHDKNYESLLQIENNDIMGYVEVPSIKV TLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNLVKKGDTFYFRVLNKVLAYKVDQILTVEP DQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKKYQQAKKAMKLVDKSRMWAEWCAAFGWIAIILVFMYS RVSAKKSK

GAS AI-3 sequences

As discussed above, a GAS AI-3 sequence is present in a M3, M18 and M5 strain isolates Examples of GAS AI-3 sequences from M3 strain isolate MGAS315 are set forth below

SpyM30097 is as a negative transcriptional regulator (Nra) An example of an amino acid sequence of SpyM30097 is set forth in SEQ ID NO 56

SEQ ID NO:56

MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKGKIICCF VKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSKNTIVGEEYRIRY LIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYD CLTRSSRQVI ENAFSLTFSQGDLEYLFLIYITTNNSFASLQWTPQHIETCCHIFEKNDTFRLLLEPILKRLPQLNHSKQDL IKALMYFSKSFLFNLQHFVIEIPSFSLPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNKQPALT WLISSNFINAKLLTDTI PRYFSDKGIHFYSFYLLRDDIYQIPSLKPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYS IASIQNLIYQLKDKKYQDFLNEQLQ

SpyM30098 is thought to be a collagen binding protein (Cpb) It contains a sortase substrate motif VPXTG (SEQ ID NO 137) shown in italics in SEQ ID NO 57

SEQ ID NO:57

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPDYSPLKT YHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRILYNGYPNDRNGIM KGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSS DKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQIEGSGFQEKIFDSNKSGEKVELPNG TYVLSELKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSVTAFNDFEEIGYLSDFNNYGKFYYAKNTNGT

TEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDKLDDATLAWHELITYAEDVTLPMTQNLDFFVPNSSRYQALIG TQYHPNELIDVISMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTL

FGLLVWLFGRKGTKK

SpyM30098 contains an ammo acid motif indicative of a cell wall anchor SEQ ID NO:184 VPPTG (shown in italics in SEQ ID NO 57, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30098 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM30098 The pilin motif sequence is underlined in SEQ ID NO 57, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 262 and 270 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus like structures Preferred fragments of SpyM30098 include at least one conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO:57

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPDYSPLKT YHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRILYNGYPNDRNGIM KGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSS DKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQIEGSGFQEKIFDSNKSGEKVELPNG NQWYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLYKYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTL TEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDKLDDATLAWHELITYAEDVTLPMTQNLDFFVPNSSRYQALIG TQYHPNELIDVISMEDKQAPI I PITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTL

FGLLVWLFGRKGTKK

An E box containing a conserved glutamic residue has been identified in SpyM30098 The E-box motif is underlined in SEQ ID NO 57, below The conserved glutamic acid (E), at amino acid residue 330, is marked in bold The E box motif in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-like structures of SpyM30098 Preferred fragments of SpyM30098 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO:57

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPDYSPLKT YHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRI EDGQLQQNILRILYNGYPNDRNGIM KGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSS DKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVI IRKYAEGDYSKLLEGATLKLAQIEGSGFQEKIFDSNKSGEKVELPNG TYVLSBLKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSVTAFNDFEEIGYLSDFNNYGKFYYAKNTNGT NQWYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLYKYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTL TEAQFRAATQLAIYYYTDSADLTTLKTYNDNKGYHGFDKLDDATLA WHELITYAEDVTLPMTQNLDFFVPNSSRYQALIG TQYHPNELIDVISMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTL

FGLLVWLFGRKGTKK

SpyM30099 is referred to as LepA An example of an amino acid sequence of SpyM30099 is set forth in SEQ ID NO 58

SEQ ID NO:58

MTNYLNRLNENPLLKAFIRL VLKISIIGFLGYILFQYVFGVMIVNTNQMSPAVSAGDGVL YYRLTDRYHINDVWYEVDDT LKVGRIAAQAGDEVNFTQEGGLL INGHPPEKEVPYLTYPHSSGPNF PYKVPTGTYFILNDYREERLDSRYYGALPINQIKG KISTLLRVRGI

SpyM30100 is thought to be a fimbπal protein An example of an ammo acid sequence of SpyM30100 is set forth in SEQ ID NO 59

SEQ ID NO:59

MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTKDGLEIK PGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKWFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYVGNKEGGGFEPKF IVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKL KNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESADEIWTNKRDTOVPTGWGTLAPF AVLSIVAIGGVIYITKRKKA

SpyM30100 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:140 QVPTG (shown in italics in SEQ ID NO 59, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30100 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in SpyM30100 The pilin motif sequences are underlined in SEQ ID NO 59, below Conserved lysine (K) residues are also marked in bold, at ammo acid residues 57 and 63 and at amino acid residues 161 and 166 The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of SpyM30100 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO:59 PGIVNGLTEQI ISYTNTDKPDSKVKSTEFDFSKWFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYVGNKEGGGFEPKF IVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKL KNGESIQLDKLPVGITYKVNEMEΆNKDGYKTTASLKEGDGQSKMYQLDMEQKTDESADEIWTNKRDTQVPTGWGTLAPF AVLSIVAIGGVIYITKRKKA

Two E boxes, each containing a conserved glutamic residue, have been identified in SpyM30100 The E-box motifs are underlined in SEQ ID NO 59, below The conserved glutamic acid (E) residues, at amino acid residues 232 and 264, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation ot oligomeπc pilus-hke structures of SpyM30100 Preferred fragments of SpyM30100 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif SEQ ID NO:59

PGIVNGLTEQI ISYTNTDKPDSKVKSTEFDFSKWFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYVGNKEGGGFEPKF IVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFBVKIGTPYKFKL KNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESADEIWTNKRDTQVPTGWGTLAPF AVLSIVAIGGVIYITKRKKA

SpyM30101 is a SrtC2 type sortase An example of an amino acid sequence of SpyM30101 is set forth in SEQ ID NO 60.

SEQ ID NO:60

MTIVQVINKAIDTLILIFCLWLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNIPGTHIDYP LVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFSKHNKAIIETKERKKL TVTIFACLKTDAFNQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIWGTIQE

SpyM30102 is referred to as a hypothetical protein An example of an amino acid sequence of SpyM30102 is set forth in SEQ ID NO 61

SEQ ID NO:61

MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQ KPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPLPLAGEVKSLLGI LSIVLLGLLVLLYVKKLKSRL

SpyM30102 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:185 LPLAG (shown in italics in SEQ ID NO 61, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30102 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM30102 The pilin motif sequence is underlined in SEQ ID NO 61, below The conserved lysine (K) residue is also marked in bold, at amino acid residue 132 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-hke structures Preferred fragments of SpyM30102 include the conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO:61

MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQ KPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPLPLAGEVKSLLGI LSIVLLGLLVLLYVKKLKSRL

Two E boxes containing conserved glutamic residues have been identified in SpyM30102 The E box motifs are underlined in SEQ ID NO 61, below The conserved glutamic acid (E) residues, at amino acid residues 52 and 122, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-like structures of SpyM30102 Preferred fragments of SpyM30102 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO:61

MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQ KPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPLPLAGEVKSLLGI LSIVLLGLLVLLYVKKLKSRL

SpyM30103 is referred to as a putative multiple sugar metabolism regulator An example of an amino acid sequence for SpyM31O3 is set forth in SEQ ID NO 62 SEQ ID NO:62

GPFYPYSLNKDYQEQLANNCLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTIHQLLQHSKQMT ADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFV

SESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI

SpyM30104 is thought to be a F2 like fibronectin binding protein An example of an amino acid sequence for SpyM30104 is set forth in SEQ ID NO 63

SEQ ID NO:63

MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNR NVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIY FKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYV KPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSR PVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GEWDTTEDTQSGMTGHSGSTTEIEDSKSSDVIIGGQGEWDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETT EDTQTGMHGDSGRKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLK SKKRLSSC

SpyM30104 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:180 LPATG (shown in italics in SEQ ID NO 63, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM30104 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in SpyM30104 The pilin motif sequences are underlined in SEQ ID NO 63, below Conserved lysine (K) residues are also marked in bold, at ammo acid residues 156 and 227 The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of SpyM30104 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence

SEQ ID NO:63

MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNR NVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIY FKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYV KPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSR PVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GEWDTTEDTQSGMTGHSGSTTEIEDSKSSDVIIGGQGEWDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETT EDTQTGMHGDSGRKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLK SKKRLSSC

An E box containing a conserved glutamic residue has been identified in SpyM30104 The E-box motif is underlined in SEQ ID NO 63, below The conserved glutamic acid (E), at amino acid residue 402, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus like structures of SpyM30104 Preferred fragments of SpyM30104 include the conserved glutamic acid residue Preferably, fragments include the E box motif SEQ ID NO:63

MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNR NVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIY FKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYV

PVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ GEWDTTEDTQSGMTGHSGSTTEIEDSKSSDVI IGGQGEWDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETT EDTQTGMHGDSGRKTEVEDTKL VQSFHFDNKEPESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLK SKKRLSSC

Examples of GAS AI-3 sequences from M3 strain isolate SSI-I are set forth below

Sps0099 is a negative transcriptional regulator (Nra) An example of an amino acid sequence for Sps0099 is set forth in SEQ ID NO 64

SEQ ID NO:64

MPYVKKKKDSFLVETYLEQSIRDKS EL VLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKGKIICCF VKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSKNTIVGEEYRIRY LIAMLYSKFGIVIYPLDHLDNQI IYRFLSQSATNLRTSPWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYD CLTRSSRQVIENAFSLTFSQGDLEYLFLIYITTNNSFASLQWTPQHIETCCHIFEKNDTFRLLLEPILKRLPQLNHSKQDL IKALMYFSKSFLFNLQHFVIEI PSFSLPTYTGNSNL YKAL KNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNKQPALT WLI SSNFINAKLLTDTI PRYFSDKGIHFYSFYLLRDDIYQI PSLKPDLVITHSRLI PFVKNDLVKGVTVAEFSFDNPDYS IASI QNL I YQLKDKKYQDFLNEQLQ

SpsOlOO is thought to be a collagen binding protein (Cbp) It contains a sortase substrate motif VPXTG shown in italics in SEQ ID NO 65

SEQ ID NO:65

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNKQSSVQDYPWYGYDSYSKGYPDYSPLKT YHNLKVNLDGSKEYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRILYNGYPNDRNGIM KGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETDLKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSS DKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQIEGSGFQEKI FDSNKSGEKVELPNG TYVLSELKPPQGYGVATPITFKVAAEKVLIKNKEGQFVENQNKEIAEPYSVTAFNDFEEIGYLSDFNNYGKFYYAKNTNGT NQWYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLYKYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTL TEAQFRAATQLAI YYYTDSADLTTLKTYNDNKGYHGFDKLDDATLAWHELITYAEDVTLPMTQNLDFFVPNSSRYQALIG TQYHPNELIDVISMEDKQAPI IPITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTL

FGLLVWLFGRKGTKK

SpsOlOl is referred to as a LepA protein An example of an amino acid sequence of SpsOlOl is set forth as SEQ ID NO 66

SEQ ID NO:66

MTNYLNRLNENPLLKAFIRLVLKISIIGFLGYILFQYVFGVMIVNTNQMSPAVSAGDGVL YYRLTDRYHINDVWYEVDDT LKVGRIAAQAGDEVNFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSRYYGALPINQIKG KISTLLRVRGI

Sps0102 is thought to be a fimbπal protein It contains a sortase substrate motif QVXTG shown in italics in SEQ ID NO 67

SEQ ID NO:67

MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKL IVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTKD GLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKWFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYVGNKEGGG FEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTP YKFKLKNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESADEIWTNKRDTQVPTGWG TLAPFAVLSIVAIGGVIYITKRKKA

Sps0103 is a SrtC2 type sortase An example of Sps0103 is set forth in SEQ ID NO 68 SEQ ID NO:68 MVMTIVQVINKAI DTLILIFCLWLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNI PGTHID YPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFSKHNKAIIETKERK KLTVTIFACLKTDAFNQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTC ENFSTDNRVIWGTIQE

Sps0104 is referred to as a hypothetical protein. It contains, a s.ortase substrate motif LPXAG shown in italics in SEQ ID NO:69.

SEQ ID NO:69

MLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQY TYRVYQKPSQNKDYQADTTVFDVL VYVTYDEDGTLVAKVI SRRAGDEEKSAITFKPKWL VKPIPPRQPNIPKTPLPLAGEV KSLLGILSIVLLGLLVLLYVKKLKSRL

Sps0105 is referred to as a putative multiple sugar metabolism regulator. An example of Sps0105 is set forth in SEQ ID NO:70.

SEQ ID NO:70

MALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTIHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKL GNPQLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAA IIHFSESLTNKSISDKRQMYNSVLHYVDSHLYSKLKVSDIAKRL YVSESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKR GIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI

SpsOlOβ is thought to be a F2 like fibronectin binding protein It contains a sortase substrate LPXTG (SEQ ID NO:122) shown in italics in SEQ ID NO:71.

SEQ ID NO:71

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKNFPTAQGVSKKL YKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWL NGLTENEKI EVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVI ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKL YRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY IYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDE VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEWDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETTEDTQ TGMHGDSGRKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKR LSSC

Examples of GAS AI-3 sequences from M5 isolate Manfredo are set forth below.

Orf 77 encodes a negative transcription regulator (Nra). An example of the nucleotide sequence encoding Nra (SEQ ID NO:88) and an Nra amino acid sequence (SEQ ID NO:89) are set forth below. SEQ ID NO:88

SEQ ID NO:89

MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKGKIICCF VKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSKNTIVGEEYRIRY LIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYD CLTRSSRQVIENAFSLMFSQGDLDYLFLIYITTNNSFASLQWTPQHIETCCHIFEKNDTFRLLLEPILKRLPQLNHSKQDL IKALMYFSKSFLFNLQHFVIEIPSFSLPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNKQPALT

WLISSNFINAKLLTDTIPRYFSDKGIHFYSFYLLRDDIYQIPSLKPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYS IASIQNLIYQLKDKKYQDFLNEQLQ

Orf 78 is thought to be a collagen binding protein (Cbp) An example of the nucleotide sequence encoding Cbp (SEQ ID NO 90) and a Cbp amino acid sequence (SEQ ID NO 91) are set forth below SEQ ID NO:90

CTATTTGGTCGTAAAGGGTTAAAAAATGAC

SEQ ID NO:91

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEKSTETKKTSVIIRKYAEGDYSKLLEGATLRLTG EDIPDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKITEPIKFRWNKKVFIVQKDGSQVENPNKELGSPYTIEAYND FDEFGLLSTQNYAKFYYGKNYDGSSQIVYCFNANLKSPPDSEDHGATINPDFTTGDIRYSHIAGSDLIKYANTARDEDPQL

SKLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVQKTWGELGDKTKGFQFELELKDKTGQPIV NTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDL VPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGLKND

Orf 78 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:184 VPPTG (shown in italics in SEQ ID NO 91, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 78 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Three E boxes containing conserved glutamic residues have been identified in Orf 78 The E-box motifs are underlined in SEQ ID NO 91, below The conserved glutamic acid (E) residues, at amino acid residues 112, 395, and 447, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-like structures of Orf 78 Preferred fragments of Orf 78 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif SEQ ID NO:91 EDIPDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKITEPIKFRWMKKVFIVQKDGSQVENPNKELGSPYTIEAYND FDEFGLLSTQNYAKFYYGKNYDGSSQIVYCFNANLKSPPDSEDHGATINPDFTTGDIRYSHIAGSDLIKYANTARDEDPQL

SKLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVQKTWGELGDKTKGFQFELELKDKTGQPIV VPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGLKND

Orf 79 is thought to be a LepA signal peptidase I An example of the nucleotide sequence encoding a LepA signal peptidase I (SEQ ID NO 92) and a LepA signal peptidase I amino acid sequence (SEQ ID NO 93) are set forth below SEQ ID NO:92

AAAATCTCAACTCTATTAAGAGTGAGAGGAATT

SEQ ID NO:93

MTNYLNRLNENi

LKVGRIVAQAGDEVSFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGKYFILNDYREERLDSRYYGAL PVNQIKG KISTLLRVRGI

Orf 80 is thought to be a fimbπal protein An example of the nucleotide sequence encoding the fimbπal protein (SEQ ID NO 94) and a fimbπal protein amino acid sequence (SEQ ID NO 95) are set forth below SEQ ID NO:94

GCT

SEQ ID NO:95

MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGWTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTASGKEGSL DIKNGIVEGLDKQVTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTVDVYVGNKANNEE GFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKI EKQITGNAGDRKKSFNFTLTLQPSEYYKTGSWKIEQDGSKKDVTIGT PYKFTLGHGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQNQKTDESADEIWTNKRDTQVPTG WGTLAPFAVLSIVAIGGVIYITKRKKA

Orf 82 contains an ammo acid motif indicative of a cell wall anchor SEQ ID NO:140 QVPTG (shown in italics in SEQ ID NO 95, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 82 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition An E box containing a conserved glutamic residue has been identified in Orf 80 The E-box motif is underlined in SEQ ID NO 95, below The conserved glutamic acid (E), at amino acid residue 270, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus- like structures of Orf 80 Preferred fragments of Orf 80 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif SEQ ID NO:95

MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGWTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTASGKEGSL DIKNGIVEGLDKQVTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTVDVYVGNKANNEE GFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQITGNAGDRKKSFNFTLTLQPSEYYKTGSWKIEQDGSKKDVTIGT PYKFTLGHGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQNQKTDESADEIWTNKRDTQVPTG WGTLAPFAVLSIVAIGGVIYITKRKKA

Orf 81 is thought to be a SrtC2 type sortase An example of the nucleotide sequence encoding the SrtC2 sortase (SEQ ID NO 96) and a SrtC2 sortase amino acid sequence (SEQ ID NO 97) are set forth below. SEQ ID NO:96

ATTCAAGAA

SEQ ID NO:97

MISQRMMMTIVQVINKAIDTLILIFCLWLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNIP GTHIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFNKHNKAIIE

IQE

Orf 82 is referred to as a hypothetical protein It contains a sortase substrate motif LPXAG shown in italics in SEQ ID NO 99. An example of the nucleotide sequence encoding the hypothetical protein (SEQ ID NO 98) and a hypothetical protein amino acid sequence (SEQ ID NO.99) are set forth below SEQ ID NO:98

GTTAAAAAACTGAAGAGTAGGCTA

SEQ ID NO:99

MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSV ALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGD EEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

Orf 82 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:185 LPLAG (shown in italics in SEQ ID NO 99, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 82 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in Orf 82 The pilm motif sequence is underlined in SEQ ID NO 99, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 173 and 188 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc pilus-hke structures Preferred fragments of Orf 82 include at least one conserved lysine residue Preferably, fragments include the pilin sequence SEQ ID NO:99

MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSV ALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGD EEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

An E box containing a conserved glutamic residue has been identified in Orf 82 The E-box motif is underlined in SEQ ID NO 99, below The conserved glutamic acid (E), at amino acid residue 163, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus- like structures of Orf 82 Preferred fragments of Orf 82 include the conserved glutamic acid residue Preferably, fragments include the E box motif SEQ ID NO:99

MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSV ALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGD EEKSAITFKPKRLVKPI P PRQPNI PKT PL PLAGEVKSLLGI L S IVLLGLLVLLYVKKLKSRL

Orf 83 is thought to be a multiple sugar metabolism regulator protein An example of a nucleotide sequence encoding the sugar metabolism regulator protein (SEQ ID NO 100) and a sugar metabolism regulator protein amino acid sequence (SEQ ID NO 101) are set forth below SEQ ID NO:100

GCTAAATACCGAGATAATATT

SEQ ID NO:101

MIQLRMGAIYQMVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLC YYQFLKHLAIPQAAQDVIFYEGLFEESFM IFPLCHYIIAIGPFYPYSLNKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTI HQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRIPLSSITSSSISALRAEKNLTVI YLTRLLEFSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNSVLHYVDSHLYSKL KVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGI PVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYL AKYRDNI Orf 84 is thought to be a F2-hke fibronectin-binding protein An example of a nucleotide sequence encoding the F2 like fibronectin-binding protein (SEQ ID NO 102) and a F2-like fibronectin-binding protein amino acid sequence (SEQ ID NO 103) are set forth below SEQ ID NO 102

TGCTCACTTATTAGTAGTGTTTTTGTAATATCACTAAAAACTAAAAAACGCCTATCATCATGT

SEQ ID NO:103

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWL NGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVI ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY

VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPK KDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC

Orf 84 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:181 LPATG (shown in italics in SEQ ID NO 103, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant Orf 84 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in Orf 84 The pilin motif sequence is underlined in SEQ ID NO 103, below A conserved lysine (K) residue is also marked in bold, at amino acid residue 270 The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of Orf 84 include the conserved lysine residue Preferably, fragments include the pilin sequence SEQ ID NO:103 MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKMFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWL

ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY

VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DSSGKTISTWI SDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVI IGGQGQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPK KDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC

An E box containing a conserved glutamic residue has been identified in Orf 84 The E-box motif is underlined in SEQ ID NO 103, below The conserved glutamic acid (E), at amino acid residue 516, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus- like structures of Orf 84 Preferred fragments of Orf 84 include the conserved glutamic acid residue Preferably, fragments include the E box motif SEQ ID NO:103

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKNFPTAQGVSKKL YKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWL NGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVI SVEPVI ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY IYSVKEVDKNGELLEPKDYIKKEDGL WTNTWKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDE VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DS SGKT I STWI SDGQVKDFYLMPGKYTFVETAAPDGYE IATAITFTVNEQGQVTVNGKATKGDAH IVMVDAYKPTKG SGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPK KDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC

Examples of GAS AI-3 sequences from Ml 8 strain isolate MGAS8232 are set forth below SpyM18_0125 is a negative transcriptional regulator (Nra) An example of SpyM18_0125 is set forth in SEQ ID NO 72

SEQ ID NO:72

MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDITIKKGKIICCF VKPVKEFYLHQLYDTSTILKLLVFFIKNGTTSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSKNTIVGEEYRIRY LIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFYNMLLALS

SpyM18_0126 is thought to be a collagen binding protein (CBP) An example of SpyM18_0126 is set forth in SEQ ID NO 73

SEQ ID NO:73

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVAL IGI VGFS I RAFGAEEQSTETKKTSVI I RKYAEGDYSKLLEGATLKLAQ IEGSGFQEQSFESSTSGQKLQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKEVAEPYSVTAYND FDDSGFINPKTFTPYGKFYYAKNANGTSQWYCFNVDLHSPPDSLDKGETIDPDFNEGKEIKYTHILGADLFSYANNPRAS TNDELLSQVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHGFGALTTEALNATKEIVAYAEDRAN LPNISNLDFWPNSNKYQSLIGTQYHPESLVDIIRMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAI

L VPPTGLTTDGAI YLWLLLLVLLGLWVWL I GRKGLKND

SpyM18_0126 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:184 VPPTG (shown in italics in SEQ ID NO 73, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM18_0126 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pihn motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM18_0126 The pihn motif sequence is underlined in SEQ ID NO 73, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 172 and 179 The pihn sequence, in particular the conserved lysine residues, are thought to be important for the formation of ohgomeπc, pilus-Iike structures Preferred fragments of SpyM18_0126 include at least one conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO.73

MQKRDKTNYGSANWKRRQTTIGLLKVFLTFVAL I GIVGFS I RAFGAEEQSTETKKTSVI I RKYAEGDYSKLLEGATLKLAQ IEGSGFQEQSFESSTSGQKLQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKEVAEPYSVTAYND FDDSGFINPKTFTPYGKFYYAKNANGTSQWYCFNVDLHSPPDSLDKGETIDPDFNEGKEIKYTHILGADLFSYANNPRAS

TNDELLSQVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHGFGALTTEALNATKEIVAYAEDRAN LPNISNLDFYVPNSNKYQSLIGTQYHPESLVDIIRMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAI

LVPPTGLTTDGAIYLWLLLLVLLGLWVWLIGRKGLKND

Three E boxes containing conserved glutamic residues have been identified in SpyM18_0126 The E-box motifs are underlined in SEQ ID NO 73, below The conserved glutamic acid (E) residues, at amino acid residues 112, 257, and 415, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeric pilus-like structures of SpyM18_0126 Preferred fragments of SpyM18_0126 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif

SEQ ID NO:73

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSTETKKTSVIIRKYAEGDYSKLLEGATLKLAQ IEGSGFQEQSFESSTSGQKLQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKEVAEPYSVTAYND FDDSGFINPKTFTPYGKFYYAKNANGTSQWYCFNVDLHSPPDSLDKGETIDPDFNEGKEIKYTHILGADLFSYANNPRAS TNDELLSQVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHGFGALTTEALNATKEIVAYAEDRAN LPNISNLDFYVPNSNKYQSLIGTQYHPESLVDIIRMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSSDGQAI

LVPPTGLTTDGAIYLWLLLLVLLGLWVWLIGRKGLKND

SpyM18_0127 is a LepA protein An example of SpyM18_0127 is shown in SEQ ID NO 74

SEQ ID NO:74

MTNYLNRLNENPLFKAFIRLVLKISIIGFLGYILFQYIFGVMIINTNVMSPALSAGDGILYYRLTDRYHINDWVYEVDNT LKVGRIVAQAGDEVSFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSRYYGALPINQIKG KISTLLRVRGI

SpyMl8_0128 is thought to be a fimbπal protein An example ofSypM18_0128 is shown in SEQ ID NO 75 SEQ ID NO:75

PGVIDGLENTKTIHYGNSDKTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVYVVNREDGGFEAK YIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGEHQRSFSFTLLLTPNECFEKGQWNILQGGETKKWIGEEYSFT LKDKESVTLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKSTDEIWTNKRDTρvPTGWGTLAPF AVLSIVAIGGVIYITKRKKA

SpyM18_0128 contains an ammo acid motif indicative of a cell wall anchor SEQ ID NO:140 QVPTG (shown in italics in SEQ ID NO 75, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM18_0128 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM18_0128 The pilin motif sequence is underlined in SEQ ID NO 75, below A conserved lysine (K) residue is also marked in bold, at amino acid residue 57 The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeric, pilus-like structures Preferred fragments of SpyM18_0128 include the conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO:75 PGVIDGLENTKTIHYGNSDKTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVYWNREDGGFEAK YIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGEHQRSFSFTLLLTPNECFEKGQWNILQGGETKKWIGEEYSFT LKDKESVTLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKSTDEI WTNKRDTQVPTGWGTLAPF AVLSIVAIGGVIYITKRKKA

An E box containing a conserved glutamic residue has been identified in SpyM18_0128 The E-box motif is underlined in SEQ ID NO 75, below The conserved glutamic acid (E), at amino acid residue 266, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-like structures of SpyM18_0128 Preferred fragments of SpyM18_0128 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO:75

MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLWKKTFPSYTDDKVLMPKADYTFKVEADDNAKGKTKDGLDIK PGVIDGLENTKTIHYGNSDKTT AKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVYWNREDGGFEAK YIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGEHQRSFSFTLLLTPNECFEKGQWNILQGGETKKWIGEEYSFT LKD KESVTLSQLPVGIEYKVTEEDVT KDGYKTSATLKDGDVTDGYNLGDSKTTDKSTDEIWTNKRDTQVPTGWGTLAPF AVLSIVAIGGVIYITKRKKA

SpyM18_0129 is a SrtC2 type sortase An example of SpyM18_0129 is shown in SEQ ID NO 76

SEQ ID NO:76

MISQRMMMTIVQVINKAIDTLILIFCLWLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNIP

TKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIWGT IQE

SpyM18_0130 is referred to as a hypothetical protein An example of SpyM18_O13O is shown in SEQ ID

NO 77

SEQ ID NO:77

MRKYWKMLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSFSVALESIDAMKTIDEITIAGSGKASFSPLTF TTVGQYTYRVYQKPSQNKDYQADTTVFDVL VYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPDIPKTPL PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

SpyM18_O13O contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:185 LPLAG (shown in italics in SEQ ID NO 77, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM18_O13O protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM18_0130 The piltn motif sequence is underlined in SEQ ID NO 77, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 144, 159, and 169 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-like structures Preferred fragments of SpyM18_0130 include at least one conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO:77

MRKYWKMLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSFSVALESIDAMKTIDEITIAGSGKASFSPLTF TTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPDIPKTPL PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

An E box containing a conserved glutamic residue has been identified in SpyM18_O13O The E-box motif is underlined in SEQ ID NO 77, below The conserved glutamic acid (E), at amino acid residue 134, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-like structures of SpyM18_O13O Preferred fragments of SpyM 18_O13O include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO:77

TTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVI SRRAGDEEKSAITFKPKRLVKPI PPRQPDI PKTPL PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

SpyM 18_0131 is referred to as a putative multiple sugar metabolism regulator An example of SpyM18_O131 is set forth in SEQ ID NO.78.

SEQ ID NO-.78

MAIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAI PQAAQDVI FYEGLFEESFMIFPLCHYIIAI GPFYPYSLNKDYQEQLANNCLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTIHQLLQHSKQMT ADPDI I HRLKHI SKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRI PLSS I TS S S I SALRAEKNLTVI YLTRLLEFS FV

SESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI

SpyM18_0132 is a F2 like fibronectin-binding protein. An example of SpyM18_0132 is set forth in SEQ ID NO:79

SEQ ID NO:79

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKNFPTAQGVSKKL YKKLSSSDEETLKQYASKYTSNRRGDTSGNL KKQIAKVLTEGYPTNKSDWL NGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVI SVEPVI ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKL YRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY IYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDE VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVI IGGQGQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPK KDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC

SpyM18_0132 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:180 LPATG (shown in italics in SEQ ID NO:79, above). In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyM18_0132 protein from the host cell. Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall. The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition.

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyM18_0132 The pilin motif sequence is underlined in SEQ ID NO:79, below A conserved lysine (K) residue is also marked in bold, at amino acid residue 270. The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeπc, pilus-like structures. Preferred fragments of SpyM18_0132 include the conserved lysine residue. Preferably, fragments include the pilin sequence

SEQ ID NO:79

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKNFPTAQGVSKKL YKKLSSSDEETLKQYASKYTSNRRGDTSGNL KKQIAKVLTEGYPTNKSDWL NGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVI ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY IYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDE VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVI IGGQGQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPK KDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC

An E box containing a conserved glutamic residue has been identified in SpyM 18_0132 The E-box motif is underlined in SEQ ID NO 79, below The conserved glutamic acid (E), at amino acid residue 516, is marked in bold The E box motif in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeric pilus-hke structures of SpyM18_θπ2 Preferred fragments of SpyM18_O112 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO:79

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFEIKKNKSQEEYNYEVYDNRNILQDGEHKLEIK RVDGTGKTYQGFCFQLTKNFPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWL NGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVI ESLPWTSLKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGΞKEVAVDDAELKQINSEGQQEISVTWTNQLVTDEKGMAY

VTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELR DSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQV IDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPK KDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC

Examples of GAS AI-3 sequences from M49 strain isolate 591 are set forth below

SpyoM01000156 is a negative transcriptional regulator (Nra) An example of SρyoM01000156 is set forth in SEQ ID NO 243

SEQ ID NO:243

MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTAVQLKYYCKELDDFFGNNLDI TIKKGKIICCFVKPVKEFYLHQLYDTSTILKLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKL LREFGLRVSKNTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTSPWLEEPFSFY NMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYDCLTRSSRQVIENAFSLTFSQGDLDYLFLIYITTNNSF ASLQWTPQHIETCCHIFEKNDTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFS LPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNKQPALTWLISSNFINAKLLT DTIPRYFSDKGIHFYSFYLLRDDIYQIPSLKPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASI QNLIYQLKDKKYQDFLNEQLQ

SpyoM01000155 is thought to be a collagen binding protein (CPA) An example of SpyoM01000155 is set forth in SEQ ID NO 244

SEQ ID NO:244

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNRQSSIQDYPWYGYDSYP KGYPDYSPLKTYHNLKVNLEGSKDYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDG QLQQNILRILYNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSNGINDQQLGLM RKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKY AEGDYSKLLEGATLKLSQIEGSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK VFIVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKNKDKSSQWYCFNADLHSPPD SYDSGETINPDTSTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQF RAATQLAIYYFTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTNLDFFVPNNSK YQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTWGELGDKTKGFQFELELKDKTGQPIVNTLK TNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT FENRKDLT/PPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND

SpyoM01000155 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:184 VPPTG (shown in italics in SEQ ID NO 244, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000155 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two pilm motifs, discussed above, containing conserved lysine (K) residues have also been identified in SpyoM01000155 The pilin motif sequence is underlined in SEQ ID NO 244, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 71 and 261 The pilin sequences, in particular the conserved lysine residues, are thought to be important for the formation of oligomeric, pilus-hke structures Preferred fragments of SpyoM01000155 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO:244

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNRQSSIQDYPWYGYDSYP KGYPDYSPLKTYHNLKVNLEGSKDYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDG QLQQNILRILYNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSNGINDQQLGLM RKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKY AEGDYSKLLEGATLKLSQIEGSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK VFIVQKDGSQVENPNKEVAEPYSVEA YNDFMDEEVLSGFTPYGKFYYAKNKDKSSQWYCFNADLHSPPD SYDSGETINPDTSTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQF RAATQLAIYYFTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTNLDFFVPNNSK YQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTWGELGDKTKGFQFELELKDKTGQPIVNTLK TNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND

Two E boxes containing conserved glutamic residues have been identified in SpyoM01000155 The E-box motifs are underlined in SEQ ID NO 244, below The conserved glutamic acid (E) residues, at amino acid residues 329 and 668, are marked m 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 SpyoM01000155 Preferred fragments of SpyoM01000155 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif

SEQ ID NO:244

MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQSVPNRQSSIQDYPWYGYDSYP KGYPDYSPLKTYHNLKVNLEGSKDYQAYCFNLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDG QLQQNILRILYNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSNGINDQQLGLM RKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLLSAEYVPDTPPKPGEEPPAKTEKTSVIIRKY AEGDYSKLLEGATLKLSQIEGSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK VFIVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKNKDKSSQWYCFNADLHSPPD SYDSGETINPDTSTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQF RAATQLAIYYFTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTNLDFFVPNNSK YQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTWGELGDKTKGFQFELELKDKTGQPIVNTLK TNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND

SpyoM01000154 is a LepA protein An example of SpyoM01000154 is shown in SEQ ID NO.245

SEQ ID NO:245

MTNYLNRLNENSLFKAFIRLVLKISIIGFLGYILFQYVFGVMIINTNDMSPALSAGDGVLYYRLADRSHI NDVWYEVDNTLKVGRIAAQAGDEVNFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILN DYREERLDSRYYGALPINQIKGKISTLLRVRGI

SpyoMO 1000153 is thought to be a fimbπal protein An example of SpyoM01000153 is shown in SEQ ID NO 246

SEQ ID NO:246

MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLWKKTFPSYTDDNVLMPKADYSFKVEADDNA KGKTKDGLDIKPGVIDGLENTKTIRYSNSDKITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITY DSQQWTVDVYWNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGEHQRLFSFTLL LTPNECFEKGQWNILQGGETKKWIGEEYSFTLKDKESVTLSQLPVGIEYKLTEEDVTKDGYKTSATLK DGEQSSTYELGKDHKTDKSADEIWTNKRDTCVPTGWGTLAPFAVLSIVAIGGVIYITKRKKA

SpyoM01000153 contains an amino acid motif indicative of a cell wall anchor. SEQ ID NO:140 QVPTG (shown in italics in SEQ ID NO-246, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000153 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in SpyoM01000153 The pilin motif sequence is underlined in SEQ ID NO 246, below A conserved lysine (K) residue is also marked in bold, at amino acid residue 57 The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeπc, pilus like structures Preferred fragments of SpyoM01000153 include the conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO:246

MKKNKLLLATAILATALGMASMSQNI KAETAGVIDGSTLWKKTFPSYTDDNVLMPKADYSFKVEADDNA

KGKTKDGLDIKPGVIDGLENTKTIRYSNSDKITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITY DSQQWTVDVYWNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGEHQRLFSFTLL LTPNECFEKGQWNILQGGETKKWIGEEYSFTLKDKESVTLSQLPVGIEYKLTEEDVTKDGYKTSATLK

DGEQS STYELGKDHKTDKSADE IWTNKRDTQVPTGWGTLAPFAVLS IVAIGGVIYITKRKKA

An E box containing a conserved glutamic residue has been identified in SpyoM01000153 The E-box motif is underlined in SEQ ID NO 246, below The conserved glutamic acid (E), at amino acid residue 265, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-like structures of SpyoM01000153 Preferred fragments of SpyoMO 1000153 include the conserved glutamic acid residue Preferably, fragments include the E box motif

SEQ ID NO:246

MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLWKKTFPSYTDDNVLMPKADYSFKVEADDNA KGKTKDGLDIKPGVIDGLENTKTIRYSNSDKITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITY DSQQWTVDVYWNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGEHQRLFSFTLL LTPNECFEKGQWNILQGGETKKWIGEEYSFTLKDKESVTLSQLPVGIEYKLTEEDVTKDGYKTSATLK DGEQSSTYELGKDHKTDKSADEIWTNKRDTQVPTGWGTLAPFAVLSIVAIGGVIYITKRKKA

SpyoM01000152 is a SrtC2 type sortase An example of SpyoM01000152 is shown in SEQ ID NO 247

SEQ ID NO:247

MMMTIVQVINKAIDTLILIFCLWLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVI GWLNIPGTHIDYPLVQGKTNLEYINKAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIP KFLKKNFFNKHNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYISKRSKQFKPV KLKHHTKFVAF STC ENF STDNRVIWGT IQE

SpyoM01000151 is referred to as a hypothetical protein An example of SpyoM01000151 is shown in SEQ ID NO 248

SEQ ID NO:248

MLFSWMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASF SPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRL VKPIPPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

SpyoM01000151 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:185 LPLAG (shown in italics in SEQ ID NO 248, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000151 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 SpyoM01000151 The pilin motif sequence is underlined in SEQ ID NO 248, below Conserved lysine (K) residues are also marked in bold, at amino acid residue 138 The pilin sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of SpyoM01000151 include the conserved lysine residue Preferably, fragments include the pilin sequence

SEQ ID NO:248 MLFSWMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASF SPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRL VKPIPPRQPDI PKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

Two E boxes containing conserved glutamic residues have been identified in SpyoM01000151 The E-box motifs are underlined in SEQ ID NO 248, below The conserved glutamic acid (E) residues, at amino acid residues 58 and 128, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of ohgomeπc pilus-like structures of SpyoM01000151 Preferred fragments of SpyoM01000151 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif

SEQ ID NO:248

MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASF SPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRL VKPIPPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL

SpyoMO 1000150 is referred to as a putative MsmRL An example of SpyoMO 1000150 is set forth in SEQ ID NO 249

SEQ ID NO:249

MVI FDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAI PQAAQDVIFYEGLFEESFM IFPLCHYIIAIGPFYPYSLNKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFD TQFETTCQQTIHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRI PLSSITSSSISALRAEKNLWIYLTRLLEFSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAII HFSESLTNKSISDKRQMYNSVLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKI KEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI

SpyoM01000149 is a F2 like fibronectm-bindmg protein An example of SpyoM01000149 is set forth in SEQ ID NO 250 SEQ ID NO:250

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFEIKKVDQNNKPLSGATFSLTP KDGKGKPVQTFTSSEEGIIDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIIS KAGSKDVSSSLQLENPKMSWSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLD RRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTFTNYIAGLDKVQLSAELSLFLENKEVLENTN ISDFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDISRLNLRKDLEAKLPQGSTQGA NKRLRIDFGENLQGKAFWKVTGKADQSGKELIVQSHLSSFNNWGSYKTLRPNSHVSFTNEIALSPSKGS GSGTSEFTKPAITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEIHFKDLTSG TYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKPHSFVKVEANKEVTIVNHKETLTFSGKKIWE NDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL GNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYAD

GVAVEGQTKFI SGSGNEWSFEFKNLKKYNGTGNDI IYSVKEVTVPTGYDVTYSANDI INTKREVITQQGP

NLEIEETLPLESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATM ELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIV MVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKPSDVIIGGQGEWDTTEDTQSGMTGHSGSTTE IEDSKSSDVIIGGQGQWETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFDNKEPESNSEIPKKDKPKSNT SI/PATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLLSC

SpyoM01000149 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:180 LPATG (shown in italics in SEQ ID NO 250, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant SpyoM01000149 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in SpyoM01000149 The pilin motif sequences are underlined in SEQ ID NO 250, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 157 and 163, and 216 and 224 The pilin sequences, in particular the conserved lysine residues are thought to be important for the formation of oligomeπc, pilus like structures Preferred fragments of SpyoM01000149 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO:250

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFEIKKVDQNNKPLSGATFSLTP KDGKGKPVQTFTSSEEGIIDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIIS KAGSKDVSSSLQLENPKMSWSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLD RRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTFTNYIAGLDKVQLSAELSLFLENKEVLENTN ISDFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDISRLNLRKDLEAKLPQGSTQGA NKRLRIDFGENLQGKAFWKVTGKADQSGKELIVQSHLSSFNNWGSYKTLRPNSHVSFTNEIALSPSKGS GSGTSEFTKPAITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEIHFKDLTSG TYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKPHSFVKVEANKEVTIVNHKETLTFSGKKIWE NDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL GNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYAD GVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGP NLEIEETLPLESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATM ELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIV MVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKPSDVIIGGQGEWDTTEDTQSGMTGHSGSTTE IEDSKSSDVIIGGQGQWETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFDNKEPESNSEIPKKDKPKSNT SLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLLSC

Two E boxes containing conserved glutamic residues have been identified in SpyoM01000149 The E-box motifs are underlined in SEQ ID NO 250, below The conserved glutamic acid (E) residues, at amino acid residues 329 and 668, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-like structures of SpyoM01000149 Preferred fragments of

SpyoMO 1000149 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif

SEQ ID NO:250

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFEIKKVDQNNKPLSGATFSLTP KDGKGKPVQTFTSSEEGIIDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIIS KAGSKDVSSSLQLENPKMSWSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLD RRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTFTNYIAGLDKVQLSAELSIIFLENKEVLENTN ISDFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDISRLNLRKDLEAKLPQGSTQGA NKRLRIDFGENLQGKAFWKVTGKADQSGKELIVQSHLSSFNNWGSYKTLRPNSHVSFTNEIALSPSKGS GSGTSEFTKPAITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEIHFKDLTSG TYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKPHSFVKVEANKEVTIVNHKETLTFSGKKIWE NDRPDQRPAKIQVQLLQNGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL GNDIFNTRETEFVFEQNNFNLEFGNAEIKGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYAD GVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGP NLEIEETLPLESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATM ELRDSSGKTI STWI SDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDAHIV MVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKPSDVIIGGQGEWDTTEDTQSGMTGHSGSTTE IEDSKSSDVI IGGQGQWETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFDNKEPESNSEIPKKDKPKSNT SLPATGEKQHNKFFWMVTSCSLISSVFVISLKSKKRLLSC

As discussed above, applicants have also determined the nucleotide and encoded amino acid sequence of fimbπal structural subunits in several other GAS AI-3 strains of bacteria Examples of sequences of these fimbrial structural subunits are set forth below

M3 strain isolate ISS 3040 is a GAS AI-3 strain of bacteria ISS3040_fimbπal is thought to be a fimbria! structural subunit of M3 strain isolate ISS 3040 An example of a nucleotide sequence encoding the ISS3040_fimbπal protein (SEQ ID NO 263) and an ISS3040_fimbπal protein amino acid sequence (SEQ ID NO 264) are set forth below SEQ ID NO-263

GTTCTTAGC SEQ ID NO:264

ETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTKDGLEIKPGIVNGLTEQIISYTNTDKPDSKVKSTE FDFSKWFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYVGNKEGGGFEPKF IVSKEQGTDVKKPVNFNNSFATTSLKVK KNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKNGESIQLDKLPVGITYKVNEMEANKDG YKTTASLKEGDGQSKMYQLDMEQKTDESADEIWTNKRDTQVPTGWGTLAPFAVLS

M44 strain isolate ISS 3776 is a GAS AI-3 strain of bacteria. ISS3776_fimbrial is thought to be a fimbrial structural subunit of M44 isolate ISS 3776. An example of a nucleotide sequence encoding the ISS3776_fimbrial protein (SEQ ID NO:253) and an ISS3776_fimbπal protein ammo acid sequence (SEQ ID NO:254) are set forth below. SEQ ID NO:253 ttggagagagaaaaaatgaaaaaaaacaaattattacttgctactgcaatcttagcaact gctttaggaacagcttctttaaatcaaaacgtaaaagctgagacggcaggggttgtaaca ggaaaatcactacaagttacaaagacaatgacttatgatgatgaagaggtgttaatgccc gaaaccgcctttacttttactatagagcctgatatgactgcaagtggaaaagaaggcagc ctagatattaaaaatggaattgtagaaggcttagacaaacaagtaacagtaaaatataag aatacagataaaacatctcaaaaaactaaaatagcacaatttgatttttctaaggttaaa tttccagctataggtgtttaccgctatatggtttcagagaaaaacgataaaaaagacgga attacgtacgatgataaaaagtggactgtagatgtttatgttgggaataaggccaataac gaagaaggtttcgaagttctatatattgtatcaaaagaaggtacttctagtactaaaaaa ccaattgaatttacaaactctattaaaactacttccttaaaaattgaaaaacaaataact ggcaatgcaggagatcgtaaaaaatcattcaacttcacattaacattacaaccaagtgaa tattataaaactggatcagttgtgaaaatcgaacaggatggaagtaaaaaagatgtgacg ataggaacgccttacaaatttactttgggacacggtaagagtgtcatgttatcgaaatta ccaattggtatcaattactatcttagtgaagacgaagcgaataaagacggctacactaca acggcaacattaaaagaacaaggcaaagaaaagagttccgatttcactttgagtactcaa aaccagaaaacagacgaatctgctgacgaaatcgttgtcacaaataagcgtgacactcaa gttccaactggtgttgtagggacccttgctccatttgcagttcttagcattgtggctatt ggtggagttatctatattacaaaacgtaaaaaagcttaa

SEQ ID NO:254

MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGWTGKSL

QVTKTMTYDDEEVLMPETAFTFTIEPDMTASGKEGSLDIKNGIVEGLDKQVTVKYKNT

DKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTVDVYVGNKANN

EEGFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQITGNAGDRKKSFNFTLTLQP

SEYYKTGSWKIEQDGSKKDVTIGTPYKFTLGHGKSVMLSKLPIGINYYLSEDEANKD

GYTTTATLKEQGKEKSSDFTLSTQNQKTDESADEIWTNKRDTQVPTGWGTLAPFAV

LSIVAIGGVIYITKRKKA

M77 strain isolate ISS4959 is a GAS AI-3 strain of bacteria. ISS4959_fimbrial is thought to be a fimbrial structural subunit of M77 strain ISS 4959. An example of a nucleotide sequence encoding the ISS4959_fimbπal protein (SEQ ID NO:271) and an ISS4959_fimbrial protein amino acid sequence (SEQ ID NO:272) are set forth below. SEQ ID NO:271 gtaacagtaaaatataagaatacagataaaacatctcaaaaaactaaaatagcacaattt gatttttctaaggttaaatttccagctataggtgtttaccgctatatggtttcagagaaa aacgataaaaaagacggaattacgtacgatgataaaaagtggacngtagatgtttatgtt gggaataaggccaataacgaagaaggtttcgaagttctatatattgtatcaaaagaaggt acttctagtnctaaaaaaccaattgaatttacaaactctattaaaactacttccttaaaa attgaaaaacaaataactggcaatgcaggagatcgtaaaaaatcattcaacttcacattn acattacanccaagtgaatattataaaactggatcagttgtgaaaatcgaacaggatgga agtaaaaaagatgtgacgataggaacgccttacaaatttactttgggacacggtaagagt gtcatgttatcgaaattnccaattggtatcaattactatcttagtgaagacgaagcgaat aaagacggntacactacancggcaacattaaaagaacaaggcaaagaaaagagttccgat ttcactttgagtactcaaaaccagaaaacagacgaatctgctg

SEQ ID NO 272

VTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKK DGITYDDKKWTVDVYVGNKANNEEGFEVLYIVSKEGTSSXKKPIEFTNSIKTTSLKIE KQITGNAGDRKKSFNFTXTLXPSEYYKTGSWKIEQDGSKKDVTIGTPYKFTLGHGKS VMLSKXPIGINYYLSEDEANKDGYTTXATLKEQGKEKSSDFTLSTQNQKTDESA

Examples of GAS AI-4 sequences from M12 strain isolate A735 are set forth below

19224133 is thought to be a RofA regulatory protein An example of a nucleotide sequence encoding the RofA regulatory protein (SEQ ID NO 104) and a RofA regulatory protein amino acid sequence (SEQ ID NO 105) are set forth below

SEQ ID NO:104

GATTTAACCAAACAATTAACATAA

SEQ ID NO:105

MTIQKRMISCQFTHPSKETYLYQLYASSNVLQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALIPLLRNFELKLSK NKIVGEEYRIRYLIALLYSKFGIKVYDLTQQDKNIIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRHQFSVTIPQTR IFQQLKKLFVYDSLKKSSRDIIETYCQLNFSAGDLDYLYLIYITANNSFASLQWTPEHIRQCCQLFEENDTFRLLLNPIIT

LLPNLKEQKASLVKALMFFSKSFLFNLQHFIPETNLFVSPYYKGNQKLYTSLKLIVEEWMAKLPGKRYLNHKHFHLFCHYV VAEISFDESILSIQELMYQVKEEKFQADLTKQLT

19224134 is thought to be a protein F fibronectin binding protein An example of a nucleotide sequence encoding the protein F fibronectin binding protein (SEQ ID NO 106) and a protein F fibronectin binding protein amino acid sequence (SEQ ID NO 107) are set forth below SEQ ID NO:106

CTTGGAATCCTTATTTTGTCAGTACTTTCTATTTTTAGCCTTTTAAAAAACAAACAAAACAATAAAGTCTGA

SEQ ID NO:107

MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACAGAIGFGQVAYAADEKTVPNFKSPDPDYPWYG YDSYRGIFARYHNLKVNLKGSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDENLDKLEKNILKFV IYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPLNDVNKMWEREVRNGEISESQVTLMREALKKLIDPNLEATAANK IPSGYRLNIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKRPDESSEPALPPLMPELDGEEVPEV

PPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTK EPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMG GQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVFHFDNNEPKV EENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQNNKV

19224134 contains an ammo acid motif indicative of a cell wall anchor SEQ ID NO:181 LPATG (shown in italics in SEQ ID NO 107, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224134 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of 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 19224134 The pilin motif sequence is underlined in SEQ ID NO 107, below Conserved lysine (K) residues are also marked in bold, at ammo acid residues 275, 285, and 299 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of 19224134 include at least one conserved lysine residue Preferably, fragments include the pilin sequence SEQ ID NO: 107

MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACAGAIGFGQVAYAADEKTVPNFKSPDPDYPWYG YDSYRGIFARYHNLKVNLKGSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDENLDKLEKNILNV IYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPLNDVNKMWEREVRNGEISESQVTLMREALKKLIDPNLEATAANK IPSGYRLNIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKRPDESSEPALPPLMPELDGEEVPEV

PPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTK EPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMG GQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVFHFDNNEPKV EENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQNNKV

Two E boxes containing conserved glutamic residues have been identified in 19224134 The E-box motifs are underlined in SEQ ID NO 107, below The conserved glutamic acid (E) residues, at amino acid residues 487 and 524, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-like structures of 19224134 Preferred fragments of 19224134 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif SEQ ID NO:107 MVSSYMFARGEICMNN KMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACAGAIGFGQVAYAADEKTVPNFKSPDPDYPWYG YDSYRGIFARYHNLKVNLKGSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDENLDKLEKNILNV IYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPLNDVNKMWEREVRNGEISESQVTLMREALKKLIDPNL EATAANK IPSGYRLNIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKRPDESSEPALPPLMPELDGEEVPEV

PPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTK EPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMG GQSESVEFTKDTQTGMSGQTTPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGFSETVT IVEDTRPKLVFHFDNNEPKV EENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQNNKV

19224135 is thought to be a capsular polysaccharide adhesin (Cpa) protein An example of a nucleotide sequence encoding the Cpa protein (SEQ ID NO: 108) and a Cpa protein amino acid sequence (SEQ ID NO 109) are set forth below. SEQ ID NO: 108

TAA

SEQ ID NO:109

MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMFLTLVSSMRGAQSIFGEEKRIEEVSVPKIKSPDDAYPWYGYDSYDSSH PYYERFKVAHDLRVNLNGSKSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESLNNKLLSIMYNAY PKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTLWASELKDGKIDFEQVKLMREAYSKLISDDLEETSKNKLPQGSK LNI FVPQDKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLEGATLRLTGEDILDFQEKVFQSNGT GEKI ELSNGTYTLTETSSPDGYKIAEPIKFRWNKKVF IVQKDGSQVENPNKEVAEPYSVEAYSDMQDSNYINPETFTPYG KFYYAKNKDKSSQWYCFNADLHSPPESEDGGGTIDPDISTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKG YNKKGDSYNGLTETQFRAATQLAIYYFTDSTDLKTLKTYNNGKGYHGFESMDEKTLAVTKELINYAQDNSAPQLTNLDFFV PNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTWGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDL VAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDL VPPTGFITDG GTYLWLLLLVPFGLLVWFFGRKGLKND

19224135 contains an amino acid motif indicative of a cell wall anchor: SEQ ID NO:184 VPPTG (shown in italics in SEQ ID NO: 109, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224135 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pilin motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224135 The pilin motif sequence is underlined in SEQ ID NO 109, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 164 and 172 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of 19224135 include at least one conserved lysine residue Preferably, fragments include the pilin sequence SEQ ID NO: 109

MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMFLTLVSSMRGAQSIFGEEKRIEEVSVPKIKSPDDAYPWYGYDSYDSSH PYYERFKVAHDLRVNLNGSKSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESLNNKLLSIMYNAY PKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTLWASELKDGKIDFEQVKLMREAYSKLISDDLEETSKNKLPQGSK LNIFVPQDKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLEGATLRLTGEDILDFQEKVFQSNGT GEKIELSNGTYTLTETSSPDGYKIAEPIKFRWNKKVF IVQKDGSQVENPNKEVAEPYSVEAYSDMQDSNYINPETFTPYG KFYYAKNKDKS SQWYCFNADLHSPPESEDGGGTIDPDISTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKG YNKKGDSYNGLTETQFRAATQLAIYYFTDSTDLKTLKTYNNGKGYHGFESMDEKTLAVTKELINYAQDNSAPQLTNLDFFV PNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTWGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDL VAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGFITDG GTYLWLLLLVPFGLLVWFFGRKGLKND

An E box containing a conserved glutamic residue has been identified in 19224135 The E-box motif is underlined in SEQ ID NO 109, below The conserved glutamic acid (E), at amino acid residue 339, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-like structures of 19224135 Preferred fragments of 19224135 include the conserved glutamic acid residue Preferably, fragments include the E box motif SEQ ID NO:109

MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMFLTLVSSMRGAQSIFGEEKRIEEVSVPKIKSPDDAYPWYGYDSYDSSH PYYERFKVAHDLRVNLNGSKSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESLNNKLLSIMYNAY PKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTLWASELKDGKIDFEQVKLMREAYSKLISDDLEETSKNKLPQGSK LNIFVPQDKSVQNLLSAEWPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLEGATLRLTGEDILDFQEKVFQSNGT GEKIELSNGTYTLTKTSSPDGYKIAEPIKFRWNKKVFIVQKDGSQVENPNKEVAEPYSVEAYSDMQDSNYINPETFTPYG KFYYAKNKDKSSQWYCFNADLHSPPESEDGGGTIDPDISTMKEVKYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVIEKG YNKKGDSYNGLTETQFRAATQLAIYYFTDSTDLKTLKTYNNGKGYHGFESMDEKTLAVTKELINYAQDNSAPQLTNLDFFV PNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTWGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDL VAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGFITDG GTYLWLLLLVPFGLLVWFFGRKGLKND

19224136 is thought to be a LepA protein An example of a nucleotide sequence encoding the LepA protein (SEQ ID NO.110) and a LepA protein amino acid sequence (SEQ ID NO 111) are set forth below SEQ ID NO:110

AAAATCTCAACTCTATTAAGAGTGAGAGGAATTTAA

SEQ ID NO:111

MTNYLNRLNENP

LKVGRIAAQAGDEVSFTQEGGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSRYYGALPINQIKG

KISTLLRVRGI

19224137 is thought to be a fimbπal protein An example of a nucleotide sequence encoding the fimbπal protein (SEQ ID NO 1 12) and a fimbπal protein amino acid sequence (SEQ ID NO 113) are set forth below SEQ ID NO:112

CTTAGCATTGTGGCTATTGGTGGAGTTATCTATATTACAAAACGTAAAAAAGCTTAA SEQ ID NO:113

GIAVNNQDIKVSYSNTDKTSGKEKQVWDFMKVTFPSVGIYRYWTENKGTAEGVTYDDTKWLVDVYVGNNEKGGLEPKYI VSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHKKAFTFTLTLQPNEYYEAS SWKI EENGQTKDVKIGEAYKFTLN DSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIWTNNRDTQVPTGWGTLAPFAV LSIVAIGGVIYITKRKKA

19224137 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:140 QVPTG (shown in italics in SEQ ID NO 113, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224137 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

A pihn motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224137 The pilin motif sequence is underlined in SEQ ID NO 113, below A conserved lysine (K) residue is also marked in bold, at amino acid residue 160 The pihn sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-hke structures Preferred fragments of 19224137 include the conserved lysine residue Preferably, fragments include the pihn sequence SEQ ID NO:113

MKKNKLLLATAILATALGTASLNQNVKAETAGWSSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKP GIAVNNQDIKVSYSNTDKTSGKEKQVWDFMKVTFPSVGIYRYWTENKGTAEGVTYDDTKWLVDVYVGNNEKGGLEPKYI VSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHKKAFTFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLN DSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIWTNNRDTQVPTGWGTLAPFAV LSIVAIGGVIYITKRKKA

An E box containing a conserved glutamic residue has been identified in 19224137 The E-box motif is underlined in SEQ ID NO 113, below The conserved glutamic acid (E), at amino acid residue 263, is marked in bold The E box motif, in particular the conserved glutamic acid residue, is thought to be important for the formation of oligomeπc pilus-hke structures of 19224137 Preferred fragments of 19224137 include the conserved glutamic acid residue Preferably, fragments include the E box motif SEQ ID NO:113

GIAVNNQDIKVSYSNTDKTSGKEKQVWD FMKVTFPSVGIYRYWTENKGTAEGVTYDDTKWL VD VYVGNNEKGGLEPKYI VSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHKKAFTFTLTLQPNEYY EASSWKIEENGQTKDVKIGEAYKFTLN DSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIWTNNRDTQVPTGWGTLAPFAV LSIVAIGGVIYITKRKKA

19224138 is thought to be a SrtC2-type sortase An example of a nucleotide sequence encoding the SrtC2 sortase (SEQ ID NO 1 14) and a SrtC2 sortase amino acid sequence (SEQ ID NO 1 15) are set forth below SEQ ID NO:114

SEQ ID NO:115

MMMTIVQVINKAIDTLILIFCLWLFLAGFGLWDSYHLYQQADASNFKKFKTAQQQPKFEDLLALNEDVIGWLNI PGTHID

KLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYISKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIWGTIQE

19224139 is an open reading frame that encodes a sortase substrate motif LPXAG shown in italics in SEQ ID NO 117 An example of a nucleotide sequence of the open reading frame (SEQ ID NO 116) and the amino acid sequence encoded by the open reading frame (SEQ ID NO 117) are set forth below SEQ ID NO:116

SEQ ID NO:117

MLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQY TYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEV KSLLGILSIVLLGLLVLLYVKKLKSKL

19224139 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:185 LPLAG (shown in italics in SEQ ID NO 117, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224139 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular 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 pihn motif, discussed above, containing a conserved lysine (K) residue has also been identified in 19224139 The pihn motif sequence is underlined in SEQ ID NO 117, below A conserved lysine (K) residue is also marked in bold, at ammo acid residue 138 The pihn sequence, in particular the conserved lysine residue, is thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of 19224139 include the conserved lysine residue Preferably, fragments include the pihn sequence SEQ ID NO:117

TYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEV KSLLGILSIVLLGLLVLLYVKKLKSKL

Two E boxes containing conserved glutamic residues have been identified in 19224139 The E-box motifs are underlined in SEQ ID NO 117, below The conserved glutamic acid (E) residues, at amino acid residues 58 and 128, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-like structures of 19224139 Preferred fragments of 19224139 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif

SEQ ID NO:117

MLFSWMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKTIEEITIAGSGKASFSPLTFTTVGQY TYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEV KSLLGILSIVLLGLLVLLYVKKLKSKL

19224140 is thought to be a MsmRL protein An example of a nucleotide sequence encoding the MsmRL protein (SEQ ID NO 118) and a MsmRL protein amino acid sequence (SEQ ID NO 119) are set forth below SEQ ID NO: 118

CATAAAATCTTTAAAAAATACACGGGTATTTCTTCAAAAGACTATCTTGCTAAATACCGAGATAATATTTAA

SEQ ID NO:119

MVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLAIPQAAQDVIFYEGLFEESFMIFPLCHYIIAI GPFYPYSLNKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTIHQLLQHSKQMT ADPDIIHRLKHISKASSQLPPVL EHLNHIMDLVKLGNPQLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFV ENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNSVLHYVDSHLYSKL KVSDIAKRL YV SESHLRSVFKKYSNVSLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI

19224141 is thought to be a protein F2 fibronectin binding protein An example of a nucleotide sequence encoding the protein F2 fibronectin binding protein (SEQ ID NO 120) and a protein F2 fibronectin binding protein amino acid sequence (SEQ ID NO 121) are set forth below SEQ ID NO:120

TAA

SEQIDNO:121

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFEIKKVDQNNKPLPGATFSLTSKDGKGTSVQTF TSNDKGIVDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSSLQLENPKMSVVS KYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLDRRLNPKGISQDIPKIIYDSANSPLAIGKYHAEN HQLIYTFTDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGS

IESYNTETGEFVWYVYVNPNRTNIPYATMNLWGFGRARSNTSDLENDANTSSAELGEIQVYEVPEGEKLPSSYGVDVTKLT LRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGKPLWQSNLASFRGASEYAAFTPVGGNVYFQNEIALS PSKGSGSGKSEFTKPSITVANLKRVAQLRFKKMSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEVHFKDLTSGTYDLYE TKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGSPHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQ NGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVNVPDGYKVSYLGNDIFNTRETEFVFEQNNFNLEFGNAEI

KEVTVPTGYDVTYSANDIINTKREVITQQGPKLEIEETLPLESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSA THIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGK ATKGDTHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDLIIGGQGEWDTTEDTQSGMTGHSGSTTEIE DSKSSDVIIGGQGQWETTEDTQTGMYGDSGCKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKPKSNTSLPATGEKQHNMF FWMVTSCSLISSVFVISLKSKKRLSSC

19224141 contains an amino acid motif indicative of a cell wall anchor SEQ ID NO:181 LPATG (shown in italics in SEQ ID NO 121, above) In some recombinant host cell systems, it may be preferable to remove this motif to facilitate secretion of a recombinant 19224141 protein from the host cell Alternatively, in other recombinant host cell systems, it may be preferable to use the cell wall anchor motif to anchor the recombinantly expressed protein to the cell wall The extracellular domain of the expressed protein may be cleaved during purification or the recombinant protein may be left attached to either inactivated host cells or cell membranes in the final composition

Two pilin motifs, discussed above, containing conserved lysine (K) residues have also been identified in 19224141 The pilin motif sequences are underlined in SEQ ID NO 121, below Conserved lysine (K) residues are also marked in bold, at amino acid residues 157 and 163 and at amino acid residues 216, 224, and 238 The pilin sequence, in particular the conserved lysine residues, are thought to be important for the formation of oligomeπc, pilus-like structures Preferred fragments of 19224141 include at least one conserved lysine residue Preferably, fragments include at least one pilin sequence SEQ ID NO:121

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFEIKKVDQNNKPLPGATFSLTSKDGKGTSVQTF TSNDKGIVDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSSLQLENPKMSWS KYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLDRRLNPKGISQDIPKIIYDSANSPLAIGKYHAEN HQLIYTFTDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGS iESYNTETGEFVWYVYVWPNRTNiPYATMNLWGFGRARSNTSDLEND ANTSSΆELGEIQVYEVPEGEKLPSSYGVDVTKLT LRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGKPLWQSNLASFRGASEYAAFTPVGGNVYFQNEIALS

TKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGSPHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQ NGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVNVPDGYKVSYLGNDIFNTRETEFVFEQNNFNLEFGNAEI

KEVTVPTGYDVTYSANDIINTKREVITQQGPKLEIEETLPLESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTI EEDSA THIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGK ATKGDTHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDLI IGGQGEWDTTEDTQSGMTGHSGSTTEIE DSKSSDVIIGGQGQWETTEDTQTGMYGDSGCKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKPKSNTSLPATGEKQHNMF FWMVTSCSLISSVFVISLKSKKRLSSC

Two E boxes containing conserved glutamic residues have been identified in 19224141 The E-box motifs are underlined in SEQ ID NO 121, below The conserved glutamic acid (E) residues, at amino acid residues 567 and 944, are marked in bold The E box motifs, in particular the conserved glutamic acid residues, are thought to be important for the formation of oligomeπc pilus-like structures of 19224141 Preferred fragments of 19224141 include at least one conserved glutamic acid residue Preferably, fragments include at least one E box motif SEQ ID NO:121

MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFEIKKVDQNNKPLPGATFSLTSKDGKGTSVQTF TSNDKGIVDAQNLQPGTYTLKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSSLQLENPKMSWS KYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETINPGDTFVLQLDRRLNPKGISQDIPKIIYDSANSPLAIGKYHAEN HQLIYTFTDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVNVLYGNESTKESNYITNGLSNVGGS IESYNTETGEFVWYVYVNPNRTNIPYATMNLWGFGRARSNTSDLENDANTSSAELGEIQVYEVPEGEKLPSSYGVDVTKLT LRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGKPLWQSNLASFRGASEYAAFTPVGGNVYFQNEIALS PSKGSGSGKSEFTKPSITVANLKRVAQLRFKKMSTDNVPLPEAAFELRSSNGNSQKLEASSNTQGEVHFKDLTSGTYDLYE TKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGSPHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQ NGQKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVNVPDGYKVSYLGNDIFNTRETEFVFEQNNFNLEFGNAEI KGQSGSKIIDEDTLTSFKGKKIWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNGTGNDIIYSV KEVWPTGYDVTYSANDIINTKREVITQQGPKLEIEETLPLESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSA THIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGK ATKGDTHIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDLI IGGQGEWDTTEDTQSGMTGHSGSTTEIE DSKSSDVIIGGQGQWETTEDTQTGMYGDSGCKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKPKSNTSLPATGEKQHNMF FWMVTSC SLI S SVFVI SLKSKKRLS SC

As discussed above, applicants have also determined the nucleotide and encoded amino acid sequence of fimbπal structural subunits in several other GAS AI-4 strains of bacteria Examples of sequences of these fimbπal structural subunits are set forth below

M12 strain isolate 20010296 is a GAS AI-4 strain of bacteria 20010296_fimbπal is thought to be a fimbπal structural subunit of M12 strain isolate 20010296 An example of a nucleotide sequence encoding the 20010296_fϊmbπal protein (SEQ ID NO 257) and a 20010296_fimbπal protein amino acid sequence (SEQ ID NO 258) are set forth below SEQ ID NO 257

AAAGATGGAGAAAAGTTATCTACTTATAACTTAGGTCAGGAACATAAAACAGACAAGACTGCTGATGAAATCGT

SEQ ID NO 258

SSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKTSGKEKQVW EVTGNTGDHKKAFNFTLTLQPNEYYEASSWKIEENGQTKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGY TTTATLKDGEKLSTYNLGQEHKTDKTADEIV

M12 strain isolate 20020069 is a GAS AI-4 strain of bacteria 20020069_fimbπal is thought to be a fimbπal structural subunit of M12 strain isolate 20020069. An example of a nucleotide sequence encoding the 20020069_fimbπal protein (SEQ ID NO:259) and a 20020069_Fimbπal protein amino acid sequence (SEQ ID NO:260) are set forth below SEQ ID NO:259

AAAGATGGAGAAAAGTTATCTACTTATAACTTAGGTCAGGAACATAAAACAGACAAGACTGCTGATGAAATCGT SEQ ID NO:260

SSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKTSGKEKQVWDFMKVT

GDHKKAFNFTLTLQPNEYYEAS SWKIEENGQTKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATL KDGEKLSTYNLGQEHKTDKTADEIV

M12 strain isolate CDC SS 635 is a GAS AI-4 strain of bacteria CDC SS 635_fϊmbrial is thought to be a fϊmbrial structural subunit of M12 strain isolate CDC SS 635. An example of a nucleotide sequence encoding the CDC SS 635_fimbrial protein (SEQ ID NO:261) and a CDC SS 635_fimbrial protein amino acid sequence (SEQ ID NO:262) are set forth below. SEQ ID NO:261

GATGAAATCGTTGTCACAAATAACCGTGACACT

SEQ ID NO:262

ETAGWSSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKTSGKEKQVW DFMKVTFPSVGIYRYWTENKGTAEGVTYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEK EVTGNTGDHKKAFNFTLTLQPNEYYEAS SWKIEENGQTKDVKIGEA YKFTLNDSQSVILSKLPVGINYKVEEAEANQGGY TTTATLKDGEKLSTYNLGQEHKTDKTADEIWTNNRDT

M5 strain isolate ISS 4883 is a GAS AI-4 strain of bacteria. ISS4883_fimbrial is thought to be a fimbria! structural subunit of M5 strain isolate ISS 4883. An example of a nucleotide sequence encoding the ISS4883_fimbrιal protein (SEQ ID NO:265) and an ISS4883_fimbrial protein amino acid sequence (SEQ ID NO:266) are set forth below. SEQ ID NO:265 ACAGACGAATCTGCTGACGAAATCGTTGTCACAAATAAGCGTGACACTCTCGAG

SEQ ID NO.266

ETAGWTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTASGKEGDLDIKNGIVEGLDKQVTVKYKNTDKTSQKTKIAQF

EKQITGNAGDRKKSFNFTLTLQPSEYYKTGSWKIEQDGSKKDVTIGTPYKFTLGHGKSVMLSKLPIGINYYLSEDEANKD GYTTTATLKEQGKEKSSDFTLSTQNQKTDESADEIWTNKRDTLE

M50 strain isolate ISS4538 is a GAS AI-4 strain of bacteria. ISS4538_fimbrial is thought to be a fimbrial structural subunit of M50 strain ISS 4538. An example of a nucleotide sequence encoding the ISS4538_fϊmbπal protein (SEQ ID NO:255) and an ISS4538_fimbπal protein amino acid sequence (SEQ ID NO 256) are set forth below. SEQ ID NO:255

CTTANCATTGNGGCTANTGGTGGNGTNATNTATNTTACAAAACGNAAAAAAGNATAA

SEQ ID NO:256

MKKNKLLLATAILATALGTASLNQNVKAETAGWSSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKP GIAVNNQDIKVSYSNTDKTSGKEKQVWDFMKVTFPSVGIYRYWT ENKGTAEGVTYDDTKWLVDVYVGNNEKGGLEPKYI VSKKGDSATKEPIQFNNSFETTSLKIEKKVTGNTGDHKKAFNFTLTLQ PNEYYEAS SWKIEENGQTKDVKIGEAYKFTLN DSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADEIWTNXRDTXVPTGWGTPPPFXV LX I XAXGGVXYXTKRKKX

Examples of GAS AI-5 sequences from M2 strain isolate 10270 are set forth below.

MGAS 10270_Spy0107 is a 33 kDa chaperonin which flanks GAS AI-5. An example of an amino acid sequence for MGAS10270_Spy0107 is shown below as SEQ ID NO:296. SEQ ID NO:296

MDKI IKS IAQSGAFRAYVLDSTETVALAQEKHNTLSSSTVALGRTLIANQILAANQKGDSKITVKVIGDSSFGHI I SVADT KGHVKGYIQNTGVDIKKTATGEVLVGPFMGNGHFVTIIDYGTGNPYTSTTPLITGEIGEDFAYYLTESEQTPSAIGLNVLL DENDKVKVAGGFMVQVLPGASEEEIARYEKRLQEMPAISYLLASKNHVD ALLEAIYGDEPYKRLSEEPLSFQCDCSRERFE AALMTLPKADLQAMIDEDKGAEIVCQFCGTKYQFNESDLEAIINDKA

MGAS 10270_Spy 108 is a transcriptional regulator (RofA) An example of an ammo acid sequence for MGAS 10270_Spy 108 is shown below as SEQ ID NO:297. SEQ ID NO:297

MISIFSLDRIEIGEYTYQRLIWLSKCRKRGPLSLIEKYLESSIESKCQLWLFFKTSYLPITEVAEKTGLTFLQINHYCEE LNAFF PGSLSMTIQKRMISCQFTHPFKETYLYQL YASSNILQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALI PL LRNFELKLSKNKIVGEEYRIRYLIALLYSKFGIKVYDLTQQDKNIIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRH QFSVTIPQTRIFQQLKKLFVYDSLKKSSRDIIETYCQLNFSAGDLDYLYLIYITANNOSFASLQWTPEHIRQCCQLFEEND TFRLLLNPI ITLLPNLKEQKASLVKALMFFSKSFLFNLQHFI PETNLFVSPYYKGNQKLYTSLKLIVEEWMAKLPGKRYLN HKHFHLFCHYVEQILRNIQPPLVWFVASNFINAHLLTDSFPRYFSDKSIDFHSYYLLQDNVYQIPDLKPDLVITHSQLIP FVHHELTKGIAVAEISFDESILSIQELMYQVKEEKFQADLTKQLT

MGAS 10270_Spy 109 is a hypothetical protein An example of an amino acid sequence for MGAS 10270_Spy 109 is shown below as SEQ ID NO 298 It contains a motif indicative of a cell wall anchor lpxTG (SEQ ID NO 133) SEQ ID NO 298

MKLSKKLLYSAWLATVAGPTVSPVAQFATSGIWRAEDTRVPSQTQPDKTTVNIYKLQGADFSKQPEGIKNENGEPIDIT KLKDTFGTAVTYLPGVKFKYYKVKNYSTSDDVLKSIKTVEQADSKTDLLDVAGAKETEATDQSGKVSIDLPSNDKVKYLFV ESSNQDTVNKVVGYTAVPFILHLPVSNSNGKGYYDEVNVYPKNTTVNEPKVDKDVTKLGKDDDTYQIGDKITWFLKSTVPS NIKTLDKFGFTDTLNKGLSFIGDKTQTVTKVQFGTTVLSPDTDYTVEILDSKLTVSLTSAGIEKVSGLVASKQLITEAEKL YKAEDNTDEAAFLSVEVNAKLNADAVMGSRIENDVELDYGHESDIYKSKVPTNEVPEVHTGGARFEKVDATNQTDKLQDAE

TTYYI KELVAPKGYWSQDIVQFDVTYSSYYKD PTKVTLGTEAGDAAPTSVKNNKRPSIPNTGGIGTAI FVAIGAAVMAFA AKGMKRRTEEN

MGAS10270_Spy0110 is a hypothetical protein An example of an amino acid sequence for MGAS10270_Spy0110 is shown below as SEQ ID NO 299 It contains a motif indicative of a cell wall anchor, IpxTG (SEQ ID NO 133) SEQ ID NO 299

MKQTLKLWSFLVMLGTMFGISQTVLAQGTHQLTIVHLEARDIDRPYPQLDIAPKEGTPTEGVLYQLYQLKSTEDGDLLAH WNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKDRNVSAFLVDLSEDKVIYPKI IWSTGELDLLKVG VDGDTKKPLAGWFELYEKDGMVPIRVKNGWSQDIDAAKRLETDSSGHIRISGLIPGDYVLKEIETRSGYQIDQSETAVT IEKSKTVTVTIKNQKIPSPKVPPRGGFIPKTGEQQAMILVIIGGILIALALRLLSKHRKNQDKH

MGAS10270_Spy0111 is a sortase An example of an amino acid sequence for MGAS10270_Spy0111 is shown below as SEQ ID NO 300 SEQ ID NO 300

MRKRSKTSLATNIRIWI FRLIFLAGFLVLAFPIVSQITYYQASHANINAFKKAVAKIDQSEINRRLELAYAYNASIAGARK TGEHPVLKDPYSAEQKQAGVIEYARMLEVKEQIGHVIIPRINQDIPIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTA HRGLPTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVISPDQLEDLYVIQGEDHITLLTCTPYMINSHRLLVRGKRIP YVKKAVQKEAETFRQKQYLTYAIWVIVGLILLSFLIWFKKTKQKKRRENEKATSQISHNNSK

MGAS10270_Spy0112 is a sortase An example of an amino acid sequence for MGAS10270_Spy0112 is shown below as SEQ ID NO 301 SEQ ID NO 301

VIDPFTRKQKEGLREYARML EVHEQIGHVAVPSIGVDIPIYAGTAESVLQKGSGHLEGTSLPVGGQSTHAVLTAHRGLPTA KCKEQAIQGYHLSLVLKILLGLLIGLFIVIMMRRWMKHRQ

MGAS10270_Spy0113 is a collagen adhesion protein An example of an amino acid sequence for MGAS10270_Spy0113 is shown below as SEQ ID NO 302 It contains a motif indicative of a cell wall anchor, FPxTG (SEQ ID NO 141) SEQ ID NO 302

MKKKQKLWRGLSVTLLILSQIPFGILVQGETQDNNPALGKVIVKKTGENAMPLGKATFVLKNDHDKSEISHETVEGSGEAA FENIKPGNYTLTEKTAPIGYKKTDKTWKVKVADNGATTIEDIDPDKVEKRKEALNGQYPESAIYEDTKESYPLVKVEDSKV GNQYKALNPINGEDGRREITEGWLSKKIKKVNELDKNKYKIELTVEGKTIVETKELNQPLDWLLLDNSNSMNNERAHNSQ

ANEVNILKSRI PKEAEHINGNRTLYQFGATFTQKALMKANEILETQSSNDRKKVIFHVTDGVPTMSYAINFNPYISTSYQN QFKSFLNKTPDRSGILQEDFI INGDDYQIVKGDGESFKLFSDRKVPVPGGTTQAAYQVPQNQLSVMSNEGYAINRGYIYLY WRD YNWVYPFDPKTKTVSATKQIKTHGEPTTLYFNGNIKPKGYDIFTVGIGVNGDPGATPLEAKEFMQSISSKTENYTNVD DTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMIEFQLKDGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVT VTYDKTSQTIKINHLNLGSGQ KWLTYDVRLKENYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKI RDVREFPVLTI SN

KPWTFTIQNGEVTNLKQDPNANKNQIGYFEEDGKHLITNTPKRPPSVFPKTGGIGTIVYILVGCTLMIVATGSFRRNQQ

MGAS10270_Spy0114 is a hypothetical protein An example of an amino acid sequence for MGAS10270_Spy0114 is shown below as SEQ ID NO 303 SEQ ID NO 303

MISSYLSLLSVIGLAKAYNASLSGTSSQATQSVLRDSYSEEQKRQGGLNTLGC

MGAS10270_Spy0115 is a sortase An example of an amino acid sequence for MGAS10270_Spy0115 is shown below as SEQ ID NO 304 SEQ ID NO 304

MDKMRRGDRFYIEHLGGKLAYQVDSIKVITPDRLDDLYLVPGKD WTLLTCTPYMINSHRLLVRGKRIPYKEGLEHKDQQL GHRGQL I TYGFVSLGLALVLGLLWYRQRKK

MGAS10270_Spy0116 is a sortase An example of an amino acid sequence for MGAS10270_Spy0116 is shown below as SEQ ID NO 305 SEQ ID NO 305

LNDPFTSKEKAGLREYARMLEVNEQIGHVAIPKIGVDLPIYAGTSAAILEKGSGHLEGTSLPIGGLSTHAVLTAHRGLPTA RLFTDLDKVKKGDYFYVTNLKETLAYQVDRIMVIEPSQLDAVSIEEGKD YVTLLTCTPYMINSHRLLVRGVRTPLTSRQAK KESLTAVRPYQYYRWLVYLAIAVLVILVMIVAKYYQKTNKSS

MGAS10270_Spy0117 is a fibronectin binding protein An example of an amino acid sequence for MGAS10270_Spy0117 is shown below as SEQ ID NO 306 It contains a motif indicative of a cell wall anchor, LPXTG (SEQ ID NO 122) SEQ ID NO 306

MSIVTNKTLHKVL VKLAAILAILFL VLPTGLTAVTVLAEQINTENLIPVETAAGKILKESDLDFEQAISTSEEALKDSSAS

WIQLTYNKVNQNLAKQDLFLRLPQGMTIEGIGQDQKLDVKTISVPKDLDLDEAASTINNTNIITGFEKIGEQDYKLSFAAT SKTLNL VFKVKGTSLNEKRELQLSDKQNLNQTSFTKILGLGIAKSDDRTSANDTTTRPSIQPRAATARETTGNQDISANLT TSNAQLNLEDKDKNNIYDVEAPDSFMFRATLSLLDSRYIVPGNYFELKLSDTIHYNMLNPTDINFPYLTQDGKVIAVPELV QDPPVDGLLRATGKIVRYRFTDQVQGLDSLTMTLNLGHSVNPNWQNNDKYDFSYQIGGHIIKQSYDVQYGKPKENGNLNV HQRLTYTDSKENLKASSLIYVNPKQTPQYAGKQTLSILRNPKTYTISEQTVPNLLELNNQTSIKVYKLKDSKGVTDAVNLD KQYLEEVINPIITKSDGKIEVSFNYTQGETSAYWAVETSLQQEGSQPVFIAQNSNLTSASTRKKVSETDATATVGSGGSA SGDQTPKGTLYLNKVDTNHKHLANATFTLSGNTTAGQFLYRTLITDTKEVSFSDLPPGSYLLKETTAPSGYQQITT PWTVT VDDKGKVTVTGNEADTQKVPEEAEWILWESTKWNYGNVQFYKDRFKELVDSVGNQNAKYTLIRYSGDTSDSAQVINQSVS SAEFNKILNSETLTASTMTNRKGMLKAYQLALQQFQASTNKRKYLLQLTNYPIYPGYREEKDFMSQSQTSFDAMKSLGWP YLLVERDPYANRNTYLDPKAEASSFGAFYPSDNIKWWSNNSSGLFTPPTNTNYTIQIQSIGSAIKQQIPESVLTVINRAS GKFSINKIDEAKKGLAGATFTLSKRTTVAVNHQVQGAFTPVSKETTAGRTTLTFDNLKPGVYDLKETKAPNAYVLDPKTYV VWQNSGKTTIVDEANFKEADYPMADNTSQFDYPTKDIANKPNKIVFTKIGDGGKSLSGAEFELRKGNEKVQTTTSGADGK VTFSKLLPGTYEVWETRVADSAYQLPQEAVATFEVKADGTFSEPMGRLFRKNIAQNNRYEIRNELVNKKTGNQKIKVIKKD

QPTVAPTARAALAAPVAEPVTGKNVSDQISIKELDITSSNEDTPRLVRPNHGENIVMRAGFTINPGSDIKAGDFFTLTLPN TIDPFGVSAPENVDFRILGPLGTLALGTYDSSTHTITYKFTDYITRYTVSSFSTISPFFIDRDRVKTNQDIDLFLKVGQVS STSYHFTVD YNPYYGTADTNNPVNVGSMITRLNQDTGDFVNYIYVNPAGQTLEQATLTFTGRGSTRIDANTRVQLFEVTNP KLQMPPSWGIQDETLREVDPANYQLIKENGRFTINF YNDLLYGRSYIVKVSGKSDKNDPDPIHTSAILTQRYFNDYPYYTP SGRYIPYGPYTESFTFTAEWKKSGESNADGSVWRLSNRKNYIDFMKSNSQGTPLEATFELRKKATNDTVTVGTPVISDK

LVAIFWDHSFRFTK

MGAS10270_Spy0118 is a hypothetical protein which flanks GAS AI-5 An example of an amino acid sequence for MGAS10270_Spy0118 is shown below as SEQ ID NO 307 SEQ ID NO:307

MKRYNKYLFTSLLAASMLFSSYKSVHAHDNIDEKGKVHLYWQGNYYVDNYVDYTKTLVDNNNSIEWTVTFNSAKEQWVYPD FSVFLPKGVKAPKEITYEHHYWDGTVDSKTRRNTKWHYDWESQQTNFNQEFDKFPGYTGWSPSLDKFYKLKNEGKFSHVLV DTYGRQSHTYFSHKMVWKFKTELEEDYKNKWDKLPFIAGIKQNNPLAASFPSYKGEFGE

Examples of GAS AI-6 sequences from M4 strain isolate 10750 are set forth below.

MGAS10750_Spy0112 is a 33 kd chaperonin which flanks GAS AI-6. An example of an amino acid sequence for MGAS10750_Spy0112 is shown below as SEQ ID NO:308. SEQ ID NO:308

MDKIIKSIAQSGAFRAYVLDSTETVALAQEKHNTLSSSTVALGRTLIANQILAANQKGDSKITVKVIGDSSFGHIISVADT KGHVKGYIQNTGVDIKKTATGEVLVGPFMGNGHFVTIIDYGTGNPYASTTPLITGEIGEDFAYYLTESEQTPSAIGLNVLL DENDKVKVAGGFMVQVLPGASEEEIARYEKRLQEMPAISHLLASKNHVDALLEAIYGDEPYKRLSEEPLSFQCDCSRERFE AALMTLPKADLQAMIDEDKGAEIVCQFCGTKYQFNESDLEALINDKA

MGAS10750_Spy0113 is a transcriptional regulator, rofA. An example of an amino acid sequence for MGAS10750_Spy0113is shown below as SEQ ID NO:309. SEQ ID NO:309

MISIFSLDRIEIGEYTYQRLIWLSKCRKRGPLYLIEKYLESSIESKCQLWLFFKTSYLPITEVAEKTGLTFLQLNHYCEE LNAFFPGSLSMTIQKRMI SCQFTHPFKETYLYQLYAS SNVLQLLAFLIKNGSHSRPLTDFARSHFLSNSSAYRMREALIPL LRNFELKLSKNKIVGEEYRIRYLIALLYSKFGIKVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWKRH QFSVTIPQTRIFQQLKKLFVYDSLKKSSHDIIETYCQLNFSAGDLDYL YLIYITANNSFASLQWTPEHTRQCCQLFEENDT FRLLLNPIITLLPNLKEQKASLVKALMFFSKSFLFNLQHFIPETNL FVSPYYKGNQKLYTSLKL IVE EWMAKL PGKRYLNH KHFHLFCHYVEQILRNIQPPLVWFVASNFINAHLLTDSFPRYFSDKSIDFHSYYLLQDNVYQIPDLKPDLVITHSQLIPF VQHELTKGIAVAEISFDESILSIQELMYQVKEEKFQADLTKQLT

MGAS10750_Spy0114 is a fibronectin binding protein. An example of an amino acid sequence for MGAS10750_Spy0114 is shown below as SEQ ID NO:310. It contains a motif indicative of a cell wall anchor, LPXTG (SEQ ID NO: 122). SEQ ID NO:310

MVSSYMFVRGEKMNNKMFLNKEAGFLAHTKRKRRFAVTLVGVFFMLLAYAGAIGFGQVAYAADEKTVPNFKSPDPDYPWYG YDAYGKGYPGYDISKYYHDLRVNLNGSQVYQVYCFNIQKIFPYNVKSVTQKWFKKVEGNSDTFGLYAMNPRVQGEELSQKL RSVMYNAYPKJMANNIMDGLDTLNAIKVTQSAVWYYSDKSEFEVDKQWESELNNHEIDQEQVTLMREALRKLISSNLEETVE KKLPENYKLNIFNPQDTSIQHLLSAE FVPENPPRPGETPEYGPKTPELDGSPIPEDPKHPDDNLEPTLPPVMLDGEEVPEV PSESLEPALPPLMPELDGQEVPEVPSESLEPALPPLMPELDGQEVPEKPSIDLPIEVPRYEFNNKDQSPLAGESGETEYIT EVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGFSETATWEDTRPKLVFHFDNNE PEVEENREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQNNKV

MGAS10750_Spy0115 is a fibronectin binding protein. An example of an amino acid sequence for MGAS10750_Spy0115 is shown below as SEQ ID NO:311. It contains a motif indicative of a cell wall anchor, FPXTG (SEQ ID NO:141). SEQ 1D NO:311

MYSRLKRELVIVINRKKKYKLIRLMVTLGLIFSQLAPPFGTLMALSGHSRSKSPVTEVKADNVSTLKTGSFKLKKFDEDGK TPIKDVTFQLTSETNPSNYKIEQITSGAGDASFANIPPGTYLLKEVAPPSGYQVMAD YYRITVSPDGYTQYTYVKVGTTTS SPTTSLPSTSGGGTGGTVFRTSKTSGWTVTDYNFTTKNKAQGNTDYTTLWATSGEFFDMSFKLKVNEGTQAGDSFTIKLS DYLSPNGIREKFISAPPLMLDKKWATGIYDESTNSYIYTFNDLINHKQNAEITVNYTFSPEAKKVDRDWYVNT YNITNII

VAFTQKNITVYRVPLSQKTSKMPYSMSGETDGLESIPFDYSSKGITFTKESFHDNETNSNTAGLLIKIKAYITADNKRSAD QIVEKYKDVQTGTEGKLILSDLDPGEYQLIETKPLDGYLVSSGPWTFTITDQGTEGTVKPSDKIIPNTKPGKQKIKVIKK

TPRTVRSVSPSATVSDKDVSRNILVKKVEFTTTNGQTPLQVKPNQGENLIARSEFELKKEIDIKKGDYFAVKLSDNIDPFG VSTGETTTFNITGPYGTLAVGKYDSKSRSIIYTFTD YVEKYEVSNFSTILPYFIDRYAVTRDADINISTSVGSQTNTARVR VLYTPYYGATDSYSPVNIGSMITKLDEKNGTFTNYIYINPMQQFIRNGKLTFQGGGSAIIDNETQVTLFKVNNSTDMPPSW KEESLKETNTVSQAYDEQYYEPVMENNSVRTVTSDSSGNVLFDKLSPGFYAIKEEKAPDG YVKQQGIVRIFQVDSSGKVIK YQYFKDKSIAGKLTEITDLETEQLKQFNEI INKKFVFPMTGGQGIALLMI IGGTMMGIAYFGHRRKQRLND

MGAS10750_Spy0116 is a cell wall surface anchor family protein An example of an amino acid sequence for MGAS10750_Spy0116 is shown below as SEQ ID NO 312 It contains a motif indicative of a cell wall anchor, IPXTG (SEQ ID NO 133) SEQ ID NO 312

EGAEELDGVSFTYWSVDKEKYKKLTKNPQNYDTVPKMKAFLQGTEKNKAL ENSSETIDGKTTGHTADKGGVKVKDLADGYY WFVENSGSNIANGETLSSSAAVPFGLELPVYKADGSTITELHVYPKNTTTKPKIDKNFSKDEKDAALAGGANYDYYQKDKG YVSRIIGSEVSYQIKTEIPAGAQYQTLRWEDTMTKGLTYKAGSLELTITTKGDGKAALNFEFQTDYKLTENQSGFVLKFTE SGLEKVKKAVQATKDAKGQVTADGHPMTVDISYKATVNSDAVVDQPDKNTVIFDYSNNPKEHKDPREKSTKPKDKQITVEK TWASQSAPTGIAVTYYLYQKGDESGKDKWDSVTLTTDYKHIFTNLDDSKDYYVKESAIGYTPEYTEAKDGKISIKNTKDD KNPDPLKPTSPAIVTHGKKFVKTSQDDERLKGATFVWDQKTNEKYLAIKADEKQSAEEKA YHDTEQKYQDEVKKATTEKP

GHKDQSGQSLEGHTQYEKGTPDFGYGQRVINKKITIPQTGGIGTVIFTWGLAIMTVTGLMMIRRNKNDKSE

MGAS10750_Spy0117 is a cell wall surface anchor family protein An example of an amino acid sequence for MGAS10750_Spy0117 is shown below as SEQ ID NO 313 It contains a motif indicative of a cell wall anchor, IPXTG (SEQ ID NO 133) SEQ ID NO 313

MGGTQLKIGRLIRLMLSICACLYFLTSPIFALQKTSSVTIHFENSDKDTQLALWQLPEGQTLPELETLFEKTDAELTRQYP QVSTVTVPKGETKLVLSNLPVGAIYYVREAEERLGVRSLAPFILKVDTDDDQAVYTKKAKAQKRGSYPFVKVSAQGGSLEG ATFEVWKQTQKQLQPVIKGSSRYLLTSSKDGSFMARDLPFGSYVLKEITAPKGYLLSKKTIPFEVTDYSEKQAPVKWNQP KIPPRIEIPYTGNAIMILWLLGFALFTLGVYLVRRNG

MGAS10750_Spy0118 is a sortase An example of an amino acid sequence for MGAS10750_Spy0118 is shown below as SEQ ID NO 314 SEQ ID NO 314

MKKQSKRHRLNQNIALIWFLIGLTILLYPQISRIYYTIESNYQSKQFDREKSTLHQEDISQRIALAKAFNASLHDVDLKD PYSDDEKTKGRAEYARMLELHEQIGHVEIPKIRVDLPIYAGTSDEIISKGSGHLEGTSLPVGGENTHTVLTSHSGLPSAKL

KQKASSGYMCYLFVLLALLTLLFGYWFYRQKKKKSQKVKREEFHAKE

MGAS10750_Spy0119 is a sortase An example of an amino acid sequence for MGAS10750_Spy0119 is shown below as SEQ ID NO 315 SEQ ID NO 315

MLKNKQSLSLRERLIQVMFPLLFLMGCLIVLYPLMSNYYYRVKQNQAVTSFESAKVIVNKDDIKRRMALARAYNATLDPGR INDPYTDLEKKGVAEYARMLELNEQIGYVEVPRFDINLPIYAGTSDDVLQKAAGHLEGTSLPIGGDSTHTVITAHTGLPQA KLFTNIHKMKKGDLFFIHNIDKTLAYKVDQILWEPDNFTPVLVKNGFDYATLLTCTPYGINSHRLLVRGYRVPYQKAFEK ADAQRPWYTKVIFLVSFLLFVILVIILLIDWHRK

MGAS10750_Spy0120 is a sortase An example of an amino acid sequence for MGAS10750_Spy0120 is shown below as SEQ ID NO 316 SEQ ID NO 316

MSKQKCVGYVLMILGLGLPLFFLTLMSLNQFQEQVAYQKFQTENRSWKNSQKEWVNRHNQEQALADRATTDPFVDAQNQLK QSPFDDNIIGYIII PKLRMAQPIRVGASERHLEKGVAQVTGTSLPIGGLGTRSVIAGHRSWYDNERFLRIAELSLGDQIVI DLGVYQLEYRVKSVEIIDAKDWRQLTAKKSQDLITLLTCNPLYPPFNERLLVMQRECCLAFLIYQLKREIRWINLN

MGAS 10750_Spy0121 is a hypothetical protein which flanks GAS AI-6 An example of an amino acid sequence for MGAS10750_Spy0121 is shown below as SEQ ID NO 317 SEQ ID NO:317

MKRCNKYLFTSLLAASMLFSSYKSVHAHDNIDEEGKVHLYWQGNYYVDNYVDYTKKLVNNSIEWTVTFNSAKEQWVYPDFS YGRQSHTYFSHKMVWKFKTELEDNYKDKWNKLPFIAGIKQNNPLAASFPSYKGEFGE

There may be an upper limit to the number of GAS proteins which will be in the compositions of the invention. Preferably, the number of GAS proteins in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 1 1 , less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3. Still more preferably, the number of GAS proteins in a composition of the invention is less than 6, less than 5, or less than 4. Still more preferably, the number of GAS proteins in a composition of the invention is 3.

The GAS proteins and polynucleotides used in the invention are preferably isolated, i.e., separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose.

Examples Other Gram positive bacterial Adhesin Island Sequences

The Gram positive bacteria AI polypeptides of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from a gram positive bacteria, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form.

The Gram positive bacteria AI proteins of the invention may include polypeptide sequences having sequence identity to the identified Gram positive bacteria proteins. The degree of sequence identity may vary depending on the amino acid sequence (a) in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more). Polypeptides having sequence identity include homologs, orthologs, allelic variants and mutants of the identified Gram positive bacteria proteins. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affinity gap search with parameters gap open penalty-l2 and gap extension penalty =1.

The Gram positive bacteria adhesin island polynucleotide sequences may include polynucleotide sequences having sequence identity to the identified Gram positive bacteria adhesin island polynucleotide sequences. The degree of sequence identity may vary depending on the polynucleotide sequence in question, but is preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more).

The Gram positive bacteria adhesin island polynucleotide sequences of the invention may include polynucleotide fragments of the identified adhesin island sequences. The length of the fragment may vary depending on the polynucleotide sequence of the specific adhesin island sequence, but the fragment is preferably at least 10 consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).

The Gram positive bacteria adhesin island amino acid sequences of the invention may include polypeptide fragments of the identified Gram positive bacteria proteins. The length of the fragment may vary depending on the amino acid sequence of the specific Gram positive bacteria antigen, but the fragment is preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferably the fragment comprises one or more epitopes from the sequence. The fragment may comprise at least one T-cell or, preferably, a B- cell epitope of the sequence T- and B-cel! epitopes can be identified empirically (e g , using PEPSCAN [Geysen et al ( 1984) PNAS USA 81 3998-4002, Carter (1994) Methods MoI Biol 36 207-223, or similar methods], or they can be predicted (e g , using the Jameson Wolf antigenic index [Jameson, BA et al 1988, CABlOS 4( 1) 1818 186], matrix-based approaches [Raddπzzani and Hammer (2000) Brief Bioinform 1(2) 179-189], TEPITOPE [De Lalla et al (199) J Immunol 163 1725-1729], neural networks [Brusic et al (1998) Bioinformatics 14(2) 121-130], OptiMer & EpiMer [Meister et al (1995) Vaccine 13(6) 581 591 , Roberts et al (1996) AIDS Res Hum Retroviruses 12(7) 593-610], ADEPT [Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3) 291-297], Tsites [Feller & de Ia Cruz ( 1991) Nature 349(6311) 720-721], hydrophilicity [Hopp (1993) Peptide Research 6 183-190], antigenic index [Welling et al (1985)FE5S Lett 188 215-218] or the methods disclosed in Davenport et al (1995) Immunogenetics 42 392-297, etc Other preferred fragments include (1) the N-terminal signal peptides of each identified Gram positive bacteria protein, (2) the identified Gram positive bacteria protein without their N-termiπal signal peptides, (3) each identified Gram positive bacteria protein wherein up to 10 amino acid residues (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the N-terminus and/or the C-terminus e g the N-termmal amino acid residue may be deleted Other fragments omit one or more domains of the protein (e g omission of a signal peptide, of a cytoplasmic domain, of a transmembrane domain, or of an extracellular domain), and (4) the polypeptides, but without their N terminal amino acid residue

As indicated in the above text, nucleic acids and polypeptides of the invention may include sequences that

(a) are identical (ι e , 100% identical) to the sequences disclosed in the sequence listing,

(b) share sequence identity with the sequences disclosed in the sequence listing,

(c) have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single nucleotide or amino acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b),

(d) when aligned with a particular sequence from the sequence listing using a pairwise alignment algorithm, a moving window of x monomers (amino acids or nucleotides) moving from start (N-terminus or 5') to end (C-terminus or 3'), such that for an alignment that extends to p monomers (where p>x) there are p-x+l such windows, each window has at least x y identical aligned monomers, where x is sleeted from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, v is selected from 050, 060, 070, 075, 0 80, 0 85, 090, 091, 092, 0 93, 094, 095, 096, 097, 098, 0 99, and if x y is not an integer then it is rounded up to the nearest integer The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [Needlman &Wunsch (1970) J MoI Biol 48, 443-453], using default parameters (e g , with Gap opening penalty = 100, and with Gap extension penalty = 05, using the EBLOSUM62 scoring matrix) This algorithm is conveniently implemented in the needle tool in the EMBOSS package [Rice et al (2000) Trends Genet 16 276-277]

The nucleic acids and polypeptides of the mention may additionally have further sequences to the N-terminus/5' and/or C-terminus/3' of these sequences (a) to (d)

All of the Gram positive bacterial sequences referenced herein are publicly available through PubMed on GenBank

Streptococcus pneumoniae Adhesin Island Sequences

As discussed above, a S pneumoniae AI sequence is present in the TIGR4 S pneumoniae genome Examples of 5 pneumoniae AI sequences are set forth below

SrtD (SpO468) is a sortase An example of an amino acid sequence of SrtD is set forth in SEQ ID NO 80 SEQ ID NO:80 MSRTKLRALLGYLLMLVACLI PI YCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDPFLAEGY EVNYQVSDDPDA VYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVG

EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

SrtC (Sp0467) is a sortase An example of an amino acid sequence of SrtC is set forth in SEQ ID NO 81

SEQ ID NO:81

MSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPA IDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVT VEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRN RRIVKGLEKQLEGRHVKD

SrtB (SP0466) is a sortase An example of an amino acid sequence of SrtB is set forth in SEQ ID NO 82

SEQ ID NO:82

MAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGDPWSEEMKKKGRAEYARMLEIHER MGHVEI PVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMA

LLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ

SpO465 is a hypothetical protein An example of an amino acid sequence of SpO465 is set forth in SEQ ID NO 83

SEQ ID NO:83

MFLPFLSASLYLQTHHFIAFPNRQSYLLRETRKSHFFLIHHPF

RrgC (SP0464) is a cell wall surface anchor family protein RrgC contains a sortase substrate motif VPXTG (SEQ ID NO 137), shown in italics in SEQ ID NO 84

SEQ ID NO:84

MISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHSWDENKL SSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTKVKLIKVDQDHNR LEGVGFKLVSVARDVSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVDH QLVTITWNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVLQNGKEWVTSGKDGRFRVEGLEYGTYYLWELQ APTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN

RrgB (SpO463) is a cell wall surface anchor protein RrgB contains a sortase substrate motif IPXTG (SEQ ID NO 133), shown in italics in SEQ ID NO 85

SEQ ID NO:85

MKSINKFLTMLi

VWTNTNNEIIDI AVPIEIELPLKΓDVVDAHVYPKNTEAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDVVEYEIVTKIPALANYATANWSDRM

GNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIPAGAEATFDLVNAQTGKΛA/QTVTLTTDKNTVTVNGLDKNTEYKFVE

RSIKGYSADYQEITTAGEIAVKNWKD ENPKPLDPTEPKWTYGKKFVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVS

QEEKQLWTTKDALDRAVAAYNALTAQQQTQQEKEKVDKAQAA YNAAVIAANNAFEWVADKDNENWKLVSDAQGRFEITG LLAGTYYLEETKQPAGYALLTSRQKFEVTATSYSATGQGIEYTAGSGKDDATKWNKKIT IPQTGGIGTIIFAVAGAAIMG IAVYAYVKNNKDEDQLA

RrgA (SpO462) is a cell wall surface anchor protein RrgA contains a sortase substrate motif YPXTG (SEQ ID NO 186), indicated in italics in SEQ ID NO 86

SEQ ID NO:86

MLNRETHMKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQR TEAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIK VDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYEQKDKSVPLDWILLDNSNSMSNIRNK

TNDKNDIVELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAP SYQNQLNAFFSKSPNKDGILLSDFITQATSGEHT IVRGDGQSYQMFTDKTVYEKGAPAAFPVKPEKYSEMKAAGYAVIGDP INGGYIWLNWRESILAYPFNSNTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQSISSKPENYT NVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELIDLQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLK NAKVLYDTTEKRIRVTGLYLGTDEKVTLTYNVRLNDEFVSNKFYDTNGRTTLHPKEVEQNTVRDFPIPKIRDVRKYPEITI SKEKKLGDIEFIKWKNDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGY KPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKRE YPfiTGGIGMLPFYLIGCMMMGGVLLYTRK HP

RIrA (SpO461) is a transcriptional regulator An example of an amino acid sequence for RIrA is set forth in SEQ ID NO 87 SEQ ID NO:87

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKL KNLFMYPILMEHCQTYL

FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRL YLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSI ILRNFTDKVASVTGYNILISPPPSEEHLTEPLI I ITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VDIRKEAFDKRVAMIAKKAHYLL

As discussed above, a S pneumoniae AI sequence is present in the S pneumoniae strain 670 genome Examples of S pneumoniae AI sequences are set forth below

Orfl_670 is a transposase An example of an amino acid sequence of orfl_670 is set forth in SEQ ID NO 171

SEQ ID NO:171

MEHINHTTLLIGIKDKNITLNKAIQHDTHIEVFATLDYHPPKCKHCKGKQIKYDFQKPSKIPFIEIGGFPSLIHLKKRRFQ CKSCRKVTVAETTLVQKNCQISEMVRQKIAQLLLNREALTHIASKLAISTSTSTVYRKLKQFHFQEDYTTLPEILSWDEFS YQKGKLAFIAQDFNTKKIMTILDNRRQTTIR^^HFFKYSKEARKKVKVVTVDMSGSYIPLIKKLFPNAKIVLDRFHIVQHMS RALNQTRINIMKQFDDKSLEYRALKYYWKFILKDSRKLSLKPFYARTFRETLTPRECLKKIFTLVPELKDYYDLYQLLLFH LQEKNTDQFWGLIQDTLPHLNRTFKTTLSTFICYKNYITNAIELPYSNAKLEATNKLIKDIKRNAFGFRNFENFKKRIFIA LNIKKERTKFVLSRA

Orf2_670 is a transcriptional regulator An example of an amino acid sequence of Orf2_670 is set forth in SEQ ID NO 172

SEQ ID NO:172

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKL KNLFMYPILMEHCQTYL

FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRL YLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VDIRKEAFDKRVAMIAKKAHYLL

Orf3_670 is a cell wall surface anchor family proten An example of an amino acid sequence of Orf3_670 is set forth in SEQ ID NO 173

SEQ ID NO:173

MLNRETHMKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQR TEAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIK VDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDWILLDNSNSMSNIRHN

TSDPTDIQTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQ SYRTQLNNF KAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYG TDLYLYWRDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVA DPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQLGADGRFDPADYTLTANDGSSLVNNVPTGG PQNDGGLLKNAK

KKLGEIEFIKINKNDKKPLRDAVFSLQKQHPDYPDI YGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPV QNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP

Orf4_670 is a cell wall surface anchor family protein An example of an amino acid sequence of orf4_670 is set forth in SEQ ID NO 174

SEQ ID NO:174

MKSINKFLTMLAALLLTASSLFSAATVF AADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLKDLTGW AEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVP NEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLNSLA VADIPESNDITYHYGNHQDHG NTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGYSAEYT

LDAAVAA YTNAADKQAAQAL VDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFA KIDDVEFWGAGSWNQGEFNYLKDVQKNDATKWNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA

Orf5_670 is a cell wall surface anchor family protein An example of an amino acid sequence of orf5_670 is set forth in SEQ ID NO 175

SEQ ID NO.175

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDDRVQIVRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSI IQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTKVKLI

KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLD TDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKWKEESGHYTPVLQNGKEVWTSGKDGRFRVEGLEYG TYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN

Orf6_670 is a sortase An example of an ammo acid sequence of orf6_670 is set forth in SEQ ID NO 176 SEQ ID NO:176

MLIKMVKTKKQKRNNLLLGWFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAE EVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA

EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ

Orf7_670 is a sortase An example of an amino acid sequence of orf7_670 is set forth in SEQ ID NO 177

SEQ ID NO:177

VSRYYYRIESNEVIKEFDEWSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPA IDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILT

RRIVKGLEKQLEEHHVKG

Orf8_670 is a sortase An example of an amino acid sequence of orf8_670 is set forth in SEQ ID NO 178

SEQ ID NO:178

MSKAKLQKLLGYLLMLVALVI PVYCFGQMVLQSLGQVKGHEIFSESVT ADSYQEQLQRSLDYNQRLDSQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSHVFFRHLDQLKVG

EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a S pneumoniae AI sequence is present in the 19A Hungary 6 S pneumoniae genome Examples of 5 pneumoniae AI sequences from 19A Hungary 6 are set forth below

ORF2_19AH is a transcriptional regulator An example of an ammo acid sequence of ORF2_19AH is set forth in SEQ ID NO 187

SEQ ID NO:187

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRL YLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSI ILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VD I RKEAFDKRVAMIAKKAHYLL

ORF3_19AH is a cell wall surface protein An example of an amino acid sequence of ORF3_19AH is set forth in SEQ ID NO 188

SEQ ID NO:188

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE

GQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDWILLDNSNSMSNIRHNHAHRAEK

QTIKDRI PSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSYRTQLN NFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYW ELNRYFYTIVNEKKSIENGTITDPMGELIDFQLGADGRFDPAD YTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTE KRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIE FIKINKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVA FQIVNGEVRDVTSIVPQDI PAGYEFTNDKHYITNEPI PPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKNP

ORF4_19AH is a cell wall surface protein. An example of an amino acid sequence of ORF4_19AH is set forth in SEQ ID NO: 189.

SEQ ID NO:189

MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLKDLTGW AEEIPNVYFELQKYNLTDGKEKENL KDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVP ALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSIGTKIPYVVNTTIPSNATFATSFWSDEMTEGLTY NEDVTITLNNVAMDQADYEVTKGXNGFNLKLTEAGLAKINGKDADQKIQITYSATLNSLAVADI PESNDITYHYGNHQDHG

VESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLENAQFWKKADSNKYIAFKSTAQQAADEKAAATAKQK KIDDVE FWGAGSWNQGEFNYLKDVQKNDATKWNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA

ORF5_19AH is a cell wall surface protein. An example of an amino acid sequence of ORF5_19AH is set forth in SEQ ID NO: 190.

SEQ ID NO:190

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDDRVQIVRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGL YYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTKVKLI

KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLD TDVQLVDHQLVTITWNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVLQNGKEVWTSGKDGRFRVEGLEYG TYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN

ORF6_19AH is a putative sortase. An example of an amino acid sequence of ORF6_19AH is set forth in SEQ ID N0:191.

SEQ ID NO:191

MLIKMVKTKKQKRNNLLLGWFFIGMAVMAYPLVSRL YYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEE EF IAANKL SHL YRYLF YVAVGL IVI LLWI I RRLRKKKKQPEKALKALKAARKEVKVEDGQQ

ORF7_19AH is a putative sortase. An example of an amino acid sequence of ORF7_19AH is set forth in SEQ ID NO: 192.

SEQ ID NO:192

MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWTAHRGL PTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAP IAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG

ORF8_19AH is a putative sortase. An example of an amino acid sequence of ORF8_19AH is set forth in SEQ ID NO: 193.

SEQ ID NO:193

MSKAKLQKLLGYLLMLVAL VI PVYCFGQMVLQSLGQVKGHEIFSESVT ADSYQEQLQRSLDYNQRLDSQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGAD YHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFMGILFVLWKLARLLRGK

As discussed above, a S. pneumoniae AI sequence is present in the 6B Finland 12 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 6B Finland 12 are set forth below.

ORF2_6BF is a transcriptional regulator. An example of an amino acid sequence of ORF2_6BF is set forth in SEQ ID NO: 194. SEQ ID NO:194 MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT

FLFGLQNLVPYYNYYEHYGiESDKPLYHi SKΆIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIILNNQAD

VNLIKSI ILRNFTDKVASVTGYNILISPPPSEEHLTEPLI I ITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VD I RKEAFDKRVAMI AKKAHYLL

ORF3_6BF is a cell wall surface protein. An example of an amino acid sequence of ORF3_6BF is set forth in SEQ ID NO: 195

SEQ ID NO:195

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE AIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIKVDGSEKN GQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDWILLDNSNSMSNIRHNHAHRAEK

QTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSYRTQLN NFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYL YW RDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGD PGTDEATATRFMQSISSSPDNYTNVADPSQILQ ELNRYFYTIVNEKKSIENGTITDPMGELIDFQLGADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTE KRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIE FIKINKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVA FQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP

ORF4_6BF is a cell wall surface protein. An example of an amino acid sequence of ORF4_6BF is set forth in SEQ ID NO: 196.

SEQ ID NO:196

MKS INKFLTMLAALLLTAS SLF SAATVFAADNVSTAPDAVTKTLT IHKLLLS EDDLKTWDTNG PKGYDGTQS SLKDLTGW

AEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVP ALVTLPLVNNNGTVIDAHVFPKNSYNKPWDKRIADTLNYNDQNGLSIGTKIPYWNTTIPSNATFATSFWSDEMTEGLTY NEDVTITLNNVAMDQADYEVTKGNNGFNL KLTEAGLAKINGKDADQKIQITYSATLNSLAVADIPESNDITYHYGNHQDHG NTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGYSAEYT VESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLENAQFWKKADSNKYIAFKSTAQQAADEKAAATAKQK LDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGWEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFA KIDDVEFWGAGSWNQGEFNYLKDVQKNDATKWNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA

ORF5_6BF is a cell wall surface protein. An example of an amino acid sequence of ORF5_6BF is set forth in SEQ ID NO: 197.

SEQ ID NO:197

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDDRVQIVRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTKVKLI KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLD TDVQLVDHQLVTITWNQKLPRGNVDFMKVDGRTNTSLQG AMFKVMKEESGHYTPVLQNGKEVWTSGKDGRFRVEGLEYG

TYYLWELQAPTGWQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN

ORF6_6BF is a putative sortase. An example of an amino acid sequence of ORF6_6BF is set forth in SEQ ID NO:198.

SEQ ID NO:198

MLIKMVKTKKQKRNNLLLGWFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVΎAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLL VRGHRI PYVAEVEE EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ

ORF7_6BF is a putative sortase. An example of an amino acid sequence of ORF7_6BF is set forth in SEQ ID NO: 199.

SEQ ID NO:199

MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGWEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWT AHRGL PTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAP IAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG

ORF8_6BF is a putative sortase An example of an amino acid sequence of ORF8_6BF is set forth in SEQ ID NO 200

SEQ ID NO:200

MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a S pneumoniae AI sequence is present in the 6B Spam 2 S pneumoniae genome Examples of S pneumoniae AI sequences from 6B Spain 2 are set forth below

ORF2_6BSP is a transcriptional regulator An example of an amino acid sequence of ORF2_6BSP is set forth in SEQ ID NO 201

SEQ ID NO:201

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRI RFLI ALLQFHFGI E IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VDIRKEAFDKRVAMIAKKAHYLL

ORF3_6BSP is a cell wall surface protein An example of an amino acid sequence of ORF3_6BSP is set forth in SEQ ID NO 202

SEQ ID NO:202

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWI KETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE AIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIKVDGSEKN

QTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSYRTQLN NFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYL YW RDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQ

KRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIE FQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLL YTRKHP

ORF4_6BSP is a cell wall surface protein An example of an amino acid sequence of ORF4_6BSP is set forth in SEQ ID NO 203

SEQ ID NO:203

MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLKDLTGW AEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVP ALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTLNYNDQNGLSIGTKIPYWNTTIPSNATFATSFWSDEMTEGLTY NEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLNSLAVADIPESNDITYHYGNHQDHG NTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGYSAEYT VESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLENAQFWKKADSNKYIAFKSTAQQAADEKAAATAKQK LDAAVAAYTNAADKQAAQAL VDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFA KIDDVEFWGAGSWNQGEFNYLKDVQKNDATKWNKKITI PQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA

ORF5_6BSP is a cell wall surface protein An example of an amino acid sequence of ORF5_6BSP is set forth in SEQ ID NO 204

SEQ ID NO:204

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDDRVQIVRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHI PNGLYYVRSI IQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTKVKLI

KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNΓLPLGNYRFKEVEPLAGYAVTTLD TYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN

ORF6_6BSP is a putative sortase. An example of an amino acid sequence of ORF6_6BSP is set forth in SEQ ID NO 205

SEQ ID NO:205

MLIKMVKTKKQKRNNLLLGWFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHD YVTLLTCTPYMINTHRLLVRGHRI PYVAEVEE EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ

ORF7_6BSP is a putative sortase. An example of an amino acid sequence of ORF7_6BSP is set forth in SEQ ID NO.206.

SEQ ID NO:206

MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWT AHRGL PTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAP IAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG

ORF8_6BSP is a putative sortase. An example of an amino acid sequence of ORF8_6BSP is set forth in SEQ ID NO:207.

SEQ ID NO:207

MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a S. pneumoniae AI sequence is present in the 9V Spain 3 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 9V Spain 3 are set forth below.

ORF2_9VSP is a transcriptional regulator. An example of an amino acid sequence of ORF2_9VSP is set forth in SEQ ID NO:208.

SEQ ID NO:208

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFI ILNNQAD VNLIKSI ILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VD I RKEAFDKRVAM I AKKAH YLL

ORF3_9VSP is a cell wall surface protein. An example of an amino acid sequence of ORF3_9VSP is set forth in SEQ ID NO:209.

SEQ ID NO:209

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTNGTTVSQRTEAQTGE AIFSNI KPGTYTLTEAQPPVGYKPSTKQRTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIKVDGSEKN GQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYERKDKSVPLDWILLDNSNSMSNIRNKNARRAER

VELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSYQNQLN AFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVYEKGAPAAFPVKPEKYSEMKAVGYAVIGDPINGGYIW LNWRESILAYPFNSNTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTK ILEQLNRYFHTIVTEKKSIENGTITDPMGELIDLQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVFYD

EIEFIKINKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKP IVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLLFYLIGCMMMGGVLLYTRKHP

ORF4_9VSP is a cell wall surface protein. An example of an amino acid sequence of ORF4_9VSP is set forth in SEQ ID NO-210 SEQ ID NO:210

VWTNTNNEIIDENGQTLGVNIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKI YEIHSLSTYVGEDGATLTGSK

AVPIEIELPLNDWDAHVΎPKNTEAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDWEYEIVTKIPALANYATANWSDRM TEGLAFNKGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKITYSATLND KAIVEVPESNDVTFNY GNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIPAGAEATFDLVNAQTGKWQTVTLTTDKNTVTVNGLDKNTEYKFVE RSIKGYSADYQEITTAGEIAVKNWKD ENPKPLDPTEPKWTYGKKFVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVS QEEKQLWTTKDALDRAVAAYNALTAQQQTQQEKEKVDKAQAAYNAAVIAANNAFEWVADKDNENWKLVSDAQGRFEITG LLAGTYYLEETKQPAGYALLTSRQKFEVTATSYSATGQGIEYTAGSGKDDATKWNKKITIPQTGGIGTIIFAVAGAVIMG IAVYAYVKNNKDEDQLA

ORF5_9VSP is a cell wall surface protein. An example of an amino acid sequence of ORF5_9VSP is set forth in SEQ ID NO:211.

SEQ ID NO:211

MTMQKMQKMQKMQKMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSY

SYDNRVQIVRDLHSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGL YYVRSI IQTDAVSYPAEFLFEMTDQTVEPLVIVAK KAD¹TVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIVVTNLPLGTYRFKEV

EPLAGYTVTTMDTDVQLVDHQLVTITWNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVLQNGKEVWASGK YLTKKTNN

ORF6_9VSP is a putative sortase. An example of an amino acid sequence of ORF6_9VSP is set forth in SEQ ID NO:212.

SEQ ID NO:212

MLIKMAKTKKQKRNNLLLGWFFIGIAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GD PWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVI EPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEE EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVEDE

ORF7_9VSP is a putative sortase. An example of an amino acid sequence of ORF7_9VSP is set forth in SEQ ID NO:213.

SEQ ID NO:213

MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWTAHRGL PTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAP IAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD

ORF8_9VSP is a putative sortase. An example of an amino acid sequence of ORF8_9VSP is set forth in SEQ ID NO:214.

SEQ ID NO:214

MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a S. pneumoniae AI sequence is present in the 14 CSR 10 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 14 CSR 10 are set forth below.

ORF2_14CSR is a transcriptional regulator. An example of an amino acid sequence of ORF2_14CSR is set forth in SEQ ID NO:215.

SEQ ID NO:215

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VDIRKEAFDKRVAMIAKKAHYLL ORF3_14CSR is a cell wall surface protein An example of an amino acid sequence of ORF3_14CSR is set forth in SEQ ID NO 216

SEQ ID NO:216

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE AIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIKVDGSEKN

QTIKDRIPSDAEELNKDKLMYQFGATFTQKALMT ADDILTKQARPNSKKVI FHITDGVPTMSYPINFKYTGTTQSYRTQLN NFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYW RDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSISSSPDNYTNVADPSQILQ ELNRYFYTIVNEKKSIENGTITDPMGELIDFQLGADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTE KRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIE FIKINKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVA FQIVNGEVRDVTS IVPQDI PAGYEFTNDKHYITNEPI PPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP

ORF4_14CSR is a cell wall surface protein An example of an amino acid sequence of ORF4_14CSR is set forth in SEQ ID NO 217

SEQ ID NO:217

MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLKDLTGW AEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVP ALVTLPLVNNNGTVI DAHVFPKNSYNKPWDKRIADTLNYNDQNGLSIGTKIPYWNTTIPSNATFATSFWSDEMTEGLTY NEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLNSLAVADIPESNDITYHYGNHQDHG NTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGYSAEYT VESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLENAQFWKKADSNKYIAFKSTAQQAADEKAAATAKQK

KIDDVE FWGAGSWNQGEFNYLKDVQKNDATKWNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA

ORF5_14CSR is a cell wall surface protein An example of an amino acid sequence of ORF5_14CSR is set forth in SEQ ID NO 218

SEQ ID NO:218

MTMQKMQKMI SRI FFVMALCF SLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDS YS YDDRVQI VRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTKVKLI KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLD TDVQLVDHQLVTITWNQKLPRGNVDFMKVDGRTNTSLQG AMFKVMKEESGHYTPVLQNGKEVWTSGKDGRFRVEGLEYG TYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN

ORF6_14CSR is a putative sortase An example of an amino acid sequence of ORF6_14CSR is set forth in SEQ ID NO 219 SEQ ID NO:219

MLIKMVKTKKQKRNNLLLGWFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAE EVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLL VRGHRI PYVAEVEE EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ

ORF7_14CSR is a putative sortase An example of an amino acid sequence of ORF7_14CSR is set forth in SEQ ID NO 220

SEQ ID NO:220

MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWTAHRGL PTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAP IAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG

ORF8_14CSR is a putative sortase An example of an amino acid sequence of ORF8_14CSR is set forth in SEQ ID NO 221 SEQ ID NO:221 MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLS I PSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGI RSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a 5 pneumoniae AI sequence is present in the 19F Taiwan 14 5 pneumoniae genome Examples of S pneumoniae AI sequences from 19F Taiwan 14 are set forth below

ORF2_19FTW is a transcriptional regulator An example of an amino acid sequence of ORF2_19FTW is set forth in SEQ ID NO 222

SEQ ID NO:222

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRL YLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VD I RKEAFDKRVAMIAKKAHYLL

ORF3_19FTW is a cell wall surface protein An example of an amino acid sequence of ORF3_19FTW is set forth in SEQ ID NO 223

SEQ ID NO:223

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWI KETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE

AIFSNI KPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIKVDGSEKN

GQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYERKDKSVPLDWILLDNSNSMSNIRNKNARRAER

AGEATRSLIDKITSDPENRVAL VTYASTIFDGTEFTVEKGVADKNGKRLNDSLFWNYDQTSFTTNTKDYSYLKLTNDKNDI

VELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSYQNQLN

AFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVYEKGAPAAFPVKPEKYSEMKAVGYAVIGDPINGGYIW

LNWRESILAYPFNSNTAKITNHGAPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTK

ILEQLNRYFHTIVTEKKSIENGTITDPMGELIDLQLGTDGRFDPAD YTLTANDGSRLENGQAVGGPQNDGGLLKNAKVFYD

TTEKRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPAITIAKEKKLG

EIEFIKINKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKP

IVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP

ORF4_19FTW is a cell wall surface protein An example of an amino acid sequence of ORF4_19FTW is set forth in SEQ ID NO 224

SEQ ID NO:224 MKSINKFLTMLAi

VWTNTNNEIIDE]

AVPIEIELPLNDWDAHVYPKNT EAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDWEYEIVTKIPALANYATANWSDRM

TEGLAFNKGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKITYSATLNDKAIVEVPESNDVTFNY

GNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIPAGAEATFDLVNAQTGKWQTVTLTTDKNTVTVNGLDKNTEYKFVE

RS I KGYSADYQEITTAGE I AVKNWKDENPKPLDPTEPKWTYGKKFVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVS

QEEKQLWTTKDALDRAVAA YNALTAQQQTQQEKEKVDKAQAAYNAAVIAANNAFEWVADKDNENWKLVSDAQGRFEITG

LLAGTYYLEETKQPAGY ALLTSRQKF EVTATSYSATGQGIEYTAGSGKDDATKWNKKITIPQTGGIGTIIFAVAGAVIMG

IAVYAYVKNNKDEDQLA

ORF5_19FTW is a cell wall surface protein An example of an amino acid sequence of ORF5_19FTW is set forth in SEQ ID NO 225

SEQ ID NO:225

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDNRVQIVRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSI IQTDAVSYPAEFLFEMTDQTVEPLVIVAKKADTVTTKVKLI KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIWTNLPLGTYRFKEVEPLAGYTVTTMD TDVQLVDHQLVTITWNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVLQNGKEVWASGKDGRFRVEGLEYG TYYLWELQAPTGYVQLTSPVSFTIGKDTRKEL VTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKTNN

ORF6_19FTW IS a putative sortase An example of an amino acid sequence of ORF6_19FTW is set forth in SEQ ID NO 226 SEQ ID NO:226

MLIKMAKTKKQKRNNLLLGWFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEE EF I AANKLSHLYRYLFYVAVGL I VI LLWI I RRLRKKKRQSERALKALKEATKEVKVEDE

ORF7_19FTW is a putative sortase. An example of an amino acid sequence of ORF7_19FTW is set forth in SEQ ID NO:227

SEQ ID NO:227

MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP

SEILDPFTDQEKKQGVSEYANMLKVHERIGYVEIPAIEQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGL

PTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGQDYATLLTCTPYMINSHRLLVRGKRIPYTAP

IAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD

ORF8_19FTW is a putative sortase. An example of an amino acid sequence of ORF8_19FTW is set forth in SEQ ED NO.-228.

SEQ ID NO:228

MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a S. pneumoniae AI sequence is present in the 23F Taiwan 15 S. pneumoniae genome. Examples of S. pneumoniae AI sequences from 23F Taiwan 15 are set forth below.

ORF2_23FTW is a transcriptional regulator. An example of an amino acid sequence of ORF2_23FTW is set forth in SEQ ID NO 229.

SEQ ID NO:229

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPL YHISKAIVQEWMTEQKIEGVIDQHRL YLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLII ITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VDIRKEAFDKRVAMIAKKAHYLL

ORF3_23FTW is a cell wall surface protein. An example of an amino acid sequence of ORF3_23FTW is set forth in SEQ ID NO230

SEQ ID NO:230

MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE AIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQIIKVDGSEKN GQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIELTVSGKTVYEQKDKSVPLDWILLDNSNSMSNIRNKNARRAER AGEATRSLIDKITSDPENRVAL VTYASTIFDGTEFTVEKGVADKNGKRLNDSLFWNYDQTSFTTNTKDYSYLKLTNDKNDI VELKNKVPTEAEDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSYQNQLN AFFSKS PNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVYEKGAPAAFPVKPEKYSEMKAAGYAVIGDPINGGYIW LNWRESILAYPFNSNTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQSISSKPENYTNVTDTTK ILEQLNRYFHTIVTEKKSIENGTITDPMGELIDLQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVLYD TTEKRIRVTGL YLGTDEKVTLTYNVRLNDEFVSNKFYDTNGRTTLHPKEVEQNTVRDFPIPKIRDVRKYPEITISKEKKLG DIEFIKWKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQWRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKP IVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP

ORF4_23FTW is a cell wall surface protein. An example of an amino acid sequence of ORF4_23FTW is set forth in SEQ ID NO:231.

SEQ ID NO:231

MKSINKFLTILAALLLTVSSLFSAATVFAAEQKTKTLTVHKLLMTDQELDAWNSDAITTAGYDGSQNFEQFKQLQGVPQGV TEISGVAFELQSYTGPQGKEQENLTNDAVWTAVNKGVTTETGVKFDTEVLQGTYRL VEVRKESTYVGPNGKVLTGMKAVPA LITLPLWQNGWENAHVYPKNSEDKPTATKTFDTAAGFVDPGEKGLAIGTKVPYIVTTTIPKNSTLATAFWSDEMTEGLD GNTPKPNKPKNGELTITKTWADAKDAPIAGVEVTFDLVNAQTGEWKVPGHETGIVLNQTNNWTFTATGLDNNTEYKFVER

TIKGYSADYQTITETGKIAVKNWKDENPEPINPEEPRVKTYGKKFVKVDQKDERLKEAQFWKNEQGKYLALKSAAQQAVN

EKAAAEis

EETKAPE

DEDQLA

ORF5_23FTW is a cell wall surface protein An example of an amino acid sequence of ORF5_23FTW is set forth in SEQ ID NO 232

SEQ ID NO:232

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDNRVQIVRDL HSWΥENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKADTVTTKVKLI KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIWTNLPLGTYRFKEVEPLAGYTVTTMD TDVQLVDHQLVTITWNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVLQNGKEVWASGKDGRFRVEGLEYG TYYLWELQAPTGWQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKTNN

ORF6_23FTW IS a putative sortase An example of an amino acid sequence of ORF6_23FTW is set forth in SEQ ID NO 233

SEQ ID NO:233

MLIKMVKTKKQKRNNLLLGWFFIGMAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGQLEGTSLPIGGNSTHAVITAHTGLPTA

EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVEDGQQ

ORF7_23FTW is a putative sortase An example of an amino acid sequence of ORF7_23FTW is set forth in SEQ ID NO 234

SEQ ID NO:234

MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWTAHRGL PTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVEPNDFEPVLIQHGKDYATLLTCTPYMINSHRLLVRGKRIPYTAP IAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG

ORF8_23FTW is a putative sortase An example of an amino acid sequence of ORF8_23FTW is set forth in SEQ ID NO 235

SEQ ID NO:235

MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTADSYQEQLQRSLDYNQRLDSQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSHVFFRHLDQLKVG DALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

As discussed above, a S pneumoniae AJ sequence is present in the 23F Poland 16 5 pneumoniae genome Examples of S pneumoniae AI sequences from 23F Poland 16 are set forth below

ORF2_23FP is a transcriptional regulator An example of an amino acid sequence of ORF2_23FP is set forth in SEQ ID NO 236

SEQ ID NO:236

MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQETFEEELTFNLDTQQVQLIEHHSHQTNYYFH QLYNQSTILKILRFFLLQGNQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQWGPEYRIRFLIALLQFHFGIE IYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHFSILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYL EPHANMTFTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKFKNILGNDISNSLSFLTALTFLTRT FLFGLQNLVPYYNYYEHYGIESDKPLYHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIILNNQAD VNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTKEYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTI VDIRKEAFDKRVAMIAKKAHYLL

ORF3_23FP is a cell wall surface protein An example of an amino acid sequence of ORF3_23FP is set forth in SEQ ID NO 237

SEQ ID NO:237

MKKVRKIFQKAVAGLCCI SQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGE AIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTPYQI IKVDGSEKN GQHKALNPNPYERVI PEGTLSKRIYQVNNLDDNQYGIELTVSGKTTVETKEASTPLDWILLDNSNSMSNIRHNHAHRAEK QTIKDRIPSDAEELNKDKLMYQFGATFTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSYRTQLN NFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYW RDSILAYPFNSSTDWITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSISSSPDNYTNV ADPSQILQ ELNRYFYTIVNEKKSIENGTITDPMGELIDFQLGADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTE KRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIE FIKINKNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVA FQIVNGEVRDVTSIVPQDI PAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKNP

ORF4_23FP is a cell wall surface protein An example of an amino acid sequence of ORF4_23FP is set forth in

SEQ ID NO 238

SEQ ID NO:238

MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWDTNGPKGYDGTQSSLKDLTGW AEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVP

NEDVTITLNNVAMDQADYEVTKGINGFNLKLTEAGLAKINGKDADQKIQITYSATLNSLAVADIPESNDITYHYGNHQDHG NTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGYSAEYT VESKGKLGVKNWKDNNPAPINLEEPRVKTYGKKFVKVDQKDTRLENAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQK LDAAVAA YTNAADKQAAQAL VDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGLAAGTYKLEEIKAPEGFA KIDDVEFWGAGSWNQGEFNYLKDVQKNDATKWNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDEDQLA

ORF5_23FP is a cell wall surface protein An example of an amino acid sequence of ORF5_23FP is set forth in SEQ ID NO 239

SEQ ID NO:239

MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDDSYSYDNRVQIVRDL HSWDENKLSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKADTVTTKVKLI KVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSSGQVGRTLYTDKNGEIWTNLPLGTYRFKEVEPLAGYAVTTMD TDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVLQNGKEVVVASGKDGRFRVEGLEYG TYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKTNN

ORF6_23FP is a putative sortase An example of an amino acid sequence of ORF6_23FP is set forth in SEQ ID NO 240

SEQ ID NO:240

MLIKMAKTKKQKRHNLLLGWFFIGIAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWS GDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRI PYVAEVEE EFIAANKLSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVEDE

ORF7_23FP is a putative sortase An example of an amino acid sequence of ORF7_23FP is set forth in SEQ ID NO 241

SEQ ID NO:241

MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKP SEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGENTHTWTAHRGL

IAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD

ORF8_23FP is a putative sortase An example of an amino acid sequence of ORF8_23FP is set forth in SEQ ID NO 242

SEQ ID NO:242

MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDPFLAEGY EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGAD YHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVG

DALYYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTK EGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK

Immunogenic compositions of the invention comprising AI antigens may further comprise one or more antigenic agents Preferred antigens include those listed below Additionally, the compositions of the present invention may be used to treat or prevent infections caused by any of the below-listed microbes Antigens for use in the immunogenic compositions include, but are not limited to, one or more of the following set forth below, or antigens derived from one or more of the following set forth below Bacterial Antigens

N menwgitides a protein antigen from N meningitides serogroup A, C, W135, Y, and/or B ( 1 7), an outer- membrane vesicle (OMV) preparation from N meningitides serogroup B (8, 9, 10, 11), a saccharide antigen, including LPS, from N meningitides serogroup A, B, C W135 and/or Y, such as the oligosaccharide from serogroup C (see PCT/US99/09346, PCT IB98/01665, and PCT IB99/00103),

Streptococcus pneumoniae a saccharide or protein antigen, particularly a saccharide from Streptooccus pneumoniae,

Streptococcus agalactiae particularly, Group B streptococcus antigens,

Streptococcus pyogenes particularly, Group A streptococcus antigens,

Enterococcus faecahs or Enterococcus faecium Particularly a tπsaccharide repeat or other Enterococcus derived antigens provided in US Patent No 6,756,361,

Helicobacter pylori including Cag, Vac, Nap, HopX, HopY and/or urease antigen,

Bordetella pertussis such as petussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B pertussis, optionally also combination with pertactin and/or agglutinogens 2 and 3 antigen.

Staphylococcus aureus including S aureus type 5 and 8 capsular polysaccharides optionally conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A, such as Staph V AX™, or antigens derived from surface proteins, invasins (leukocidin, kinases, hyaluronidase), surface factors that inhibit phagocytic engulfmeπt (capsule, Protein A), carotenoids, catalase production, Protein A, coagulase, clotting factor, and/or membrane-damaging toxins (optionally detoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxm, leukocidin),

Staphylococcus epidermis particularly, S epidermidis slime-associated antigen (SAA),

Staphylococcus saprophyticus (causing urinary tract infections) particularly the 160 kDa hemagglutinin of S saprophyticus antigen,

Pseudomonas aeruginosa particularly, endotoxin A, Wzz protein, P aeruginosa LPS, more particularly LPS isolated from PAOl (05 serotype), and/or Outer Membrane Proteins, including Outer Membrane Proteins F (OprF) (Infect Immuπ 2001 May, 69(5) 3510-3515);

Bacillus anthracis (anthrax) such as B anthracis antigens (optionally detoxified) from A-compoπents (lethal factor (LF) and edema factor (EF)), both of which can share a common B component known as protective antigen (PA),

Moraxella catarrhalis (respiratory) including outer membrane protein antigens (HMW-OMP), C-antigen, and/or LPS,

Yersinia pestis (plague) such as Fl capsular antigen (Infect lmmun 2003 Jan, 71(1)) 374-383, LPS (Infect lmmun 1999 Oct, 67(10) 5395), Yersinia pestis V antigen (Infect lmmun 1997 Nov, 65(11) 4476-4482),

Yersinia enterocohtica (gastrointestinal pathogen) particularly LPS (Infect lmmun 2002 August, 70(8) 4414),

Yersinia pseudotuberculosis gastrointestinal pathogen antigens,

Mycobacterium tuberculosis such as lipoproteins, LPS, BCG antigens, a fusion protein of antigen 85B ( Ag85B) and/or ESAT-6 optionally formulated in cationic lipid vesicles (Infect lmmun 2004 October, 72(10) 6148), Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associated antigens (Proc Natl Acad Sci U S A 2004 Aug 24, 101(34) 12652), and/or MPT51 antigens (Infect lmmun 2004 July, 72(7) 3829),

Legionella pneumophila (Legionnairs' Disease) L pneumophila antigens — optionally derived from cell lines with disrupted asd genes (Infect lmmun 1998 May, 66(5) 1898),

Rickettsia including outer membrane proteins, including the outer membrane protein A and/or B (OmpB) (Biochim Biophys Acta 2004 Nov 1 ,1702(2) 145), LPS, and surface protein antigen (SPA) (J Autoimmun 1989 Jun,2 Suppl δl), E coh including antigens from enterotoxigenic E coh (ETEC) enteroaggregative E coli (EAggEC) diffusely adhering E coh (DAEC), enteropathogenic E coh (EPEC), and/or enterohemorrhagic E coli (EHEC),

Vibrio cholerae including proteinase antigens, LPS, particularly lipopolysacchaπdes of Vibrio cholerae II, Ol Inaba O specific polysaccharides, V cholera 0139, antigens of IEM 108 vaccine (Infect lmnutn 2003 Oct,71( 10) 5498- 504), and/or Zonula occludens toxin (Zot),

Salmonella typhi (typhoid fever) including capsular polysaccharides preferably conjugates (Vi, i e vax-TyVi),

Salmonella typhimurium (gastroenteritis) antigens derived therefrom are contemplated for microbial and cancer therapies, including angiogenesis inhibition and modulation of flk.

Listeria monocytogenes (sytemic infections in immunocompromised or elderly people, infections of fetus) antigens derived from L monocytogenes are preferably used as carriers/vectors for intracytoplasmic delivery of conjugates/associated compositions of the present invention,

Porphyromonas gingivals particularly, P gingivahs outer membrane protein (OMP),

Tetanus such as tetanus toxoid (TT) antigens, preferably used as a carrier protein in conjunction/conjugated with the compositions of the present invention,

Diphtheria such as a diphtheria toxoid, preferably CRMi₉₇, additionally antigens capable of modulating, inhibiting or associated with ADP πbosylation are contemplated for combination/co-administration/conjugation with the compositions of the present invention, the diphtheria toxoids are preferably used as carrier proteins,

Borrelia burgdorferi (Lyme disease) such as antigens associated with P39 and P13 (an integral membrane protein, Infect Immun. 2001 May, 69(5) 3323-3334), VIsE Antigenic Variation Protein (J CIm Microbiol 1999 Dec, 37(12) 3997),

Haemophilus influenzae B such as a saccharide antigen therefrom,

Klebsiella such as an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid,

Neiserna gonorrhoeae including, a Por (or poπn) protein, such as PorB (see Zhu et al , Vaccine (2004) 22 660

- 669), a transferring binding protein, such as TbpA and TbpB (See Price et al , Infection and Immunity (2004) 71(1) 277

- 283), a opacity protein (such as Opa), a reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see Plante et al , J Infectious Disease (2000) 182 848 - 855), also see e g WO99/24578, WO99/36544, WO99/57280, WO02/079243),

Chlamydia pneumoniae particularly C pneumoniae protein antigens,

Chlamydia trachomatis including antigens derived from serotypes A, B, Ba and C are (agents of trachoma, a cause of blindness), serotypes L,, L₂ & L₃ (associated with Lymphogranuloma venereum), and serotypes, D-K, Treponema pallidum (Syphilis) particularly a TmpA antigen, and Haemophilus ducreyi (causing chancroid) including outer membrane protein (DsrA)

Where not specifically referenced, further bacterial antigens of the invention may be capsular antigens, polysaccharide antigens or protein antigens of any of the above Further bacterial antigens may also include an outer membrane vesicle (OMV) preparation Additionally, antigens include live, attenuated, split, and/or purified versions of any of the aforementioned bacteria The bacterial or microbial derived antigens of the present invention may be gram- negative or gram-positive and aerobic or anaerobic

Additionally, any of the above bacterial-derived saccharides (polysaccharides, LPS, LOS or oligosaccharides) can be conjugated to another agent or antigen, such as a carrier protein (for example CRM₁₉₇) Such conjugation may be direct conjugation effected by reductive animation of carbonyl moieties on the saccharide to amino groups on the protein, as provided in US Patent No 5,360,897 and Can J Biochem Cell Biol 1984 May,62(5) 270-5 Alternatively, the saccharides can be conjugated through a linker, such as, with succinamide or other linkages provided in Btoconjugate Techniques, 1996 and CRC Clieniistn of Protein Conjugation and Cioss Linking, 1993 Viral Antigens

Influenza including whole viral particles (attenuated), split or subunit comprising hemagglutinin (HA) and/or neuraminidase (NA) surface proteins, the influenza antigens may be derived from chicken embryos or propogated on cell culture, and/or the influenza antigens are derived from influenza type A, B, and/or C, among others,

Respiratory syncytial virus (RSV) including the F protein ot the A2 strain of RSV (J Gen Virol 2004 Nov, 85(Pt 11) 3229) and/or G glycoprotein,

Parainfluenza virus (PIV) including PIV type 1, 2, and 3, preferably containing hemagglutinin, neuraminidase and/or fusion glycoproteins,

Pohovirus including antigens from a family of picomaviπdae, preferably pohovirus antigens such as OPV or, preferably IPV,

Measles including split measles virus (MV) antigen optionally combined with the Protolhn and or antigens present in MMR vaccine,

Mumps including antigens present in MMR vaccine,

Rubella including antigens present in MMR vaccine as well as other antigens from Togaviπdae, including dengue virus,

Rabies such as lyophilized inactivated virus (RabAvert™),

Flavivindae viruses such as (and antigens derived therefrom) yelow fever virus, Japanese encephalitis virus, dengue virus (types 1, 2, 3, or 4), tick borne encephalitis virus, and West Nile virus,

Cahciviridae, antigens therefrom,

HlV including HIV-I or HIV-2 strain antigens, such as gag (p24gag and p55gag), env (gplόO and gp41), pol, tat, nef, rev vpu, miniprotems, (preferably p55 gag and gpl40v delete) and antigens from the isolates HIV_11n,, HIV_SF2, HIV_LAV, HIV_LA), HIV_MN, HIV-1_CM235, HIV-1_US4, HIV-2, simian immunodeficiency vims (SIV) among others,

Rotavirus including VP4, VP5, VP6, VP7, VP8 proteins (Protein Expr Purif 2004 Dec,38(2) 205) and/or NSP4,

Pestivirus such as antigens from classical porcine fever virus, bovine viral diarrhoea virus, and/or border disease virus,

Parvovirus such as parvovirus B 19,

Coronavirus including SARS virus antigens, particularly spike protein or proteases therefrom, as well as antigens included in WO 04/92360,

Hepatitis A virus such as inactivated virus,

Hepatitis B virus such as the surface and/or core antigens (sAg), as well as the presurface sequences, pre-Sl and pre-S2 (formerly called pre-S), as well as combinations of the above, such as sAg/pre-Sl, sAg/pre-S2, sAg/pre-Sl/ρre-S2, and pre-S l/pre-S2, (see, e g , AHBV Vaccines - Human Vaccines and Vaccination, pp 159-176, and U S Patent Nos 4,722,840, 5,098,704, 5,324,513, Beames et al , 7 Virol (1995) 69 6833-6838, Birnbaum et al , J Virol (1990) 64 3319- 3330, and Zhou et al , J Virol (1991) 65 5457-5464),

Hepatitis C virus such as El, E2, E1/E2 (see, Houghton et al , Hepatology (1991) 14 381), NS345 polyprotein, NS 345-core polyprotein, core, and/or peptides from the nonstructural regions (International Publication Nos WO 89/04669, WO 90/11089, and WO 90/14436),

Delta hepatitis virus (HDV) antigens derived therefrom, particularly δ-antigen from HDV (see, ej . U S Patent No 5,378,814), Hepatitis E virus (HEV) antigens derived therefrom,

Hepatitis G virus (HGV), antigens derived therefrom,

Varcicella zoster urus antigens derived from varicella zoster virus (VZV) (J Gen Virol (1986) 67 1759),

Epslein-Barr virus antigens derived from EBV (Baer et al , Nature ( 1984) 310 207),

Cytomegalovirus CMV antigens, including gB and gH (Cytomegaloviruses (J K McDougall, ed , Springer Verlag 1990) pp 125 169),

Herpes simplex virus including antigens from HSV 1 or HSV-2 strains and glycoproteins gB, gD and gH (McGeoch et al , J Gen Virol (1988) 69 1531 and U S Patent No 5,171,568),

Human Herpes Virus antigens derived from other human herpesviruses such as HHV6 and HHV7, and

HPV including antigens associated with or derived from human papillomavirus (HPV), for example, one or more of El - E7, Ll, L2, and fusions thereof, particularly the compositions of the invention may include a virus-like particle (VLP) comprising the Ll major capsid protein, more particular still, the HPV antigens are protective against one or more of HPV serotypes 6, 11, 16 and/or 18

Further provided are antigens, compostions, methods, and microbes included in Vaccines, 4^th Edition (Plotkin and Orenstein ed 2004), Medical Microbiology 4^th Edition (Murray et al ed 2002), Virology, 3rd Edition (W K Joklik ed 1988), Fundamental Virology, 2nd Edition (B N Fields and D M Knipe, eds 1991), which are contemplated in conjunction with the compositions of the present invention

Additionally, antigens include live, attenuated, split, and/or purified versions of any of the aforementioned viruses

Funeal Antieens

Fungal antigens for use herein, associated with vaccines include those described in U S Pat Nos 4,229,434 and 4,368,191 for prophylaxis and treatment of tπchopytosis caused by Trichophyton mentagrophytes, U S Pat Nos 5,277,904 and 5,284,652 for a broad spectrum dermatophyte vaccine for the prophylaxis of dermatophyte infection in animals, such as guinea pigs, cats, rabbits, horses and lambs, these antigens comprises a suspension of killed T equinum, T mentagrophytes (var granulare), M cams and/or M gypseum in an effective amount optionally combined with an adjuvant, U S Pat Nos 5,453,273 and 6,132,733 for a ringworm vaccine comprising an effective amount of a homogenized, formaldehyde-killed fungi, i e , Microsporum cams culture in a carrier, U S Pat No 5,948,413 involving extracellular and intracellular proteins for pythiosis Additional antigens identified within antifungal vaccines include Ringvac bovis LTF- 130 and Bioveta

Further, fungal antigens for use herein may be deπved from Dermatophytres, including Epidermophyton floccusum, Microsporum audouim, Microsporum cams, Microsporum distortum, Microsporum equinum Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton galhnae, Trichophyton gypseum, Trichophyton megnint, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T verrucosum var album, var discoides, var ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme

Fungal pathogens for use as antigens or in derivation of antigens in conjunction with the compositions of the present invention comprise Aspergillus fumigatus, Aspergillus flavus Aspergillus niger, Aspergillus nidulans Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans Candida enolase, Candida tropicalis Candida glabrata, Candida krusei, Candida parapsilosis Candida stellatoidea Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi Cladosporium carrwnii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum Histoplasma capsulatum Klebsiella pneumoniae Paracoccidioides brasihensis Pneumocystis cannii Pythiumn insidiosum Pityrosporum ovale Sacharomyces cerevisae Saccharomyces boulardu Saccharomvces pombe Scedosporium apiosperum Sporothrn ichenckii, Trichosporon beigelu Toxoplasma gondii, Penicillitim marneffet, Malassezia spp , Fonsecaea spp , Wangiella spp , Sporothπx spp , Basidiobolus spp , Conidiobolus spp , Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp Cunninghamella spp, and Saksenaea spp

Other fungi from which antigens are derived include Alternaπa spp, Curvulaπa spp, Helminthospoπum spp, Fusaπum spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctoma spp, Paecilomyces spp, Pithomyces spp, and Cladospoπum spp

Processes for producing a fungal antigens are well known in the art (see US Patent No 6,333,164) In a preferred method a solubihzed fraction extracted and separated from an insoluble fraction obtainable from fungal cells of which cell wall has been substantially removed or at least partially removed, characterized in that the process comprises the steps of obtaining living fungal cells, obtaining fungal cells of which cell wall has been substantially removed or at least partially removed, bursting the fungal cells of which cell wall has been substantially removed or at least partially removed, obtaining an insoluble fraction, and extracting and separating a solubihzed fraction from the insoluble fraction STD Antigens

In particular embodiments, microbes (bacteria, viruses and/or fungi) against which the present compositions and methods can be implement include those that cause sexually transmitted diseases (STDs) and/or those that display on their surface an antigen that can be the target or antigen composition of the invention In a preferred embodiment of the invention, compositions are combined with antigens derived from a viral or bacterial STD Antigens derived from bacteria or viruses can be administered in conjunction with the compositions of the present invention to provide protection against at least one of the following STDs, among others chlamydia, genital herpes, hepatitis (particularly HCV), genital warts, gonorrhoea, syphilis and/or chancroid (See, WOOO/15255)

In another embodiment the compositions of the present invention are co-administered with an antigen for the prevention or treatment of an STD

Antigens derived from the following viruses associated with STDs, which are described in greater detail above, are preferred for co-administration with the compositions of the present invention hepatitis (particularly HCV), HPV, HIV, or HSV

Additionally, antigens derived from the following bacteria associated with STDs, which are described in greater detail above, are preferred for co-administration with the compositions of the present invention Neiserna gonorrhoeae, Chlamydia pneumoniae, Chlamydia trachomatis, Treponema pallidum, or Haemophilus ducreyi Respiratory Antigens

The antigen may be a respiratory antigen and could further be used in an immunogenic composition for methods of preventing and/or treating infection by a respiratory pathogen, including a virus, bacteria, or fungi such as respiratory syncytial virus (RSV), PIV, SARS virus, influenza, Bacillus anthracis, particularly by reducing or preventing infection and/or one or more symptoms of respiratory virus infection A composition comprising an antigen described herein, such as one derived from a respiratory virus, bacteria or fungus is administered in conjunction with the compositions of the present invention to an individual which is at risk of being exposed to that particular respiratory microbe, has been exposed to a respiratory microbe or is infected with a respiratory virus, bacteria or fungus The composιtιon(s) of the present invention is/are preferably co-administered at the same time or in the same formulation with an antigen of the respiratory pathogen Administration of the composition results in reduced incidence and/or severity of one or more symptoms of respiratory infection Pediatric/Genatric Antigens

In one embodiment the compositions of the present invention are used in conjunction with an antigen for treatment of a pediatric population, as in a pediatric antigen In a more particular embodiment the pediatric population is less than about 3 years old, or less than about 2 years, or less than about 1 years old In another embodiment the pediatric antigen (in conjunction with the composition of the present invention) is administered multiple times over at least 1 2, or 3 years

In another embodiment the compositions of the present invention are used in conjunction with an antigen for treatment of a geriatric population, as in a geriatric antigen Other Antigens

Other antigens for use in conjunction with the compositions of the present include hospital acquired (nosocomial) associated antigens

In another embodiment, parasitic antigens are contemplated in conjunction with the compositions of the present invention Examples of parasitic antigens include those derived from organisms causing malaria and/or Lyme disease

In another embodiment, the antigens in conjunction with the compositions of the present invention are associated with or effective against a mosquito born illness In another embodiment, the antigens in conjunction with the compositions of the present invention are associated with or effective against encephalitis In another embodiment the antigens in conjunction with the compositions of the present invention are associated with or effective against an infection of the nervous system

In another embodiment, the antigens in conjunction with the compositions of the present invention are antigens transmissible through blood or body fluids Antigen Formulations

In other aspects of the invention, methods of producing microparticles having adsorbed antigens are provided The methods comprise (a) providing an emulsion by dispersing a mixture comprising (i) water, (ii) a detergent, (in) an organic solvent, and (iv) a biodegradable polymer selected from the group consisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydπde, and a polycyanoacrylate The polymer is typically present in the mixture at a concentration of about 1% to about 30% relative to the organic solvent, while the detergent is typically present in the mixture at a weight-to-weight detergent-to- polymer ratio of from about 000001 1 to about 0 1 1 (more typically about 00001 1 to about 0 1 1, about 0001 1 to about 0 1 1, or about 0005 1 to about 0 1 1), (b) removing the organic solvent from the emulsion, and (c) adsorbing an antigen on the surface of the microparticles In certain embodiments, the biodegradable polymer is present at a concentration of about 3% to about 10% relative to the organic solvent

Microparticles for use herein will be formed from materials that are steπlizable, non-toxic and biodegradable Such materials include, without limitation, poly(α-hydroxy acid), polyhydroxybutyπc acid, polycaprolactone, polyorthoester, polyanhydπde, PACA, and polycyanoacrylate Preferably, microparticles for use with the present invention are derived from a poly(α-hydroxy acid), in particular, from a poly(lactide) ("PLA") or a copolymer of D,L-lactide and glycolide or glycolic acid, such as a poly(D,L-lactide-co- glycohde) ("PLG' or "PLGA' ), or a copolymer of D,L-lactide and caprolactone The microparticles may be derived from any of various polymeric starting materials which have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide glycolide ratios, the selection of which will be largely a matter of choice, depending in part on the coadministered macromolecule These parameters are discussed more fully below

Further antigens may also include an outer membrane vesicle (OMV) preparation Additional formulation methods and antigens (especially tumor antigens) are provided in U S Patent Serial No81 772

Antigen References

The following references include antigens useful in conjunction with the compositions of the present invention

1 International patent application WO99/24578

2 International patent application WO99/36544

3 International patent application WO99/57280

4 Internationa] patent application WO00/22430

5 Tettelm et al (2000) Science 287 1809-1815

6 International patent application WO96/29412

7 Pizza et al (2000) Science 287 1816-1820

8 PCT WO 01/52885

9 Bjune et al (1991) Lancet 338(8775)

10 Fuskasawa et al (1999) Vaccine 17 2951-2958

1 1 Rosenqist et al (1998) Dev Biol Strand 92 323-333

12 Constantino et al (1992) Vaccine 10 691-698

13 Constantino et al (1999) Vaccine 17 1251-1263

14 Watson (2000) Pediatr Infect Dis J 19 331-332

15 Rubin (20000) Pediatr Clin North Am 47 269-285,v

16 Jedrzejas (2001) Microbiol MoI Biol Rev 65 187 207

17 International patent application filed on 3^rd July 2001 claiming priority from GB-0016363 4,WO 02/02606, PCT IB/01/00166

18 Kalman et al (1999) Nature Genetics 21 385-389

19 Read et al (2000) Nucleic Acids Res 28 1397-406

20 Shirai et al (2000) J Infect Dis 181(Suppl 3) S524-S527

21 International patent application WO99/27105

22 International patent application WO00/27994

23 International patent application WO00/37494

24 International patent application WO99/28475

25 Bell (2000) Pediatr Infect Dis J 19 1187-1188

26 Iwarson (1995) APMIS 103 321 326

27 Gerlich et al (1990) Vaccine 8 Suppl S63-68 & 79-80

28 Hsu et al (1999) Clin Liver Dis 3 901-915

29 Gastofsson et al (1996) N Engl J Med 334- 349-355

30 Rappuoli et al (199I) TIBTECH 9 232-238

31 Vaccines (1988) eds Plotkin & Mortimer ISBN 0-7216-1946-0

32 Del Guidice et al (1998) Molecular Aspects of Medicine 19 1 -70

33 International patent application WO93/018150

34 International patent application WO99/53310

35 International patent application WO98/04702

36 Ross et al (2001) Vaccine 19 135-142

37 Sutter et al (2000) Pediatr Clin North Am 47 287-308

38 Zimmerman & Spann (1999) Am Fan Physician 59 113-118, 125 126

39 Dreensen ( 1997) Vaccine 15 Suppl"S2-6

40 MMWR Morb Mortal WkIy rep 1998 Jan 16 47(1) 12, 9

41 McMichael (2000) Vaccinel9 Suppl 1 S101-107

42 Schuchat (1999) Lancer 353(9146) 51-6

43 GB patent applications 0026333 5, 0028727 6 & 01056407

44 Dale (1999) Infect Disclin North Am 13 227-43, vin

45 Ferretti et al (200I) PNAS USA 98 4658-4663

46 Kuroda et al (2001) Lancet 357(9264) 1225-1240, see also pages 1218-1219

47 Ramsay et al (2001) Lancet 357(9251) 195 196

48 Lindberg (1999) Vaccine 17 Suppl 2 S28-36

49 Buttery & Moxon (2000) J R Coil Physicians Long 34 163 168

50 Ahmad & Chapnick (1999) Infect Dis Clin North Am 13 113-133, vii

51 GoIdblatt ( 1998) J Med Microbiol 47 663-567

52 European patent 0477 508

53 U S Patent No 5,306,492

54 International patent application WO98/42721

55 Conjugate Vaccines (eds Cruse et al ) ISBN 3805549326, particularly vol 10 48 114 56 Hermanson ( J996) Bioconjugate Techniques ISBN 012323368 & 012342335X

57 European patent application 0372501

58 European patent application 0378881

59 European patent application 0427347

60 International patent application WO93/17712

61 International patent application WO98/58668

62 European patent application 0471177

63 International patent application WO00/56360

64 International patent application WO00/67161

The contents of all of the above cited patents, patent applications and journal articles are incorporated by reference as if set forth fully herein

There may be an upper limit to the number of Gram positive bacterial proteins which will be in the compositions of the invention Preferably, the number of Gram positive bacterial proteins in a composition of the invention is less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 1 1, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3 Still more preferably, the number of Gram positive bacterial proteins in a composition of the invention is less than 6, less than 5, or less than 4 Still more preferably, the number of Gram positive bacterial proteins in a composition of the invention is 3

The Gram positive bacterial proteins and polynucleotides used in the invention are preferably isolated, / e , separate and discrete, from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose Fusion Proteins GBS AI sequences

The GBS AI proteins used in the invention may be present in the composition as individual separate polypeptides, but it is preferred that at least two (ι e 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) of the antigens are expressed as a single polypeptide chain (a "hybrid" or "fusion" polypeptide) Such fusion polypeptides offer two principal advantages first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable fusion partner that overcomes the problem, second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful

The fusion polypeptide may comprise one or more AI polypeptide sequences Preferably, the fusion comprises an AI surface protein sequence Preferably, the fusion polypeptide includes one or more of GBS 80, GBS 104, and GBS 67 Most preferably, the fusion peptide includes a polypeptide sequence from GBS 80 Accordingly, the invention includes a fusion peptide comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a GBS AI surface protein or a fragment thereof Preferably, the first and second amino acid sequences in the fusion polypeptide comprise different epitopes

Hybrids (or fusions) consisting of ammo acid sequences from two, three, four, five, six, seven, eight, nine, or ten GBS antigens are preferred In particular, hybrids consisting of amino acid sequences from two, three, four, or five GBS antigens are preferred

Different hybrid polypeptides may be mixed together in a single formulation Within such combinations, a GBS antigen may be present in more than one hybrid polypeptide and/or as a non-hybrid polypeptide It is preferred, however, that an antigen is present either as a hybrid or as a non hybrid, but not as both

Hybrid polypeptides can be represented by the formula NH₂-A-{-X-L-},,-B-COOH, wherein X is an amino acid sequence of a GBS AI protein or a fragment thereof, L is an optional linker amino acid sequence, A is an optional N-termmal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 If a X- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein In some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-terminus of the hybrid protein / e the leader peptide of Xi will be retained, but the leader peptides of X₂ X_n will be omitted This is equivalent to deleting all leader peptides and using the leader peptide of X| as moiety -A-

For each n instances of { X-L }, linker amino acid sequence -L- may be present or absent For instance, when «=2 the hybrid may be NH₂-X₁-L₁-X₂-L₂ COOH, NH₂-X₁-X₂-COOH, NH₂-Xi-L, X₂ COOH, NH₂-X₁-X₂-L₂-COOH, etc Linker amino acid sequence(s) L will typically be short {e g 20 or fewer ammo acids ; e 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples comprise short peptide sequences which facilitate cloning, poly-glycine linkers (i e comprising GIy_n where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (J e HiS_n where n = 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable linker amino acid sequences will be apparent to those skilled in the art A useful linker is GSGGGG, with the Gly-Ser dipeptide being formed from a BamHl restriction site, thus aiding cloning and manipulation, and the (Gly)₄ tetrapeptide being a typical poly glycine linker

-A- is an optional N-terminal amino acid sequence This will typically be short (e g 40 or fewer amino acids i e 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e g histidine tags i e HiS_n where n - 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art If Xi lacks its own N-terminus methionine, -A- is preferably an oligopeptide (e g with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine

-B- is an optional C-terminal amino acid sequence This will typically be short (e g 40 or fewer amino acids t e 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e g comprising histidine tags i e HiS_n where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art

Most preferably, Λ is 2 or 3

In some embodiment the GBS hybrid proteins of the invention may comprise first -X- moiety (-X_a-) and a second -X- moiety (-X_b-) The -X_a- moiety has one of the following amino acid sequences SEQ ID NO 16, SEQ ID NO 126, SEQ ID NO 2, SEQ ID NO 11, SEQ ID NO 21, SEQ ID NO 27, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 36

The -X_b- moiety is related to -X_a- such that (i) -X_b- has sequence identity to -X_a-, and/or (j) -X_b- comprises a fragment of -X_a- Examples of this second type of hybrid protein include proteins in which two or more -X- moieties are identical, or in which they are variants of the same protein e g two polymorphic forms may be expressed as X_a X_b , and three polymorphic forms may be expressed as -X_a-X_b-X_<;- etc The -Xa- and -Xb- moieties may be in either order from N- terminus to C-terminus

The degree of 'sequence identity' referred to in (i) is preferably greater than 50% (ea 60%, 70%, 80%, 90%, 95%, 99% or more, up to 100%) This includes mutants, homologs, orthologs, allelic variants etc Identity is preferably determined by the Smith- Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an 30 affine gap search with parameters gap open penalty=12 and gap extension penalty=l, Typically, 50% identity or more between two proteins is considered as an indication of functional equivalence The 'fragment' referred to in (j) should consist of least m consecutive amino acids from an amino acid sequence from SEQ ID NO 16, SEQ ID NO 126, SEQ ID NO 2, SEQ ID NO 11, SEQ ID NO 21, SEQ ID NO 27, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 36 and, depending on the particular sequence, m is 7 or more (ea 8, 10,&rsqb,2, 14, 16, 18 20, 25, 30, 35 40, 50, 60, 70, 80, 90, 100, 150, 200 or more) Preferably the fragment comprises an epitope from an amino acid sequence from SEQ ID NO 16, SEQ ID NO 126, SEQ ID NO 2, SEQ ID NO 11, SEQ ID NO 21, SEQ ID NO 27, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 36

Fusion Proteins Gram positive bacteria AI sequences

The Gram positive bacteria AI proteins used in the invention may be present in the composition as individual separate polypeptides, but it is preferred that at least two (i e 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18) of the antigens are expressed as a single polypeptide chain (a "hybrid" or "fusion" polypeptide) Such fusion polypeptides offer two principal advantages first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable fusion partner that overcomes the problem, second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful

The fusion polypeptide may comprise one or more AI polypeptide sequences Preferably, the fusion comprises an AI surface protein sequence Accordingly, the invention includes a fusion peptide comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second ammo acid sequences are selected from a Gram positive bacteria AI protein or a fragment thereof Preferably, the first and second amino acid sequences in the fusion polypeptide comprise different epitopes

Hybrids (or fusions) consisting of ammo acid sequences from two, three, four, five, six, seven, eight, nine, or ten Gram positive bacteria antigens are preferred In particular, hybrids consisting of ammo acid sequences from two, three, four, or five Gram positive bacteria antigens are preferred

Different hybrid polypeptides may be mixed together in a single formulation Within such combinations, a Gram positive bacteria AI sequence may be present in more than one hybπd polypeptide and/or as a non-hybrid polypeptide It is preferred, however, that an antigen is present either as a hybπd or as a non-hybrid, but not as both

Hybrid polypeptides can be represented by the formula NH₂-A-I-X-L-I_n-B-COOH, wherein X is an amino acid sequence of a Gram positive bacteria AI sequence or a fragment thereof, L is an optional linker amino acid sequence, A is an optional N-terminal ammo acid sequence, B is an optional C-terπunal amino acid sequence, and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15

If a -X- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein In some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-termmus of the hybπd protein i e the leader peptide of Xi will be retained, but the leader peptides of X₂ X_n will be omitted This is equivalent to deleting all leader peptides and using the leader peptide of Xi as moiety -A-

For each n instances of {-X-L-}, linker amino acid sequence -L- may be present or absent For instance, when n=2 the hybπd may be NH₂-X₁-L₁-X₂-L₂-COOH, NH₂-X₁-X₂-COOH, NH₂-X₁-Li-X₂-COOH, NH₂-X₁-X₂-L₂-COOH, etc Linker amino acid sequence(s) -L- will typically be short (e g 20 or fewer ammo acids i e 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples comprise short peptide sequences which facilitate cloning, poly-glycine linkers (ι e comprising GIy_n where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (ι e HiS_n where n = 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable linker amino acid sequences will be apparent to those skilled in the art A useful linker is GSGGGG, with the Gly-Ser dipeptide being formed from a BarήΑl restriction site, thus aiding cloning and manipulation, and the (GIy)₄ tetrapeptide being a typical poly glycine linker

-A- is an optional N-terminal amino acid sequence This will typically be short (e g 40 or fewer amino acids i e 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e g histidine tags i e His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art If X₁ lacks its own N-terminus methionine -A- is preferably an oligopeptide (e g with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine

-B- is an optional C-terminal amino acid sequence This will typically be short (e g 40 or fewer amino acids f e 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e g comprising histidine tags i e HiS_n where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art

Most preferably, n is 2 or 3 Antibodies GBS AI sequences

The GBS AI proteins of the invention may also be used to prepare antibodies specific to the GBS AI proteins The antibodies are preferably specific to the an oligomeric or hyper oligomeπc form of an AI protein The invention also includes combinations of antibodies specific to GBS AI proteins selected to provide protection against an increased range of GBS serotypes and strain isolates For example, a combination may comprise a first and second antibody, wherein said first antibody is specific to a first GBS AI protein and said second antibody is specific to a second GBS AI protein Preferably, the nucleic acid sequence encoding said first GBS AI protein is not present in a GBS genome comprising a polynucleotide sequence encoding for said second GBS AI protein Preferably, the nucleic acid sequence encoding said first and second GBS AI proteins are present in the genomes of multiple GBS serotypes and strain isolates

The GBS specific antibodies of the invention include one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a GBS polypeptide. The antibodies of the invention include antibodies which specifically bind to a GBS AI protein The invention includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following hybrid (chimeric) antibody molecules (see, for example, Winter et al (1991) Nature 349 293-299, and US Patent No 4,816,567, F(ab')₂ and F(ab) fragments, F_v molecules (non-covalent heterodimers, see, for example, Inbar et al (1972) Proc Natl Acad Sci USA 69 2659-2662, and Ehrlich et al (1980) Biochem 19 4091 -4096) , single-chain Fv molecules (sFv) (see, for example, Huston et al (1988) Proc Natl Acad Sci USA 85 5897 5883), dimeric and tπmeπc antibody fragment constructs, minibodies (see, e g , Pack et al. (1992) Biochem 31 1579-1584, Cumber et al (1992) J Immunology 149B 120-126), humanized antibody molecules (see, for example, Riechmann et al (1988) Nature 332 323-327, Verhoeyan et al (1988) Science 239 1534-1536, and U K Patent Publication No GB 2,276,169, published 21 September 1994), and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule The invention further includes antibodies obtained through non-conventional processes, such as phage display

Preferably, the GBS specific antibodies of the invention are monoclonal antibodies Monoclonal antibodies of the invention include an antibody composition having a homogeneous antibody population Monoclonal antibodies of the invention may be obtained from murine hybπdomas, as well as human monoclonal antibodies obtained using human rather than murine hybπdomas See, e g , Cote, et al Monoclonal Antibodies and Cancer Therapy, Alan R Liss, 1985, p 77

The antibodies of the invention may be used in diagnostic applications, for example, to detect the presence or absence of GBS in a biological sample The antibodies of the invention may also be used in the prophylactic or therapeutic treatment of GBS infection Antibodies Gram positive bacteria AI sequences

The Gram positive bacteria AI proteins of the invention may also be used to prepare antibodies specific to the Gram positive bacteria AI proteins The antibodies are p/eferably specific to the an oligomeric or hyper-oligomeπc form of an AI protein. The invention also includes combinations of antibodies specific to Gram positive bacteria AI proteins selected to provide protection against an increased range of Gram positive bacteria genus, species, serotypes and strain isolates.

For example, a combination may comprise a first and second antibody, wherein said first antibody is specific to a first Gram positive bacteria AI protein and said second antibody is specific to a second Gram positive bacteria AI protein. Preferably, the nucleic acid sequence encoding said first Gram positive bacteria AI protein is not present in a Gram positive bacterial genome comprising a polynucleotide sequence encoding for said second Gram positive bacteria AI protein. Preferably, the nucleic acid sequence encoding said first and second Gram positive bacteria AI proteins are present in the genomes of multiple Gram positive bacteria genus, species, serotypes or strain isolates.

As an example of an instance where the combination of antibodies provides protection against an increased range of bacteria serotypes, the first antibody may be specific to a first GAS AI protein and the second antibody may be specific to a second GAS AI protein. The first GAS AI protein may comprise a GAS AI-I surface protein, while the second GAS AI protein may comprise a GAS AI-2 or AI-3 surface protein.

As an example of an instance where the combination of antibodies provides protection against an increased range of bacterial species, the first antibody may be specific to a GBS AI protein and the second antibody may be specific to a GAS AI protein. Alternatively, the first antibody may be specific to a GAS AI protein and the second antibody may be specific to a S. pneumoniae AI protein.

The Gram positive specific antibodies of the invention include one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a Gram positive bacteria AI polypeptide. The antibodies of the invention include antibodies which specifically bind to a Gram positive bacteria AI protein. The invention includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, for example. Winter et al. (1991) Nature 349: 293-299; and US Patent No. 4,816,567; F(ab')₂ and F(ab) fragments; F_v molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5897-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g.. Pack et al. (1992) Biochem 3J.: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, for example, Riechmann et al. (1988) Nature 332:323- 327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 September 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule. The invention further includes antibodies obtained through non-conventional processes, such as phage display.

Preferably, the Gram positive specific antibodies of the invention are monoclonal antibodies. Monoclonal antibodies of the invention include an antibody composition having a homogeneous antibody population. Monoclonal antibodies of the invention may be obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. See, e.g., Cote, et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p 77.

The antibodies of the invention may be used in diagnostic applications, for example, to detect the presence or absence of Gram positive bacteria in a biological sample. The antibodies of the invention may also be used in the prophylactic or therapeutic treatment of Gram positive bacteria infection. Nucleic Acids

The invention provides nucleic acids encoding the Gram positive bacteria sequences and/or the hybrid fusion polypeptides of the invention. The invention also provides nucleic acid encoding the GBS antigens and/or the hybrid fusion polypeptides of the invention Furthermore, the invention provides nucleic acid which can hybridise to these nucleic acids, preferably under "high stringency" conditions (e g 65⁰C in a 0 IxSSC, 05% SDS solution)

Polypeptides of the invention can be prepared by various means (e g recombinant expression, purification from cell culture, chemical synthesis, etc ) and in various forms (e g native, fusions, non-glycosylated, lipidated, etc ) They are preferably prepared in substantially pure form (ι e substantially free from other GAS or host cell proteins)

Nucleic acid according to the invention can be prepared in many ways (e g by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc ) and can take various forms (e g single stranded, double stranded, vectors, probes, etc ) They are preferably prepared in substantially pure form (ι e substantially free from other GBS or host cell nucleic acids)

The term "nucleic acid" includes DNA and RNA, and also their analogues, such as those containing modified backbones (e g phosphorothioates, etc ), and also peptide nucleic acids (PNA), etc The invention includes nucleic acid comprising sequences complementary to those described above (e g for antisense or probing purposes)

The invention also provides a process for producing a polypeptide of the invention, comprising the step of culturing a host cell transformed with nucleic acid of the invention under conditions which induce polypeptide expression

The invention provides a process for producing a polypeptide of the invention, comprising the step of synthesising at least part of the polypeptide by chemical means

The invention provides a process for producing nucleic acid of the invention, comprising the step of amplifying nucleic acid using a primer-based amplification method (e g PCR)

The invention provides a process for producing nucleic acid of the invention, comprising the step of synthesising at least part of the nucleic acid by chemical means Purification and Recombinant Expression

The Gram positive bacteria AI proteins of the invention may be isolated from the native Gram positive bacteria, or they may be recombinantly produced, for instance in a heterologous host. For example, the GAS, GBS, and S pneumoniae antigens of the invention may be isolated from Streptococcus agalactiae, S pyogenes, S pneumoniae, or they may be recombinantly produced, for instance, in a heterologous host Preferably, the GBS antigens are prepared using a heterologous host

The heterologous host may be prokaryotic (e g a bacterium) or eukaryotic It is preferably E coli, but other suitable hosts include Bacillus subtihs, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cmerea, Mycobacteria (e g M tuberculosis), S gordonn, L. lactis, yeasts, etc

Recombinant production of polypeptides is facilitated by adding a tag protein to the Gram positive bacteria AI sequence to be expressed as a fusion protein comprising the tag protein and the Gram positive bacteria antigen For example, recombinant production of polypeptides is facilitated by adding a tag protein to the GBS antigen to be expressed as a fusion protein comprising the tag protein and the GBS antigen Such tag proteins can facilitate purification, detection and stability of the expressed protein Tag proteins suitable for use in the invention include a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmoduhn-binding peptide, cellulose-binding domain, SBP-tag,, chitin-binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination anti-terminiantion factor (NusA), E coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA) Preferred tag proteins include His-tag and GST A full discussion on the use of tag proteins can be found at Terpe et al , "Overview of tag protein fusions from molecular and biochemical fundamentals to commercial systems, " Appl Microbiol Biotechnol (2003) 60 523 - 533 After purification, the tag proteins may optionally be removed from the expressed fusion protein, i e , by specifically tailored enzymatic treatments known in the art Commonly used proteases include enterokinase tobacco etch virus (TEV), thrombin, and factor X_d GBS polysaccharides

The compositions of the invention may be further improved by including GBS polysaccharides Preferably, the GBS antigen and the saccharide each contribute to the immunological response in a recipient The combination is particularly advantageous where the saccharide and polypeptide provide protection from different GBS serotypes

The combined antigens may be present as a simple combination where separate saccharide and polypeptide antigens are administered together, or they may be present as a conjugated combination, where the saccharide and polypeptide antigens are covalently linked to each other

Thus the invention provides an immunogenic composition comprising (i) one or more GBS AI proteins and (11) one or more GBS saccharide antigens The polypeptide and the polysaccharide may advantageously be covalently linked to each other to form a conjugate

Between them, the combined polypeptide and saccharide antigens preferably cover (or provide protection from) two or more GBS serotypes (e g 2, 3, 4, 5, 6, 7, 8 or more serotypes) The serotypes of the polypeptide and saccharide antigens may or may not overlap For example, the polypeptide might protect against serogroup II or V, while the saccharide protects against either serogroups Ia, Ib, or III Preferred combinations protect against the following groups of serotypes (1) serotypes Ia and Ib, (2) serotypes Ia and II, (3) serotypes Ia and III, (4) serotypes Ia and IV, (5) serotypes Ia and V, (6) serotypes Ia and VI, (7) serotypes Ia and VII, (8) serotypes Ia and VIII, (9) serotypes Ib and II, (10) serotypes Ib and III, (11) serotypes Ib and IV, (12) serotypes Ib and V, (13) serotypes Ib and VI, (14) serotypes Ib and VII, (15) serotypes Ib and VIII, 16) serotypes II and III, (17) serotypes II and IV, (18) serotypes II and V, (19) serotypes II and VI, (20) serotypes II and VII, (21) serotypes II and VII, (22) serotypes III and IV, (23) serotypes III and V, (24) serotypes III and VI, (25) serotypes III and VII, (26) serotypes III and VIII, (27) serotypes IV and V, (28) serotypes IV and VI, (29) serotypes IV and VII, (30) serotypes IV and VIII, (31) serotypes V and VI, (32) serotypes V and VII, (33) serotypes V and VIII, (34) serotypes VI and VII, (35) serotypes VI and VIII, and (36) serotypes VII and VIII

Still more preferably, the combinations protect against the following groups of serotypes (1) serotypes Ia and II, (2) serotypes Ia and V, (3) serotypes Ib and II, (4) serotypes Ib and V, (5) serotypes HI and II, and (6) serotypes III and V Most preferably, the combinations protect against serotypes III and V

Protection against serotypes II and V is preferably provided by polypeptide antigens Protection against serotypes Ia, Ib and/or III may be polypeptide or saccharide antigens Immunogenic compositions and medicaments

Compositions of the invention are preferably immunogenic compositions, and are more preferably vaccine compositions The pH of the composition is preferably between 6 and 8, preferably about 7 The pH may be maintained by the use of a buffer The composition may be sterile and/or pyrogen-free The composition may be isotonic with respect to humans

Vaccines according to the invention may either be prophylactic (ι e to prevent infection) or therapeutic (/ e to treat infection), but will typically be prophylactic Accordingly, the invention includes a method for the therapeutic or prophylactic treatment of a Gram positive bacteria infection in an animal susceptible to such gram positive bacterial infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic composition of the invention For example, the invention includes a method for the therapeutic or prophylactic treatment of a Streptococcus agalactiae, S pyogenes, or 5 pneumoniae infection in an animal susceptible to streptococcal infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic compositions of the invention

The invention also provides a composition ot the invention for use of the compositions described herein as a medicament The medicament is preferably able to raise an immune response in a mammal (i e it is an immunogenic composition) and is more preferably a vaccine

The invention also provides the use of the compositions of the invention in the manufacture of a medicament for raising an immune response in a mammal The medicament is preferably a vaccine

The invention also provides kits comprising one or more containers of compositions of the invention Compositions can be in liquid form or can be lyophihzed, as can individual antigens Suitable containers for the compositions include, for example, bottles, vials, syringes and test tubes Containers can be formed from a variety of materials, including glass or plastic A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The composition may comprise a first component comprising one or more Gram positive bacteria AI proteins Preferably, the AI proteins are surface AI proteins Preferably, the AI surface proteins are in an oligomeπc or hyperoligomeric form For example, the first component comprises a combination of GBS antigens or GAS antigens, or S pneumoniae antigens Preferably said combination includes GBS 80 Preferably GBS 80 is present in an oligomeπc or hyperoligomeric form

The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution It can also contain other materials useful to the end- user, including other buffers, diluents, filters, needles, and syringes The kit can also comprise a second or third container with another active agent, for example an antibiotic

The kit can also comprise a package insert containing written instructions for methods of inducing immunity against S agalactiae andor S pyogenes and/or S pneumoniae or for treating S agalactiae andor S pyogenes and/or S pneumoniae infections The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body

The invention also provides a delivery device pre-filled with the immunogenic compositions of the invention

The invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity This immune response will preferably induce long lasting {e g , neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to one or more GBS and/or GAS and/or 5 pneumoniae antigens The method may raise a booster response

The invention provides a method of neutralizing GBS, GAS, or S pneumoniae infection in a mammal comprising the step of administering to the mammal an effective amount of the immunogenic compositions of the invention, a vaccine of the invention, or antibodies which recognize an immunogenic composition of the invention

The mammal is preferably a human Where the vaccine is for prophylactic use, the human is preferably a female (either of child bearing age or a teenager) Alternatively, the human may be elderly (e g , over the age of 50, 55, 60, 65, 70 or 75) and may have an underlying disease such as diabetes or cancer. Where the vaccine is for therapeutic use, the human is preferably a pregnant female or an elderly adult

These uses and methods are preferably for the prevention and/or treatment of a disease caused by Streptococcus agalactiae, or S pyogenes, or S pneumoniae The compositions may also be effective against other streptococcal bacteria The compositions may also be effective against other Gram positive bacteria

One way of checking efficacy of therapeutic treatment involves monitoring Gram positive bacterial infection after administration of the composition of the invention One way of checking efficacy of prophylactic treatment involves monitoring immune responses against the Gram positive bacterial antigens in the compositions of the invention after administration of the composition

One way or checking efficacy of therapeutic treatment involves monitoring GBS infection after administration of the composition of the invention One way of checking efficacy of prophylactic treatment involves monitoring immune responses against the GBS antigens in the compositions of the invention after administration of the composition

A way of assessing the immunogenicity of the component proteins of the immunogenic compositions of the present invention is to express the proteins recombinantly and to screen patient sera or mucosal secretions by immunoblot A positive reaction between the protein and the patient serum indicates that the patient has previously mounted an immune response to the protein in question- that is, the protein is an immunogen This method may also be used to identify immunodominant proteins and/or epitopes

Another way of checking efficacy of therapeutic treatment involves monitoring GBS or GAS or S pneumoniae infection after administration of the compositions of the invention One way of checking efficacy of prophylactic treatment involves monitoring immune responses both systemically (such as monitoring the level of IgGl and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the GBS and/or GAS and/or 5 pneumoniae antigens m the compositions of the invention after administration of the composition Typically, GBS and/or GAS and/or S pneumoniae serum specific antibody responses are determined post-immunization but pre-challenge whereas mucosal GBS and/or GAS and/or S pneumoniae specific antibody body responses are determined post- lmmumzation and post-challenge

The vaccine compositions of the present invention can be evaluated in in vitro and in vivo animal models prior to host, e g , human, administration

The efficacy of immunogenic compositions of the invention can also be determined in vivo by challenging animal models of GBS and/or GAS and/or S pneumoniae infection, e g , guinea pigs or mice, with the immunogenic compositions The immunogenic compositions may or may not be derived from the same serotypes as the challenge serotypes Preferably the immunnogenic compositions are derivable from the same serotypes as the challenge serotypes More preferably, the immunogenic composition and/or the challenge serotypes are derivable from the group of GBS and/or GAS and/or S pneumoniae serotypes

In vivo efficacy models include but are not limited to (i) A murine infection model using human GBS and/or GAS and/or S pneumoniae serotypes, (n) a murine disease model which is a murine model using a mouse-adapted GBS and/or GAS and/or S pneumoniae strain, such as those strains outlined above which is particularly virulent in mice and (HI) a primate model using human GBS or GAS or S pneumoniae isolates

The immune response may be one or both of a THl immune response and a TH2 response

The immune response may be an improved or an enhanced or an altered immune response

The immune response may be one or both of a systemic and a mucosal immune response

Preferably the immune response is an enhanced system and/or mucosal response

An enhanced systemic and/or mucosal immunity is reflected in an enhanced THl and/or TH2 immune response Preferably, the enhanced immune response includes an increase in the production of IgGl and/or IgG2a and/or IgA

Preferably the mucosal immune response is a TH2 immune response Preferably, the mucosal immune response includes an increase in the production of IgA

Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-IO A TH2 immune response may result in the production of IgGl, IgE, IgA and memory B cells for future protection A TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-IO), or an increase in the production of IgGl, IgE, IgA and memory B cells. Preferably, the enhanced TH2 immune resonse will include an increase in IgGl production.

A THl immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with- a THl immune response (such as IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. Preferably, the enhanced THl immune response will include an increase in IgG2a production.

Immunogenic compositions of the invention, in particular, immunogenic composition comprising one or more GAS antigens of the present invention may be used either alone or in combination with other GAS antigens optionally with an immunoregulatory agent capable of eliciting a ThI and/or Th2 response.

Compositions of the invention will generally be administered directly to a patient. Certain routes may be favored for certain compositons, as resulting in the generation of a more effective immune response, preferably a CMI response, or as being less likely to induce side effects, or as being easier for administration. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intradermally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g. see WO 99/27961) or transcutaneous (e.g. see WO 02/074244 and WO 02/064162), intranasal (e.g. see WO03/028760), ocular, aural, pulmonary or other mucosal administration.

The invention may be used to elicit systemic and/or mucosal immunity.

In one particularly preferred embodiment, the immunogenic composition comprises one or more GBS or GAS or S pneumoniae antigen(s) which elicits a neutralising antibody response and one or more GBS or GAS or S pneumoniae antigen(s) which elicit a cell mediated immune response. In this way, the neutralising antibody response prevents or inhibits an initial GBS or GAS or S pneumoniae infection while the cell-mediated immune response capable of eliciting an enhanced ThI cellular response prevents further spreading of the GBS or GAS or S pneumoniae infection. Preferably, the immunogenic composition comprises one or more GBS or GAS or S pneumoniae surface antigens and one or more GBS or GAS or S pneumoniae cytoplasmic antigens. Preferably the immunogenic composition comprises one or more GBS or GAS or S pneumoniae surface antigens or the like and one or other antigens, such as a cytoplasmic antigen capable of eliciting a ThI cellular response.

Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.

The compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, such as antibiotics, as needed. By 'immunologically effective amount, ' it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention, or increases a measurable immune response or prevents or reduces a clinical symptom This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e g non human primate primate, etc ) the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor s assessment of the medical situation, and other relevant factors It is expected that the amount will fall in a relatively broad range that can be determined through routine trials Further Components of the Composition

The composition of the invention will typically, in addition to the components mentioned above, comprise one or more 'pharmaceutically acceptable carriers ' which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric ammo acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes) Such carriers are well known to those of ordinary skill in the art The vaccines may also contain diluents, such as water, saline, glycerol, etc Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present A thorough discussion of pharmaceutically acceptable excipients is available in Gennaro (2000) Remington The Science and Practice of Pharmacy 20th ed , ISBN 0683306472 Adjuvants

Vaccines of the invention may be administered in conjunction with other immunoregulatory agents In particular, compositions will usually include an adjuvant Adjuvants for use with the invention include, but are not limited to, one or more of the following set forth below

A Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminum salts and calcium salts The invention includes mineral salts such as hydroxides (e g oxyhydroxides), phosphates (e g hydroxyphosphates, orthophosphates), sulfates, etc (e g see chapters 8 & 9 of Vaccine Design (1995) eds Powell & Newman ISBN 030644867X Plenum ), or mixtures of different mineral compounds (e g a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e g gel, crystalline, amorphous, etc ), and with adsorption to the salt(s) being preferred The mineral containing compositions may also be formulated as a particle of metal salt (WO 00/23105)

Aluminum salts may be included in vaccines of the invention such that the dose of Al³⁺ is between 02 and 1 0 mg per dose

B Od Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% Squalene, 05% Tween 80, and 05% Span 85, formulated into submicron particles using a microfluidizer) See WO90/14837 See also, Podda, 'The adjuvanted influenza vaccines with novel adjuvants experience with the MF59- adjuvanted vaccine " Vaccine (2001) .19 2673-2680, Frey et al , "Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non elderly adults, ' Vaccine (2003) 214234-4237 MF59 is used as the adjuvant in the FLU AD™ influenza virus tπvalent subunit vaccine

Particularly preferred adjuvants for use in the compositions are submicron oil-in-water emulsions Preferred submicron oil in water emulsions for use herein are squalene/ water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oιl-in-water emulsion containing 4-5% w/v squalene, 025 1 0% w/v Tween 80 ™ (polyoxyelthylenesorbitan monooleate), and/or 025-1 0% Span 85™ (sorbitan trioleate), and, optionally, N acetylmuramyl-L-alanyl-D isogluatminyl L-alanine-2-( l -2 -dipalmitoyl-.stt-glycero-3-huydroxyphosphophoryloxy)- ethylamine (MTP-PE), for example, the submicron oil-in-water emulsion known as MF59" (International Publication No WO 90/14837, US Patent Nos 6,299,884 and 6,451 325, incorporated herein by reference in their entireties, and Ott et al , "MF59 — Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines ' in Vaccine Design The Subunit and Adjuvant Approach (Powell, M F and Newman, M J eds ) Plenum Press, New York, 1995, pp 277-296) MF59 contains 4-5% w/v Squalene (e g 4 3%), 025-05% w/v Tween 80™, and 0 5% w/v Span 85™ and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 11OY microfluidizer (Microfluidics, Newton, MA) For example, MTP-PE may be present in an amount of about 0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100 μg/dose As used herein, the term "MF59-0" refers to the above submicron oil-m-water emulsion lacking MTP-PE, while the term MF59-MTP denotes a formulation that contains MTP-PE For instance, "MF59-100" contains 100 μg MTP-PE per dose, and so on MF69, another submicron oil in water emulsion for use herein, contains 4 3% w/v squalene, 0 25% w/v Tween 80™, and 075% w/v Span 85™ and optionally MTP-PE Yet another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 04% Tween 80™, 5% pluronic-blocked polymer L 121, and thr-MDP, also microfluidized into a submicron emulsion MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 μg MTP-PE per dose

Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No WO 90/14837 and US Patent Nos 6,299,884 and 6,45 1,325, incorporated herein by reference in their entireties

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention

C Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention Saponins are a heterologous group of sterol glycosides and tπterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species Saponin from the bark of the Quillaia saponana Molina tree have been widely studied as adjuvants Saponin can also be commercially obtained from Smilax ornata (sarsapπlla), Gypsophilla paniculata (brides veil), and Saponana officinalis (soap root) Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs

Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-LC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C Preferably, the saponin is QS21 A method of production of QS21 is disclosed in US Patent No 5,057,540 Saponin formulations may also comprise a sterol, such as cholesterol (see WO96/33739)

Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulating Complexs (ISCOMs) ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine Any known saponin can be used in ISCOMs Preferably, the ISCOM includes one or more of Quil A, QHA and QHC ISCOMs are further described m EP0109942, WO 96/11711 and WO 96/33739 Optionally, the ISCOMS may be devoid of additional detergent See WO 00/07621

A review of the development of saponin based adjuvants can be found at Barr, et al , "ISCOMs and other saponin based adjuvants, ' Advanced Drug Delivery Reviews ( 1998) 32 247-271 See also Sjolander, et al , "Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines, Advanced Drug Delivery Reviews (1998) 32 321- 338 D Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the invention These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome The viral proteins may be recombinantly produced or isolated from whole viruses These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HlV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi) VLPs are discussed further in WO 03/024480, WO 03/024481, and Nπkura et al , "Chimeric Recombinant Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle Presenting Foreign Epitopes, " Virology (2002) 293 273-280, Lenz et al , "Papillomaπvurs-Like Particles Induce Acute Activation of Dendritic Cells, ' Journal of Immunology (2001) 5246-5355, Pinto, et al , "Cellular Immune Responses to Human Papillomavirus (HPV)-16 Ll Healthy Volunteers Immunized with Recombinant HPV-16 Ll Virus-Like Particles, " Journal of Infectious Diseases (2003) 188 327-338, and Gerber et al , "Human Papillomavπsu Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Entertoxin Mutant R192G or CpG, " Journal of Virology (2001) 75(10) 4752-4760 Virosomes are discussed further in, for example, Gluck et al , "New Technology Platforms in the Development of Vaccines for the Future, " Vaccine (2002) 20 BlO -B 16 Immunopotentiating reconstituted influenza virosomes (IRIV) are used as the subunit antigen delivery system in the intranasal trivalent INFLEXAL™ product {Mischler & Metcalfe (2002) Vaccine 20 Suppl 5 B 17-23} and the INFLUVAC PLUS™ product

E Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as

(1) Non-toxic derivatives of enterobacterial lipopob/ saccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL) 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains A preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454 Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0 22 micron membrane (see EP 0 689 454) Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e g RC-529 See Johnson et al. (1999) Bworg Med Chem Lett 9 2273-2278

(2) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174 OM-174 is described for example in Meraldi et al , "OM-174, a New Adjuvant with a Potential for Human Use, Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242-310 from the circumsporozoite protein of Plasmodium berghei, " Vaccine (2003) 2J_. 2485-2491, and Pajak, et al , 'The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo, " Vaccine (2003) 21 836-842

(3) lmmuno stimulatory oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosme and linked by a phosphate bond) Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded Optionally, the guanosme may be replaced with an analog such as 2'-deoxy-7- deazaguanosine See Kandimalla, et al , "Divergent synthetic nucleotide motif recognition pattern design and development of potent immunomodulatory oligodeoxyπbonucleotide agents with distinct cytokine induction profiles, ' Nucleic Acids Research (2003) 31(9) 2393 2400, WO02/26757 and WO99/62923 for examples of possible analog substitutions The adjuvant effect of CpG oligonucleotides is further discussed in Kπeg, 'CpG motifs the active ingredient in bacterial extracts⁹, ' Nature Medicine (2003) 9(7) 831-835, McCluskie, et al , "Parenteral and mucosal prime boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA, " FEMS Immunology and Medical Microbiology (2002) 32 179-185, WO98/40100, US Patent No 6,207,646, US Patent No 6,239,116 and US Patent No 6,429,199

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT See Kandimalla, et al , 'Toll-like receptor 9 modulation of recognition and cytokine induction by novel synthetic CpG DNAs, ' Biochemical Society Transactions (2003) 31 (part 3) 654-658 The CpG sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN CpG-A and CpG-B ODNs are discussed in Blackwell, et al , "CpG-A-lnduced Monocyte IFN-gamma-Inducible Protein- 10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha, " J Immunol (2003) 170(81 4061-4068. Kπeg, "From A to Z on CpG, ' TRENDS in Immunology (2002) 23(2) 64-65 and WO01/95935 Preferably, the CpG is a CpG-A ODN

Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers" See, for example, Kandimalla, et al , "Secondary structures in CpG oligonucleotides affect immunostimulatory activity, " BBRC (2003) 306 948-953, Kandimalla, et al , 'Toll-like receptor 9 modulation of recognition and cytokine induction by novel synthetic GpG DNAs, ' Biochemical Society Transactions (2003) 31(part 3) 664-658, Bhagat et al , "CpG penta and hexadeoxyπbonucleotides as potent immunomodulatory agents" BBRC (2003) 300 853-861 and WO 03/035836

(4) ADP-rώosylating toxins and detoxified derivatives thereof

Bacterial ADP-πbosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention Preferably, the protein is derived from E coli (ι e , E coli heat labile enterotoxm "LT), cholera ("CT"), or pertussis ("PT") The use of detoxified ADP-πbosylating toxins as mucosal adjuvants is described m WO95/17211 and as parenteral adjuvants in WO98/42375 Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G The use of ADP-πbosylating toxins and detoxified deπvaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety Beignon, et al , 'The LTR72 Mutant of Heat-Labile Enterotoxm of Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin, " Infection and Immunity (2002) 70(6) 3012 3019, Pizza, et al , "Mucosal vaccines non toxic derivatives of LT and CT as mucosal adjuvants, ' Vaccine (2001) 19 2534-2541, Pizza, et al , "LTK63 and LTR72, two mucosal adjuvants ready for clinical trials" Int J Med Microbiol (2000) 290(4-5) 455-461, Scharton-Kersten et al , 'Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits and Unrelated Adjuvants, " Infection and Immunity (2000) 68(9) 5306- 5313, Ryan et al , "Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine Differential Effects of the Nontoxic AB Complex and Enzyme Activity on ThI and Th2 Cells" Infection and Immunity (1999) 67(12) 6270-6280, Partidos et al , "Heat labile enterotoxm of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides, " Immunol Lett (1999) 67(3) 209-216, Peppoloni et al , "Mutants of the Escherichia coli heat-labile enterotoxm as safe and strong adjuvants for intranasal delivery of vaccines Vaccines (2003) 2(2) 285-293, and Pine et al , (2002) "Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxm from Escherichia coli (LTK63)" J Control Release (2002) 85(1 3) 263 270 Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP πbosylating toxins set forth in Domenighini et al , MoI Microbiol (1995) 15(6) 1 165 1 167, specifically incorporated herein by reference in its entirety

F Bιoadhesι\ e_'< and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in the invention Suitable bioadhesives include esteπfied hyaluronic acid microspheres (Singh et al (2001) J Cont ReIe 70267-276) or mucoadhesives such as cross- linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrolhdone, polysaccharides and carboxymethylcellulose Chitosan and derivatives thereof may also be used as adjuvants in the invention E g WO99/27960

G Microparticles

Microparticles may also be used as adjuvants in the invention Microparticles (i e a particle of -lOOnm to ~150μm m diameter, more preferably ~200nm to ~30μm in diameter, and most preferably ~500nm to ~10μm in diameter) formed from materials that are biodegradable and non-toxic (« j a poly(α-hydroxy acid), a polyhydroxybutyπc acid, a polyorthoester, a polyanhydπde, a polycaprolactone, etc ), with poly(lactide-co-glycohde) are preferred, optionally treated to have a negatively charged surface (e g with SDS) or a positively charged surface (e g with a cationic detergent, such as CTAB)

H Liposomes

Examples of liposome formulations suitable for use as adjuvants are described in US Patent No 6,090,406, US Patent No 5,916,588, and EP 0 626 169

/ Polyoxyethylene ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters WO99/52549 Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152)

Preferred polyoxyethylene ethers are selected from the following group polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene- 35-lauryl ether, and polyoxyethylene-23-lauryl ether

J Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andπanov et al , "Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions, ' Biomateπals (1998) 12(1-3) 109-115 and Payne et al , "Protein Release from Polyphosphazene Matrices, ' Adv Drug Delivery Review (1998) 11(3) 185-196

K Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-1- aIanyl-d-isoglutaminyl-l-alanine-2-( l'-2'-dipal mi toyl-sn-glycero-3-hydroxyphosphoryloxy)-ethyIamine MTP-PE)

L. Imidazoquinolone Compounds

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues, described further in Stanley, "Imiquimod and the lmidazoquinolones mechanism of action and therapeutic potential" Clin Exp Dermatol (2002) 27(7) 571-577 and Jones, "Resiquimod 3M " Curr Opin Investig Drugs (2003) 4(2) 214-218

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above For example, the following adjuvant compositions may be used in the invention ( 1) a saponin and an oil-in-water emulsion (WO 99/11241)

(2) a saponin (e g , QS21) + a non-toxic LPS derivative (e g 3dMPL) (see WO 94/00153),

(3) a saponin (e g , QS21) + a non-toxic LPS derivative (e g 3dMPL) + a cholesterol,

(4) a saponin (e g QS21) + 3dMPL + IL-12 (optionally + a sterol) (WO 98/57659),

(5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231),

(6) SAF, containing 10% Squalane, 04% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion

(7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 02% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™),

(8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dPML)

(9) one or more mineral salts (such as an aluminum salt) + an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif) Combination No (9) is a preferred adjuvant combination

M Human lmmunomodulators

Human lmmunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e g EL-I, IL-2, EL-4, IL-5, EL-6, IL-7, IL-12, etc ), interferons (e g interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor

Aluminum salts and MF59 are preferred adjuvants for use with injectable influenza vaccines Bacterial toxins and bioadhesives are preferred adjuvants for use with mucosally-dehvered vaccines, such as nasal vaccines

The immunogenic compositions of the present invention may be administed in combination with an antibiotic treatment regime In one embodiment, the antibiotic is administered prior to administration of the antigen of the invention or the composition comprising the one or more of the antigens of the invention

In another embodiment, the antibiotic is administered subsequent to the adminstration of the one or more antigens of the invention or the composition comprising the one or more antigens of the invention Examples of antibiotics suitable for use in the treatment of the Steptococcal infections of the invention include but are not limited to penicillin or a derivative thereof or clindamycin or the like Further antigens

The compositions of the invention may further comprise one or more additional Gram positive bacterial antigens which are not associated with an AI Preferably, the Gram positive bacterial antigens that are not associated with an AI can provide protection across more than one serotype or strain isolate For example, a first non-AI antigen, in which the first non-AI antigen is at least 90% (ι e , at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) homologous to the ammo acid sequence of a second non-AI antigen, wherein the first and the second non-AI antigen are derived from the genomes of different serotypes of a Gram positive bacteria, may be further included in the compositions The first non-AI antigen may also be homologous to the amino acid sequence of a third non-AI antigen, such that the first non AI antigen, the second non-AI antigen, and the third non-AI antigen are derived from the genomes of different serotypes of a Gram positive bacteria The first non-AI antigen may also be homologous to the amino acid sequence of a fourth non-AI antigen, such that the first non-AI antigen, the second non-AI antigen, the third non-AI antigen, and the fourth non AI antigen are derived from the genomes of different serotypes of a Gram positive bacteria

The first non-AI antigen may be GBS 322 The amino acid sequence of GBS 322 across GBS strains from serotypes Ia, Ib, II, III, V, and VIII is greater than 90% Alternatively, the first non-AI antigen may be GBS 276 The amino acid sequence of GBS 276 across GBS strain from serotypes Ia, Ib, II, III, V, and VIII is greater than 90% Table 13 provides the percent amino acid sequence identity of GBS 322 and GBS 276 across different GBS strains and serotypes. Table 13: Conservation of GBS 322 and GBS 276 amino acid sequences

As an example, inclusion of a non-AI protein, GBS 322, in combination with AI antigens GBS 67, GBS 80, and GBS 104 provided protection to newborn mice in an active maternal immunization assay.

Table 14: Active maternal immunization assay for a combination of fragments from GBS 322, GBS 80, GBS 104, and GBS 67

In fact, the non-AI GBS 322 antigen may itself provide protection to newborn mice in an active maternal immunization assay. Table 16: Active maternal immunization assay for each of GBS 80 and GBS 322 antigens

Thus, inclusion of a non-AI protein in an immunogenic composition of the invention may provide increased protection a mammal.

The immunogenic compositions comprising S. pneumoniae AI polypeptides may further secondary SP protein antigens which include (a) any of the SP protein antigens disclosed in WO 02/077021 or U.S. provisional application , filed April 20, 2005 (Attorney Docket Number 002441.00154), (2) immunogenic portions of the antigens comprising at least 7 contiguous amino acids, (3) proteins comprising amino acid sequences which retain immunogenicity and which are at least 95% identical to these SP protein antigens (e.g., 95%, 96%, 97%, 98%, 99%, or 99.5% identical), and (4) fusion proteins, including hybrid SP protein antigens, comprising (l)-(3).

Alternatively, the invention may include an immunogenic composition comprising a first and a second Gram positive bacteria non-AI protein, wherein the polynucleotide sequence encoding the sequence of the first non-AI protein is less than 90% (i.e., less than 90, 88, 86, 84, 82, 81, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35, or 30 percent) homologous than the corresponding sequence in the genome of the second non-AI protein.

The compositions of the invention may further comprise one or more additional non-Gram positive bacterial antigens, including additional bacterial, viral or parasitic antigens. The compositions of the invention may further comprise one or more additional non-GBS antigens, including additional bacterial, viral or parasitic antigens.

In another embodiment, the GBS antigen combinations of the invention are combined with one or more additional, non-GBS antigens suitable for use in a vaccine designed to protect elderly or iπununocomprised individuals. For example, the GBS antigen combinations may be combined with an antigen derived from the group consisting of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitides, influenza, and Parainfluenza virus ('PIV).

Where a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier protein in order to enhance immunogenicity { e.g. Ramsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond 34: 163-168; Ahmad & Chapnick (1999) Infect Dis Clin North Am 13: 113-133, vii.; Goldblatt (1998) J. Med. Microbiol 47:563-567; European patent 0 477 508; US Patent No. 5,306,492; International patent application WO98/42721; Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114; and Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X}. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRMi₉₇ diphtheria toxoid is particularly preferred {Research Disclosure, 453077 (Jan 2002)}. Other carrier polypeptides include the N. meningitidis outer membrane protein (EP-A-0372501), synthetic peptides (EP-A-0378881; EP-A-0427347), heat shock proteins (WO 93/17712; WO 94/03208), pertussis proteins (WO 98/58668; EP A 0471 177), protein D from H. influenzae (WO 00/56360), cytokines (WO 91/01 146), lymphokines, hormones, growth factors, toxin A or B from C difficile (WO00/61761), iron uptake proteins (WO01/72337), etc Where a mixture comprises capsular saccharides from both serogroups A and C, it may be preferred that the ratio (w/w) of MenA saccharide MenC saccharide is greater than 1 (e g 2 1, 3 1, 4 1, 5 1, 10 1 or higher) Different saccharides can be conjugated to the same or different type of carrier protein Any suitable conjugation reaction can be used, with any suitable linker where necessary

Toxic protein antigens may be detoxified where necessary e g detoxification of pertussis toxin by chemical and/or genetic means

Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens

Antigens in the composition will typically be present at a concentration of at least lμg/ml each In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen

As an alternative to using protein antigens in the composition of the invention, nucleic acid encoding the antigen may be used {e g refs Robinson & Torres (1997) Seminars in Immunology 9 271-283, Donnelly et al (1997) Annu Rev Immunol 15 617-648, Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9 471-480, Apostolopoulos & Plebanski (2000) Curr Opin MoI Ther 2 441-447, Ilan (1999) Curr Opin MoI Ther 1 116-120, Dubensky et al (2000) MoI Med 6 723-732, Robinson & Pertmer (2000) Adv Virus Res 55 1-74, Donnelly et al (2000) Am J Respir Crit Care Med 162(4 Pt 2) S190-193, and Davis (1999) Mt Sinai J Med 66 84-90} Protein components of the compositions of the invention may thus be replaced by nucleic acid (preferably DNA e g in the form of a plasmid) that encodes the protein Definitions

The term "comprising" means "including" as well as "consisting" e g a composition "comprising" X may consist exclusively of X or may include something additional e g X + Y

The term "about" in relation to a numerical value x means, for example, Λ+10%

References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7 7 18 of Current Protocols in Molecular Biology (F M Ausubel et al , eds , 1987) Supplement 30 A preferred alignment is determined by the Smith Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62 The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv Appl Math 2 482-489

The invention is further illustrated, without limitation, by the following examples EXAMPLE 1: Binding of an Adhesin Island surface protein, GBS 80, to Fibrinogen and Fibronectin.

This example demonstrates that an Adhesin Island surface protein, GBS 80 can bind to fibrinogen and fibronectin

An enzyme-linked immunosorbent assay (ELISA) was used to analyse the in vitro binding ability of recombinant GBS 80 to immobilized extra-cellular matrix (ECM) proteins but not to bovine serum albumin (BSA) Microtiter plates were coated with ECM proteins (fibrinogen, fibronectin, laminin, collagen type IV) and binding assessed by adding varying concentrations of a recombinant form of GBS 80, over-expressed and purified from E coli (FIG 5A) Plates were then incubated sequentially with a) mouse anti GBS 80 primary antibody, b) rabbit anti-mouse AP-conjugated secondary antibody, c) pNPP coloπmetπc substrate Relative binding was measured by monitoring absorbance at 405 nm, using 595 nm as a reference wavelength FIG 5b shows binding of recombinant GBS 80 to immobilized ECM proteins ( 1 μg) as a function of concentration of GBS 80 BSA was used as a negative control Data points represent the means of OD₄₀₅ values ± standard deviation for 3 wells

Binding of GBS 80 to the tested ECM proteins was found to be concentration dependent and exhibited saturation kinetics As is also evident from FIG 5, binding of GBS 80 to fibronectin and fibrinogen was greater than binding to laminin and collagen type IV at all the concentrations tested EXAMPLE 2: GBS 80 is required for surface localization of GBS 104.

This example demonstrates that co-expression of GBS 80 is required for surface localization of GBS 104

The polycistronic nature of the Adhesin Island I mRNA was investigated through reverse transcπptase-PCR (RT-PCR) analysis employing primers designed to detect transcripts arising from contiguous genes Total RNA was isolated from GBS cultures grown to an optical density at 600 nm (OD₆₀₀) of 0 3 in THB (Todd-Hewitt broth) by the RNeasy Total RNA isolation method (Qiagen) according to the manufacturer's instructions The absence of contaminating chromosomal DNA was confirmed by failure of the gene amplification reactions to generate a product detectable by agarose gel electrophoresis, in the absence of reverse transcriptase RT-PCR analysis was performed with the Access RT-PCR system (Promega) according to the manufacturer's instructions, employing PCR cycling temperatures of 6O⁰C for annealing and 7O⁰C for extension Amplification products were visualized alongside 100-bp DNA markers in 2% agarose gels after ethidium bromide staining

FIG 5 shows that all the genes are co-transcribed as an operon A schematic of the AI-I operon is shown above the agarose gel analysis of the RT-PCR products Large rectangular arrows indicate the predicted transcript direction Primer pairs were selected such as "1-4" cross the 3'finish-5'start of successive genes and overlap each gene by at least 200 bp Additionally, "1" crosses a putative rho-independent transcriptional terminator "5" is an internal GBS 80 control and "6" is an unrelated control from a highly expressed gene Lanes "a" RNA plus RTase enzyme, "b" RNA without RTase, "c" genomic DNA control

In the effort to elucidate the functions of the AI-I proteins, in frame deletions of all of the genes within the operon have been constructed and the resulting mutants characterized with respect to surface exposure of the encoded antigens (see FIG 8)

Each in-frame deletion mutation was constructed by splice overlap extension PCR (SOE-PCR) essentially as decπbed by Horton et al [Horton R M , Z L Cai, S N Ho, L R Pease (1990) Biotechniques 8 528-35] using suitable primers and cloned into the temperature sensitive shuttle vector pJRS233 to replace the wild type copy by allelic exchange [Perez-Casal, J , J A Price, et al (1993) MoI Microbiol 8(5) 809-19 ] All plasmid constructions utilized standard molecular biology techniques, and the identities of DNA fragments generated by PCR were verified by sequencing Following SOE-PCR, the resulting mutant DNA fragments were digested with Xhol and EcoRI, and ligated into a similarly digested pJRS233 The resutmg vectors were introduced by electroporation into the chromosome of 2603 and COHl GBS strains in a three-step process, essentially as described in Framson et al [Framson, P E , A Nittayajarn, J Merry, P Youngman, and C E Rubens (1997) Appl Environ Microbiol 63(9) 3539-47] Briefly, the vector pJRS233 contains an erm gene encoding erythromycin resistance and a temperature sensitive gram-positive replicon that is active at 3O⁰C but not at 37°C Initially, the constructs are electroporated into GBS electro-competent cells prepared as described by Frameson et al , and transformants containing free plasmid are selected by their ability to grow at 30°C on Todd-Hewitt Broth (THB) agar plates containing 1 μg/ml erythromycin The second step includes a selection step for strains in which the plasmid has integrated into the chromosome via a single recombination event over the homologous plasmid insert and chromosome sequence by their ability to grow at 37°C on THB agar medium containing 1 mg/ml erythromycin In the third step, GBS cells containing the plasmid integrated within the chromosome (integrants) are serially passed in broth culture in the absence of antibiotics at 30⁰C Plasmid excision from the chromosome via a second recombination event over the duplicated target gene sequence either completed the allelic exchange or reconstituted the wild-type genotype Subsequent loss of the plasmid in the absence of antibiotic selection pressure resulted in an erythromycin-sensitive phenotype In order to assess gene replacement a screening of erythromycin-sensitive colonies was performed by analysis of the target gene PCR amplicons

FIG 7 reports a schematic of the IS-I operon for each knock-out strain generated, along with the deletion position within the amino acidic sequence Most data presented here concern the COHl deletion strains, in which the expression of each of the antigens is higher by DNA microarray analysis as well as detectable by FACS analysis (see FIG 8) The double mutant in 2603 Δ80, Δ104 double mutant was constructed by sequential allelic exchanges of the shown alleles

Immunization protocol

Immune sera for FACS experiments were obtained as follows

Groups of 4 CD 1 outbred female mice 6-7 weeks old (Charles River Laboratories, Calco Italy) were immunized with the selected GBS antigens, (20 μg of each recombinant GBS antigen), suspended in 100 μl of PBS Each group received 3 doses at days 0, 21 and 35 Immunization was performed through intra-peritoneal injection of the protein with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses In each immunization scheme negative and positive control groups are used Immune response was monitored by using serum samples taken on day 0 and 49

FACS analysis

Preparation of paraformaldehyde treated GBS cells and their FACS analysis were carried out as follows

GBS serotype COHl strain cells were grown in Todd Hewitt Broth (THB, Difco Laboratories, Detroit, Mich ) to ODβOOnm = 05 The culture was centπfuged for 20 minutes at 5000 rpm and bacteria were washed once with PBS, resuspended in PBS containing 0 05% paraformaldehyde, and incubated for 1 hours at 37 ⁰C and then overnight at 4°C 50μl of fixed bacteria (OD600 0 1) were washed once with PBS, resuspended in 20μl of Newborn Calf Serum, (Sigma) and incubated for 20 min at room temperature The cells were then incubated for 1 hour at 4°C in lOOμl of preimmune or immune sera, diluted 1 200 in dilution buffer (PBS, 20% Newborn Calf Serum, 0 1% BSA) After centrifugation and washing with 200μl of washing buffer (0 1% BSA in PBS), samples were incubated for 1 hour at 4°C with 50μl of R- Phicoerytπn conjugated F(ab)2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc ), diluted 1 100 in dilution buffer Cells were washed with 200μl of washing buffer and resuspended in 200μl of PBS Samples were analysed using a FACS Calibur apparatus (Becton Dickinson, Mountain View, Calf) and data were analyzed using the Cell Quest Software (Becton Dickinson) A shift in mean fluorescence intensity of > 75 channels compared to preimmune sera from the same mice was considered positive This cutoff was determined from the mean plus two standard deviations of shifts obtained with control sera raised against mock purified recombinant proteins from cultures of £ colt carrying the empty expression vector and included in every experiment Artifacts due to bacterial lysis were excluded using antisera raised against 6 different known cytoplasmic proteins all of which were negative

FACS data on COHl single KO mutants for GBS 104 and GBS 80 indicated that GBS 80 is required for surface localization of GBS 104

As shown in FIG 8, GBS 104 is not surface exposed in the Δ80 strain (second column, bottom), but is present in the whole protein extracts (see FlG 10) Mean shift values suggest that GBS 104 is partially responsible for GBS 80 surface exposure (Mean shift of GBS 80 is reduced to -60% wild-type levels in Δ104), and that GBS 80 is over-expressed in the complemented strain (mean shift value -200% wild-type level) The Δ80/pGBS 80 strain contains the GBS 80 orf cloned in the shuttle-vector pAM401 (Wirth, R , F Y An, et al (1986) J Bacteπol 165(3) 831-6) The vector alone does not alter the secretion pattern of GBS 104 (right column) FACS was performed on mid-log fixed bacteria with mouse polyclonal antibodies as indicated at left Black peak is pre-immune sera, colored peaks are sera from immunized animals

EXAMPLE 3: Deletion of GBS 80 causes attenuation in vivo.

This example demonstrates that deletion of GBS 80 causes attenuation in vivo, suggesting that this protein contributes to bacterial virulence

By using a mouse animal model, we studied the role of GBS 80 and GBS 104 in the virulence of S agalactiae Groups of ten outbred female mice 5-6 week weeks old (Charles River Laboratories, Calco Italy) were inoculated intrapeπtoneally with different dilutions of the mutant strains and LD50 (lethal dose 50) were calculated according to the method of Reed and Muench [Reed, L J and H Muench (1938) The American Journal of Hygiene 27(3) 493-7] As presented in Table 3 below the number of colony forming units (cfu) counted for both the Δ80 and the Δ80, Δ104 double mutants is about 10 fold higher when compared to the wild type strain suggesting that inactivation of GBS 80 but not GBS 104 is responsible for an attenuation in virulence This finding indicates that GBS 80 gene in the AI-I might contribute to virulence

Table 3 Lethal dose 50% analysis of AI-I mutants m the 2603 strain background LD50s were performed by IP injection of female CDI mice at an age of 5-6 weeks LD50s were calculated by the method of Reed and Muench (8)

EXAMPLE 4: Effect of Adhesin Island Sortase Deletions on Surface Antigen Presentation

This example demonstrates the effect of adhesin island sortase deletions on surface antigen presentation

FACS analysis results set forth in FIG 9 show that a deletion in sortase SAG0648 prevented GBS 104 from reoching the surface and slightly reduced the surface exposure of GBS 80 (fourth panel, mean shift value -60% wild-type COHl) In the double sortase knock-out strain, neither antigen was surface exposed (far right panel) Either sortase alone was sufficient for GBS 80 to arrive at the bacterial surface (third and fourth columns, top) No effect was seen on surface exposure of antigens GBS 80 or GBS 104 in the ΔGBS 52 strain Antibodies derived from purified GBS 52 were either non-specific or were FACS negative for GBS 52 FACS analysis was performed as described above (see EXAMPLE 2)

As shown in FIG 10, inactivation of GBS 80 has no effect on GBS 104 expression as much as GBS 104 knock out doesn't change the total amount GBS 80 expressed The Western blot of whole protein extracts (strains noted above lanes) probed with anti-GBS 80 antisera is shown in panel A Arrow indicates expected size of GBS 80 (60 kDa) GBS 80 antibodies recognize a doublet, the lower band is not present in ΔGBS 80 strains Panel B shows a Western blot of whole protein extracts probed with anti-GBS 104 antisera Arrow indicates expected size of GBS 104 (99 4 kDa) Protein extracts were prepared from the same bacterial cultures used for FACS (FIGS 8 and 9) In conclusion, although GBS 104 does not arrive at the surface in the Δ80 strain by FACS (FIG 8, second column), it is present at approximately wild- type levels in the whole protein preps (B, second lane) Approximately 20 μg of each protein extract was loaded per lane

Western blot analysis

Ahquots of total protein extract mixed with SDS loading buffer (Ix 60 mM TRIS-HCl pH 6 8, 5% w/v SDS, 10% v/v glycerin, 0 1% Bromophenol Blue, 100 mM DTT) and boiled 5 minutes at 95° C, were loaded on a 12 5% SDS- PAGE precast gel (Biorad) The gel is run using a SDS-PAGE running buffer containing 250 mM TRIS, 2 5 mM Glycine and 0 1 %SDS The gel is electroblotted onto nitrocellulose membrane at 200 mA for 60 minutes The membrane is blocked for 60 minutes with PBS/005 % Tween-20 (Sigma), 10% skimmed milk powder and incubated O/N at 4° C with PBS/0 05 % Tween 20, 1% skimmed milk powder, with the appropriate dilution ot the sera After washing twice with PBS/0 05 % Tween, the membrane is incubated for 2 hours with peroxidase-conjugated secondary anti-mouse antibody (Amersham) diluted 1 4000 The nitrocellulose is washed three times for 10 minutes with PBS/005 % Tween and once with PBS and thereafter developed by Opti-4CN Substrate Kit (Biorad)

Example 5: Binding of Adhesiπ Island proteins to epithelial cells and effect of Adhesin Island proteins on capacity of GBS to adhere to epithelial cells.

This example illustrates the binding of AI proteins to epithelial cells and the effect of AI proteins on the capacity of GBS to adhere to epithelial cells

Applicants analysed whether recombinant AI surface proteins GBS 80 or GBS 104 would demonstrate binding to various epithelial cells in a FACS analysis Applicants also analysed whether deletion of AI surface proteins GBS 80 or GBS 104 would effect the capacity of GBS to adhere to and invade ME180 cervical epithelial cells

As shown in FIG 28, deletion of GBS 80 sequence from GBS strain isolate 2603 (serotype V) did not affect the capacity of the mutated GBS to adhere to and invade ME180 cervical epithelial cells Here ME180 cervical carcinoma epithelial cells were infected with wild type GBS 2603 or GBS 2603 Δ80 isogenic mutant After two hours of infection, non-adherent bacteria were washed off and infection prolonged for a further two hours and four hours In invasion experiments, after each time point, was followed by a two hour antibiotic treatment Cells were then lysed with 1% saponin and lysates platedon TSA plates As shown in FIG 28, there was little difference between the percent invasion or percent adhesion of wild type and mutant strains up to the four hour time point

FIG 30 repeats this experiment with both Δ104 and Δ 80 mutants from a different strain isolate Here, ME 180 cervical carcinoma epithelial cells were infected with GBS strain isolate COH (serotype III) wild type or COHl ΔGBS 104 or COHl Δ80 isogenic mutant After one hour of infection, non-adherent bacteria were washed off and the cells were lysed with 1% saponin The lysates were plated on TSA plates As shown in HG 30, while there was little difference in the percent invasion, there was a significant decrease in the percent association of the Δ104 mutant compared to both the wild type and Δ80 mutant

The affect of AI surface proteins on the ability of GBS to translocate through an epithelial monolayer was also analysed As shown in FIG 31, a GBS 80 knockout mutant strain partially loses the ability to translocate through an epithelial monolayer Here epithelial monolayers were inoculated with wildtype or knockout mutant in the apical chamber of a transwell system for two hours and then non-adherent bacteria were washed off Infection was prolonged for a further two and four hours Samples were taken from the media of the basolateral side and the number of colony forming unties measured Transepithelial electrical resistance measured prior to and after infection gave comparable values, indicating the maintenance of the integrity of the monolayer By the six hour time point, the Δ80 mutants demonstrated a reduced percent transcytosis

A similar experiment was conducted with GBS 104 knock out mutants Here, as shown in FIG 22, the Δ104 mutants also demonstrated a reduced percent transcytosis, indicating that the mutant strains translocate through an epithelial monolayer less efficiently than their isogenic wild type counterparts

Applicants also studied the effect of AI proteins on the capacity of a GBS strain to invade J774 macrophage-like cells Here, J774 cells were infected with GBS COHl wild type or COHl ΔGBS104 or COHl ΔGBS80 isogenic mutants After one hour of infection, non adherent bacteria were washed off and intracellular bacteria were recovered at two, four and six hours post antibiotic treatment At each time point, cells were lysed with 025% Triton X-100 and lysates plated on TSA plates As shown in FIG 32, the Δ104 mutant demonstrated a significantly reduced percent invasion compared to both the wild type and Δ80 mutant Example 6: Hyperoligomeric structures comprising AI surface proteins GBS 80 and GBS 104.

This example illustrates hyperoligomeric structures comprising AI surface proteins GBS 80 and GBS 104 A GBS isolate COHl (serotype III) was adapted to increase expression of GBS 80 FIG. 34 presents a regular negative stain electron micrograph of this mutant; no pilus or hyperoligomeric structures are distinguishable on the surface of the bacteria. When the EM stain is based on anti-GBS 80 antibodies labelled with 10 or 20 nm gold particles, the presence of GBS 80 throughout the hyperoligomeric structure is clearly indicated (FIGS. 36, 37 and 38). EM staining against GBS 104 (anti-GBS 104 antibodies labelled with 10 nm gold particles) also reveals the presence of GBS 104 primarily on or near the surface of the bacteria or potentially associated with bacterial peptidoglycans (FIG. 39). Analysis of this same strain (over-expressing GBS 80) with a combination of both anti-GBS 80 (using 20 nm gold particles) and anti-GBS 104 (using 10 nm gold particles) reveals the presence of GBS 104 on the surface and within the hyperoligomeric structures (see FIGS. 40 and 41).

Example 7: GBS 80 is necessary for polymer formation and GBS 104 and sortase SAG0648 are necessary for efficient pili assembly

This example demonstrates that GBS 80 is necessary for formation of polymers and that GBS 104 and sortase SAG0648 are necessary for efficient pili assembly. GBS 80 and GBS 104 polymeric assembly was systematically analyzed in Cohl strain single knock out mutants of each of the relevant coding genes in AI-I (GBS 80, GBS 104, GBS 52, sagO647, and sagO648). FIG. 41 provides Western blots of total protein extracts (strains noted above lanes) probed with either anti-GBS 80 (left panel) sera or anti-GBS 104 sera (right panel) for each of these Cohl and Cohl knock out strains. (Cohl, wild type Cohl; Δ80, Cohl with GBS 80 knocked out; Δ104, Cohl with GBS 104 knocked out; Δ52, Cohl with GBS 52 knocked out; Δ647, Cohl with SAG0647 knocked out; Δ648, Cohl with SAG0648 knocked out, Δ647-8, Cohl with SAGO647 and SAG0648 knocked out; Δ8O/pGBS8O, Cohl with GBS 80 knocked out but complemented with a high copy number plasmid expressing GBS 80. Asterisks identify the monomer of GBS 80 and GBS 104 )

The smear of immunoreactive material observed in the wild type strain, along with its disappearance in Δ80 and Δ104 mutants, is consistent with the notion that such high molecular weight structures are composed of covalently linked (SDS-resistant) GBS 80 and GBS 104 subunits. The immunoblotting with both anti-GBS 80 (α-GBS 80) and anti-GBS 104 (α-GBS 104) revealed that deletion of sortase SAG0648 also interferes with the assembly of high molecular weight species, whereas the knock out mutant of the second sortase (SAG0647), even if somehow reduced, still maintains the ability to form polymeric structures.

Total extracts form GBS were prepared as follows. Bacteria were grown in 50 ml of Todd-Hewitt broth (Difco) to an OD_δoo_πm ⁰^ 0.5-0.6 and successively pelleted. After two washes in PBS the pellet was resuspended and incubated 3 hours at 37°C with mutanolisin. Cells were then lysed with at least three freezing-thawing cycles in dry ice and a 37°C bath. The lysate was then centrifuged to eliminate the cellular debris and the supernatant was quantified. Approximately 40 μg of each protein extract was separated on SDS-PAGE. The gel was then subjected to immunoblotting with mice antisera and detected with chemiluminescence.

Example 8: GBS 80 is polymerized by an AI-2 sortase

This example illustrates that GBS 80 can be polymerized not only by AI-I sortases, but also by AI-2 sortases FIG. 42 shows total cell extract immunoblots of GBS 515 strain, which lacks AI-I . The left panel, where an anti-GBS 67 sera was used, shows that GBS 67 from AI-2 is assembled into high-molecular weight-complexes, suggesting the formation of a second type of pilus The same high molecular structure is observed when GBS 80 is highly expressed by reintroducing the gene within a plasmid (pGBS 80) By using anti-GBS 80 (right panel) sera on the same extracts, again it is observed that, with GBS 80 over expression (515/pGBS 80), a high-molecular weight structure is assembled This implies that, in the absence of AI-I sortases A] 2 sortases (SAG 1405 and SAG 1406) can complement the lacking function, still being able to assemble GBS 80 in a pilus structure

Example 9: Cohl produces a high molecular weight molecule, the GBS 80 pilin

This example illustrates that Cohl produces a high molecular weight molecule, greater than 1000 IcDa, which is the GBS 80 pilin FIG 43 provides silver-stained electrophoretic gels that show that Cohl produces two macromolecules One of these macromolecules disappears in the Cohl GBS 80 knock out cells, but does not disappear in the Cohl GBS 52 knock out mutant cells The last two lanes on the right were loaded with 15 times the amount loaded in the other lanes This was done in order to be able to count the bands By doing this, a conservative size estimate of the top bands was calculated by starting at 240 kDa and considering each of 14 higher bands as the result of consecutive additions of a GBS 80 monomer

Cohl, wild type Cohl, Δ80, Cohl cells with GBS 80 knocked out, Δ52, Cohl cells with GBS 52 knocked out, Δ80/pGBS 80, Cohl cells with GBS 80 knocked out and complemented with a high copy number construct expressing GBS 80

Example 10. GBS 52 is a minor component of the GBS pilus

This example illustrates that GBS 52 is present in the GBS pilus and is a minor component of the pilus FIG 45 shows an immunoblot of total cell extracts from a GBS Cohl strain and a GBS Cohl strain knocked out for GBS 52 (Δ52) The total cell extracts were immunoblotted anti-GBS 80 antisera (left) and anti-GBS 52 antisera (right) Immunoblotting was performed using a 3-8% Tπs-acetate polyacrylamide gel (Invitrogen) which provided excellent separation of large molecular weight proteins (see FIG 41) When the gel was incubated with anti-GBS 80 sera, the bands from the Cohl wild-type strain appeared shifted when compared to the Δ52 mutant This observation indicated a different size of the pilus polymeric components in the two strains When the same gel was stripped and incubated with anti GBS 52 sera the high-molecular subunits in the Cohl wild-type strain showed similar molecular size of those in the correspondent lane in the left panel These findings confirmed that GBS 52 is indeed associated with GBS 80 macro- molecular structures but represents a minor component of the GBS pilus

Example 11 : Pilus structures are present in the supernatant of GBS bacterial cultures

This example illustrates that the pilus structure assembled in Cohl GBS is present in the supernatant of a bacterial cell culture FIG 46 shows an immunoblot where the protein extract of the supernatant from cultures of different GBS mutant strains (117 = Cohl GBS 80 knockout, 159= Cohl GBS 104 knockout, 202= Cohl GBS 52 knockout, 206= Cohl GBS sagO647 knockout, 208= Cohl GBS sagO648 knockout, 197= Cohl GBS sag0647/sag0648 knockout, 179= Cohl GBS 80 knockout complemented with a high copy plasmid expressing GBS 80) GBS 80 antisera detects the presence of pilus structures in the appropriate Coh 1 strains

The protein extract was prepared as follows Bacteria were grown in THB to an OD^o_nm of 0 5-06 and the supernatant was separated from the cells by centπfugation The supernatant was then filtered (0 02 μm) and 1 ml was added with 60% TCA for protein precipitation

GBS pili were also extracted from the fraction of surface-exposed proteins in Cohl strain and its GBS 80 knock out mutant as described hereafter Bacteria were grown to an OD_60On-, of 0 6 in 50 ml of THB at 37°C Cells were washed once with PBS and the pellet was then resuspended in 0 1 M KPO4 pH 6 2, 40% sucrose, 10 mM MgCI2, 400U/ml miitanolysin and incubated 3 hours at 37°C Protoplasts were separated by centπfugation and the supernatant was recovered and its protein content measured

In order to study the dynamics of pilus production during different growth phases, 1 ml supernatant of a culture at different OD₆₀o_nm was TCA precipitated and loaded onto a 3-8% SDS-PAGE as described before FIG 47 shows the corresponding Western blot with GBS 80 anti-sera The first group of lanes (left five sample lanes) refer to a Cohl strain growth (OD₆oonm ^are noted above the lanes) whereas the second group of lanes (right five samples) are from a GBS 80 knock out strain over expressing GBS 80. The experiment shows that pilus macromolecular structures can be found in the supernatant in all of the growth phases tested

Example 12: In GBS strain Cohl, only GBS 80 and a sortase (sagO647 or sagO648) is required for polymerization

This example describes requirements for pilus formation in Cohl. FIG. 48 shows a Western blot of total protein extracts (prepared as described before) using anti-GBS 80 sera on Cohl clones (Cohl, wild type Cohl; Δ104, Cohl knocked out for GBS 104, Δ647, Cohl knocked out for sagO647, Δ648, Cohl knocked for sagO648, Δ647-8, Cohl knocked out for sagO647 and sagO648; 515, wild type bacterial strain 515, which lacks an AI-I ; p80 a high copy number plasmid which expresses GBS 80.) The data show that only the double sortase mutant is unable to polymerize GBS 80 indicating that the 'conditw sine qua non for pilus polymerization is the co-existence of GBS 80 with at least one sortase This result leads to a reasonable assumption that SAG 1405 and SAG1406 are responsible for polymerization in this strain.

Example 13: GBS 80 can be expressed in L. lactis under its own promoter and terminator sequences

This example demonstrates that L lactis, a non-pathogenic bacterium, can express GBS AI polypeptides such as GBS 80 L lactis M1363 (/. Bacterial. 154 (1983):l-9) was transformed with a construct encoding GBS 80. Briefly, the construct was prepared by cloning a DNA fragment containing the gene coding for GBS 80 under its own promoter and terminator sequences into plasmid pAM401 (a shuttle vector for E. coli and other Gram positive bacteria; J. Bacterial 163 (1986):831-836). Total extracts of the transformed bacteria in log phase were separated on SDS-PAGE, transferred to membranes, and incubated with antiserum against GBS 80. A polypeptide corresponding to the molecular weight of GBS 80 was detected in the lanes containing total extracts of L lactis transformed with the GBS 80 construct. See FIGS 133A and 133B, lanes 6 and 7. This same polypeptide was not detected in the lane containing total extracts of L. lactis not transformed with the GBS 80 construct, lane 9. This example shows that L lactis can express GBS 80 under its own promoter and terminator.

Example 14: L. lactis modified to express GBS AI-I under the GBS 80 promoter and terminator sequences expresses GBS 80 in polymeric structures

This example demonstrates the ability of L lactis to express GBS AI-I polypeptides and to incorporate at least some of the polypeptides into oligomers. L lactis was transformed with a construct containing the genes encoding GBS AI-I polypeptides. Briefly, the construct was prepared by cloning a DNA fragment containing the genes for GBS 80, GBS 52, SAG0647, SAG0648, and GBS 104 under the GBS 80 promoter and terminator sequences into construct pAM401. The construct was transformed into L. lactis M1363. Total extracts of log phase transformed bacteria were separated on reducing SDS-PAGE, transferred to membranes, and incubated with antiserum against GBS 80. A polypeptide with a molecular weight corresponding to the molecular weight of GBS 80 was detected in the lanes containing L. lactis transformed with the GBS AI-I encoding construct. See FIG 134, lane 2. In addition, the same lane also showed immunoreactivity of polypeptides having higher molecular weights than the polypeptide having the molecular weight ot GBS 80 These higher molecular weight polypeptides are likely oligomers of GBS 80 Oligomers of similar molecular weights were also observed on a Western blot of the culture supernatant of the transformed L lactis See lane 4 of FIG 135 Thus, this example shows that L lactis transformed to express GBS Al-I can efficiently polymerize GBS 80 in the form of a pilus This pilus structure can likely be purified from either the cell culture supernatant or cell extracts

Example 15: Cloning and Expression of S pneumoniae SpO462

This example describes the production of a clone encoding a SpO462 polypeptide and expression of the clone To produce a clone encoding SpO462, the open reading frame encoding SpO462 was amplified using primers that annealed within the full-length SpO462 open reading frame sequence FIG 150A provides a 893 amino acid sequence of SpO462 The primers used to produce a clone encoding the SpO462 polypeptide are shown in FIG 150B These primers annealed to the nucleotide sequences encoding the amino acid residues indicated by underlining in FIG 150A Amplification of the open reading frame encoding SpO462 using these primers produced the amplicon shown at lane 2 of the agarose gel provided in FIG 160 The SpO462 clone encodes ammo acid residues 38-862 of the 893 ammo acid residue SpO462 protein, the italicized residues in FIG 150A were eliminated FIG 15 IA provides a schematic depiction of the recombinant SpO462 polypeptide FIG 151B shows a schematic depiction of the full-length SpO462 polypeptide Both the recombinant SpO462 encoded by the clone and the full-length SpO462 protein have two collagen binding protein type B (Cna B) domains and a von Hillebrand factor A (vWA) domain The cloned recombinant SpO462 lacks the LPXTG motif present in the full-length SpO462 protein Western blot analysis for expression of the SpO462 clone did not result in detection of polypeptides with serum obtained from S pne«/nonjαe-infected patients (FIG 152A) or GBS 80 antiserum (FIG 152B)

Example 16: Cloning and Expression of S. pneumoniae SpO463

This example describes the production of a clone encoding a SpO463 polypeptide and detection of recombinant SpO463 polypeptide expressed from the clone To produce a clone encoding SpO463, the open reading frame encoding SpO463 was amplified using primers that annealed within the full-length SpO463 open reading frame sequence FIG 153A provides a 665 amino acid sequence of SpO463 The primers used to produce the clone encoding SpO463 polypeptide are shown in FIG 153B These primers annealed to the nucleotide sequences encoding the ammo acid residues indicated by underlining in FIG 153 A Amplification of the open reading frame encoding SpO463 using these primers produced the amplicon shown at lane 3 of the agarose gel provided in FlG 160 The SpO463 clone encodes amino acid residues 23-627 of the 665 amino acid residue SpO463 protein, the italicized residues in FIG 153A were eliminated FIG 154A provides a schematic depiction of the recombinant SpO463 polypeptide FIG 154B shows a schematic depiction of the full-length SpO463 polypeptide Both the recombinant SpO463 encoded by the clone and the full-length SpO463 protein have a Cna B domain and an E box motif The cloned recombinant SpO463 lacks the LPXTG motif present in the full-length SpO463 protein Expression of the SpO463 clone resulted in the detection of a 60 kD polypeptide, the expected molecular weight of the recombinant SpO463 polypeptide, by Western blot analysis See FIG 155

Example 17: Cloning and Expression of 5. pneumoniae SpO464

This example describes the production of a clone encoding a SpO464 polypeptide and detection of recombinant SpO464 polypeptide expressed from the clone To produce a clone encoding SpO464, the open reading frame encoding SpO464 was amplified using primers that annealed either within the full-length SpO464 open reading frame sequence FIG 157A provides a 393 amino acid sequence of SpO464 The primers used to produce a clone encoding the SpO464 polypeptide are shown in FIG 157B These primers annealed to the nucleotide sequences encoding the amino acid residues indicated by underlining in FIG 157A Amplification of the open reading frame encoding SpO464 using these primers produced the amplicon shown at lane 4 of the agarose gel provided in FIG 160 The SpO464 clone encodes amino acid residues 19-356 of the 393 amino acid residue SpO464 protein, the italicized residues in FIG 157A were eliminated FIG 158A provides a schematic depiction of the recombinant SpO464 polypeptide FIG 158B shows a schematic depiction of the full-length SpO464 polypeptide Both the recombinant SpO464 encoded by the clone and the full-length SpO464 protein have two Cna B domains The cloned recombinant SpO464 lacks the LPXTG motif present in the full length SpO464 protein Expression of the SpO464 clone resulted in the detection of a 38 kD polypeptide, the expected molecular weight of the recombinant SpO464 polypeptide, by Western blot analysis See FIG 159

Example 18: Intranasal Immunization of Mice with Recombinant L. lactis Expressing GBS 80 and Subsequent Challenge

This example describes a method of intranasally immunizing mice using L lactis that express GBS 80 Intranasal immunization consisted of 3 doses at days 0, 14 and 28, each dose administered in three consecutive days Each day, groups of 3 CD-I outbred female mice 6-7 weeks old (Charles River Laboratories, Calco Italy) were immunized intranasally with 10⁹ or lθ'° CFU of the recombinant Lactococcus lactis suspended in 20 μl of PBS In each immunization scheme negative (wild-type L lactis) and positive (recombinant GBS80) control groups were used The immune response of the dams was monitored by using serum samples taken on day 0 and 49 The female mice were bred 2-7 days after the last immunization (at approximately t= 36 - 37), and typically had a gestation period of 21 days Within 48 hours of birth, the pups were challenged via I P with GBS in a dose approximately equal to an amount which would be sufficient to kill 90 % of immunized pups (as determined by empirical data gathered from PBS control groups) The GBS challenge dose is preferably administered in 50ml of THB medium Preferably, the pup challenge takes place at 56 to 61 days after the first immunization The challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB prior to use Survival of pups was monitored for 5 days after challenge

Example 19: Subcutaneous Immunization of Mice with Recombinant L. lactis Expressing GBS 80 and Subsequent Challenge

This example describes a method of subcutaneous immunization mice using L lactis that express GBS 80 Subcutaneous immunization consists of 3 doses at days 0, 14 and 28 Groups of 3 CD-I outbred female mice 6-7 weeks old (Charles River Laboratories, Calco Italy) were injected subcutaneously with 10⁹ or lθ'° CFU of the recombinant Lactococcus lactis suspended in 100 μl of PBS In each immunization scheme, negative (wild-type Z-. lactis) and positive (recombinant GBS80) control groups were used The immune response of the dams was monitored by using serum samples taken on day 0 and 49 The female mice were bred 2-7 days after the last immunization (at approximately t= 36 - 37), and typically had a gestation period of 21 days Within 48 hours of birth, the pups were challenged via I P with GBS in a dose approximately equal to an amount which would be sufficient to kill 90 % of immunized pups (as determined by empirical data gathered from PBS control groups) The GBS challenge dose is preferably administered in 50ml of THB medium Preferably, the pup challenge takes place at 56 to 61 days after the first immunization The challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB prior to use Survival of pups was monitored for 5 days after challenge

Example 20: Immunization of Mice with GAS AI polypeptides and Subsequent Intranasal Challenge This example describes a method of immunizing mice with GAS AI polypeptides and subsequently intranasally challenging the mice with GAS bacteria Groups of 10 CDl female mice aged between 6 and 7 weeks are immunized with a combination of GAS antigens of the invention GAS 15, GAS 16, and GAS 18, ( 15 μg of each recombinant antigen, derived from Ml strain SF370) or L lactis expressing the Ml strain SF370 adhesin island, suspended in 100 μl of suitable solution Each group receives 3 doses at days 0, 21 and 45 Immunization is performed through subcutaneous or intraperitoneal injection for the GAS 15, GAS 16, GAS 18 protein combination The protein combination is administered with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses Immunization is performed intranasally for the L lactis expressing the Ml strain SF37O adhesin island In each immunization scheme negative and positive control groups are used

The negative control group for the mice immunized with the GAS 15, GAS 16, GAS 18 protein combination included mice immunized with PBS The negative control group for the mice immunized with L. lactis expressing the Ml strain SF370 adhesin island, included mice immunized with either wildtype L lactis or L lactis transformed with the pAM401 expression vector lacking any cloned adhesin island sequence

The positive control groups included mice immunized with purified Ml strain SF370 M protein

Immunized mice are then anaesthetized with Zoletil and challenged intranasally with a 25 μL suspension containing 1 2 x 10⁶ or 1 2 x 10^s CFU of ISS 3348 in THB Animals are observed daily and checked for survival

Example 21: Active Maternal Immunization Assay

As used herein, an Active Maternal Immunization assay refers to an in vivo protection assay where female mice are immunized with the test antigen composition The female mice are then bred and their pups are challenged with a lethal dose of GBS Serum titers of the female mice during the immunization schedule are measured as well as the survival time of the pups after challenge Mouse immunization

Specifically, groups of 4 CD-I outbred female mice 6-8 weeks old (Charles River Laboratories, Calco Italy) are immunized with one or more GBS antigens, (20 μg of each recombinant GBS antigen), suspended in 100 μl of PBS Each group receives 3 doses at days 0, 21 and 35 Immunization is performed through lntra-peπtoneal injection of the protein with an equal volume of Complete Freund's Adjuvant (CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for the following two doses In each immunization scheme negative and positive control groups are used Immune response is monitored by using serum samples taken on day 0 and 49 The sera are analyzed as pools from each group of mice Active maternal immunization

A maternal immunization/neonatal pup challenge model of GBS infection was used to verify the protective efficacy of the antigens in mice. The mouse protection study was adapted from Rodewald et al (Rodewald et al J Infect Diseases 166, 635 (1992)) In brief, CD-I female mice (6-8 weeks old) were immunized before breeding, as described above The mice received 20 μg of protein per dose when immunized with a single antigen and 60 μg of protein per dose (15 μg of each antigen) when immunized with the combination of antigens Mice were bred 2-7 days after the last immunization Within 48 h of birth, pups were injected intraperitoneal Iy with 50 μl of GBS culture Challenge inocula were prepared starting from frozen cultures diluted to the appropriate concentration with THB before use In preliminary experiments (not shown), the challenge doses per pup for each strain tested were determined to cause 90% lethality Survival of pups was monitored for 2 days after challenge Protection was calculated as (percentage deadControl minus percentage deadVaccine) divided by percentage deadControl multiplied by 100 Data were evaluated for statistical significance by Fisher's exact test Example 22: GBS 59 isoforms cross-reactivity

In some instances GBS 59 polypeptides of different isoforms may be cross-reactive as well as GBS59 polypeptides of the same isoform may not be cross reactive In fact GBS 59 polypeptides are usually covalently linked in a macromolecular structure (i e the pilus), combined to other polypeptides such as GBS 67 and GBS 150, which show themselves some variability Therefore the immunologic reactivity of such complex structures may not be predictable based on the sequence of single GBS 59 polypeptides For instance in flow cytometry, where the readout is typically an average of different epitopes being recognized on these multimeπc structures on the surface of the bacteria, some cross- reactivity is expected, even in the presence of different isoforms Table 52 summarizes the results of experiments where three GBS 59 recombinant polypeptides from three different strains (CJBl 11, 515 and 2603) were used to immunize mice that were then challenged with homologous and heterologous strains With the exception of 2603 strain, the protein is well expressed on the surface (i e the Δ-mean is greater than 200 channels) and confers protection against homologous challenge In the case of mice immunized with the GBS 59^CJB1" variant, the challenge with the heterologous strain 515 resulted in a low survival rate, confirming that the two polypeptides, although representing the same isoform, are not cross-protective in the animal model Table 52 GBS cross-reactivity.

Example 23: PiIi are immunogenic in humans during infection

Human sera from 9 patients diagnosed with pneumococcal disease were analyzed by FACS for their ability to recognize whole cell pneumococcal preparations of the serotype 4 S pneumoniae strain TIGR4 All 9 sera were able to recognize TIGR4 bacteria, while a serum from a healthy donor did not produce appreciable positivity (FIG 241A) To find out whether the antibodies induced during infection recognize pili, we tested the human sera by western blot against a TIGR4 pilus-enπched mutanolysin preparation, which in SDS-PAGE forms a typical ladder of high molecular mass pilus polymers All 9 individual patient sera recognized the pilus ladder, while the serum from a healthy donor was negative (FIG 241B) To investigate whether, in addition to the ladder, the human sera recognized the individual pilus subunits, a pool of sera from the 9 patients was tested by western blot against recombinant pilus subunits RrgA and RrgB were recognized, while there was no detectable recognition of RrgC

Serum antibodies against each of the three pilus subunits were quantified by ELISA, presenting marked differences in their relative abundance The highest specific IgG level was directed against RrgB , followed by RrgA and RrgC (FIG 24IC) These results might reflect, at least in part, the relative abundance of the three subunits in the pilus since RrgB which constitutes the backbone, is the most abundant pilus component, followed by RrgA and RrgC (Barocchi 2006) Differential epitope exposure of the three subunits in the assembled native pilus could also contribute to preferentially direct the immune response to one subunit rather than another, a possibility that cannot be excluded and requires further investigation for clarification In the healthy donor serum, very low but still measurable levels of specific IgG against pilus antigens were detected by ELISA (FIG 241C) This may be reasonably ascribed to an immune response previously developed against S pneumoniae by this subject A larger study with sera from patients with pneumococcal disease and from healthy people could be used to identify the negativity threshold for ELISA detection of specific IgG against pilus antigens Example 24 Native and recombinant pilus subunits are immunogenic in mice.

Mice vaccinated with heat-inactivated TIGR4, containing native pilus structures, generated serum antibodies against recombinant pilus antigens, as evaluated by ELISA on sera obtained after the third immunization The highest response was detected against the main pilus subunit RrgB , followed by RrgA and RrgC (FIG 242), similarly to that observed for the human sera

In order to find out whether such a difference m antibody response was due to the pilus structure or to the intrinsic immunogenicity of the pilus subunits, serum IgG response was also quantified by ELISA in mice that were immunized with recombinant pilus subunits (FIG 242) Immunization with the individual recombinant pilus antigens (20 μg each) elicited high IgG response, sera becoming titrable at 1 50,000 - 1 100,000 dilution, and the antibody titers to the three pilus subunits were comparable Immunization with the combination of RrgA+B+C also elicited high IgG levels against each of the three antigens, with titers slightly reduced, consistently with the lower antigen dose used (10 μg each) (FIG 242) Specific IgG titers were undetectable in control groups (adjuvant plus saline)

When mice were immunized with the combined pilus antigens RrgA+B+C and AI(OH)₃ as an adjuvant, high IgG response was also induced, even though slightly lower than that obtained with Freund's adjuvant These results indicate that each of the three pilus subunits has similar immunogenicity Thus, the differences of IgG levels against each of the pilus subunits observed both in infected humans and in TIGR4-immunized mice should be most likely ascribed to the composition and structure of the native pilus Example 25: Immunization with recombinant pilus antigens is protective in mice

Mice were immunized intraperitoneally with recombinant pilus antigens, alone or in combination, then challenged intraperitoneally with 10² CFU of TIGR4 per mouse, a dose previously observed to cause high levels of bacteremia 24 h post-challenge and early death in naive mice Bacteria in the blood were quantified 24 h post-challenge As shown in FIG 243, in this model, control animals, receiving adjuvant plus saline, had a geometric mean of >10⁴ CFU/ml, including 7 mice with >10^δ CFU/ml and 5 mice with no detectable bacteremia (FIG 243A), 9 out of 16 mice did not survive at 10 days (FIG 243B) In marked contrast, no bacteremia was detected in any of the mice vaccinated with the whole TIGR4 bacteria (FIG 243A), and all mice of this group were alive at 10 days (FIG 243B) All groups of mice vaccinated with recombinant pilus antigens showed lower bacteremia and increased survival, as compared with the control groups receiving adjuvant plus saline The best efficacy was shown by RrgB , which afforded a protection similar to the whole TIGR4 bacteria, with only 1/8 mice bacteremic and 100% survival at the endpoint The groups vaccinated with RrgA or with the combination RrgA+B+C also resulted protected, with only 1/8 mice bacteremic and 7/8 mice surviving challenge in each group Finally, the mice vaccinated with RrgC showed only limited protection Both in terms of bacteremia and survival, all groups immunized with pilus antigens gave results not statistically different (P > 0 1) from those of the group vaccinated with heat-inactivated TIGR4, which resulted completely protected The combination RrgA+B+C showed similar protective efficacy when Freund adjuvant was replaced by AI(OHh (FIG 243)

Interestingly, in vaccinated groups, infection and death correlated with low specific antibody titers against the three pilus subunits, suggesting the relevance of antibody response in the observed protection

In order to further investigate whether the protective efficacy of pilus subunits is antibody-dependent, we tested mouse antisera raised against recombinant pilus antigens for their protective ability by passive transfer Immune sera were intraperitoneal^ injected in mice prior to challenge with 10² CFU of 5 pneumoniae TIGR4 per mouse As shown in FIG 243A, 24 h post-challenge, control animals presented a geometric mean of >10⁵ bacteria per ml of blood, with 10/16 mice having values >10⁵ CFU/ml, one mouse <10⁵ CFU/ml, and 5 mice with no detectable bacteremia Ten days post- challenge, 8/16 control mice were still alive (FIG 243B) All 8 mice receiving anti-TIGR4 serum presented undetectable bacteremia and survived at 10 days (FIG 243B) All groups that received antisera against recombinant pilus antigens showed reduced bacteremia and increased survival time, as compared with the control group

The passive transfer of anti-RrgA+B+C serum resulted in undetectable bacteremia at 24 h (FIG 243A) and survival at the endpoint (FIG 243B) for all 8 mice Also, after passive transfer of either anti-RrgA or anti-RrgB serum, only 1 and 2 mice, respectively, were found bacteremic 24 h post-challenge (FIG 243A), and 8/8 mice in each group survived lethal challenge (FIG 243B)

Finally, passive transfer of anti-RrgC serum resulted in 5/8 mice with no detectable bacteremia (FIG 243A), and 7/8 mice survived challenge (FIG 243B) Similarly to that obtained with active immunization, all groups that received antisera raised against pilus antigens showed bacteremia and survival not statistically different (P > 0 1) from those of the group that received anti-TIGR4 antiserum, which resulted completely protected These results indicate that antibodies play a relevant role in the protective effect these antigens elicit

The observation that, both by active and passive immunization, RrgA and RrgB are much more effective than RrgC in protecting mice against lethal challenge, even though all three antigens elicit comparable specific antibody titers, can be explained also in this case by the different relative abundance of these antigens in the native pilus In fact, the efficacy of high antibody titers to RrgC can be hampered by the relatively low availability of their target in the infecting bacteria, that is not the case for the more abundant RrgB and RrgA

Moreover, passive transfer of mouse immune serum raised against RτgA+B+C_6B was able to protect mice against heterologous challenge with 10² CFU of TIGR4 All 8 mice receiving anti-RrgA+B+C_6B antiserum were not bacteremic 24 h post-challenge and were still alive at 10 days (FlG 243) These preliminary results suggest the possible cross-protective ability of pilus subunits against different S pneumoniae serotypes

FIG 254 demonstrates that that passive transfer of antisera to TIGR4 native pilus protects against TIGR4 challenge

These examples provide evidence that the three S pneumoniae pilus subunits, RrgA, RrgB and RrgC, are naturally immunogenic, and that immunization of mice with the three recombinant proteins elicits high antibody titers Both active immunization with the three recombinant pilus components and passive transfer of antisera against these antigens is protective in mice against subsequent lethal challenge, RrgB and the combination of RrgA+B+C showing the best overall efficacy, followed by RrgA and RrgC Although pilus structures are not universal in pneumococcal strains, the ability of the pilus recombinant proteins to protect mice against infection suggests their use as potential components of a multi-protein vaccine as an alternative capsule-independent strategy to protect against 5 pneumoniae Example 26: Cloning, expression and purification of RrgA, RrgB and RrgC (Examples 23-25).

Standard recombinant techniques were used for nucleic acid cloning and restriction analyses. Briefly, genomic DNA from TIGR4 S. pneumoniae strain was prepared using the Wizard genomic DNA purification Kit (Promega). PCR was carried out with Expand High fidelity PCR system (Roche) according to the manufacturer's instructions. Primers were as follows: rrgA:

5'-AGTTGCTGCT AGCGAAACGCCTGAAACC-3'(forward)(SEQ ID NO:467),

5^>-CAGTTCGCTCGAGTTCTCTCTTTGGAGG3'(reverse) (SEQ ID NO:468); rrgB:

5'-GTGCGTGCT AGCGCTGCAAC AGTTTTTGCGGCTGGG-3'(forward) (SEQ ID NO:469),

5'-C AGCGTCTCGAGAGTGATTTTTTTGTTGACT ACTTT-3'(reverse) (SEQ ID NO:470); rrgC:

5'-GTGCGTGCTAGCCATGCAGTCCAAGCGCAAGAAGAT-3'(forward) (SEQ ID NO:471),

5'- CAGCGTCTCGAGATCAATCCGTGGTCGCTTGTT ATT-3'(reverse) (SEQ ID NO:472).

The amplification products were purified, digested with the appropriate enzymes (Ndel and Xhol) and ligated in a His6 expression vector, pet21b+ (Novagen). The resulting plasmids were introduced into E. coli DH5α for sequence analysis and in E. coli strain BL21 star (DE3) for protein expression.

IPTG-induced recombinant E. coli cultures, expressing His-tagged RrgA, RrgB and RrgC proteins, were harvested and subjected to lysis by lysozyme in a BugBuster (Novagen), Benzonase Nuclease (Novagen) solution containing proteinase inhibitors. After centrifugation at 100,000 rcf for Ih at 4⁰C, the soluble fraction was subjected to metal chelate affinity chromatography on His-Trap HP columns (GE Healthcare) equilibrated and eluted according to manufacturer's instructions. Purity was evaluated by scanning densitometry of Coomassie Blue-stained SDS-PAGE: fractions corresponding to > 90% purity were used. Pooled fractions were dialysed overnight against 0.9% NaCl and stored at -80⁰C until further use. Protein concentration was determined by scanning densitometry of Coomassie Blue- stained SDS-PAGE using a BSA standard and measuring Absorbance at 280 nm of the protein solution (NanoDrop).

Bacterial culture. Bacteria were grown at 37°C under 5% CO₂ on Tryptic Soy Agar (Becton Dickinson) with 5% sheep blood, inoculated into Tryptic Soy Broth (Becton Dickinson), and further cultured until reaching OD_60O = 0.2 (= 10⁷ CFU/ml).

Protein expression and purification. Genomic DNA was prepared from TIGR4 or 6B strains using the Wizard Genomic DNA Purification Kit (Promega). PCR was done with Expand High Fidelity PCR System (Roche). Primers are listed in Table 1. PCR products were digested with Ndel and Xhol (New England Biolabs), ligated in pET21b+ (Novagen), and the plasmids introduced into E. coli BL21 Star (DE3). Soluble recombinant pilus subunits corresponding to the sequence of TIGR4 (RrgA , RrgB , RrgC ) or 6B (RrgA _6B, RrgB _6B, RrgC _6B) were purified by His-Trap HP (GE Healthcare). Protein purity and concentration were determined by SDS-PAGE scanning densitometry.

Mice and study design. Animal experiments were done in compliance with the current law. Six-week-old specific-pathogen-free female BALB/c mice (Charles River) were immunized intraperitoneally (i.p) on day 0, 14 and 28 with RrgA , RrgB , RrgC (20 μg), a combination RrgA+B+C or RrgA+B+C₆B (10 μg each), or heat-inactivated bacteria (10⁸ CFU), along with Freund's adjuvant. The combination RrgA+B+C was also given i.p. on day 0, 10 and 20, with 200 μg Al(OH)₃. Controls received an identical course of saline plus the adjuvant. Two weeks after the last immunization, each mouse was i.p. challenged with 10² CFU of TIGR4 (LD_I00 in naive mice). For passive immunization, 10-week-old mice received i.p. 50 Gl of pooled mouse immune sera 15 min before lethal challenge with TIGR4 as above or with 10⁶ CFU of 6B Bacteremia was quantified at 24 (TIGR4) or 5 h (6B), and the survival monitored for 10 days (TIGR4) or 15 days (6B) post-challenge

FACS Analysis. TIGR4 bacteria were incubated on ice for 30 min with human sera diluted 1 50 Antibody binding was revealed by FITC labeled anti-human IgG (Jackson ImmunoResearch) and samples analyzed by FACSCAN (Becton Dickinson)

Western blot. TIGR4 mutanolysin preparation was run on 3-8% NuPage Novex Bis Tπs Gel (Invitrogen) and blotted onto 045 μm nitrocellulose Human sera were added at 1 3,000 dilution followed by alkaline-phosphatase conjugated anti-human IgG (Promega) Immunoreactive bands were visualized by the Western Blue Stabilized Substrate (Promega)

ELISA. Serial dilutions of human or mouse sera were dispensed in Maxisorp 96 well plates (Nalge Nunc Int ) coated with recombinant RrgA, RrgB or RrgC 02 μg/well Antibody binding was revealed by alkaline phosphatase- conjugated anti-human (Sigma) or anti-mouse (Southern Biotechnology Ass ) IgG, followed by p-nitrophenyl-phosphate (Sigma) Absorbance was measured at 405 nm Mouse sera were titrated using a reference line calculation program, by comparison with the reference curves Reference consisted of pooled anti-RrgA, -RrgB or -RrgC mouse sera, which tested by ELISA at 1 100,000 dilution gave similar A₄₀₅ values, and to which the titer of 50,000 was assigned

Statistics. Data were evaluated by one-tailed Mann-Whitney U test P values < 005 were considered and referred to as significant

Example 27: Pilus like structures promote cell auto aggregation and biofilm formation in group A Streptococcus pyogenes (GAS)

Bacterial Strains, Media, and Growth Conditions. GAS Ml strain SF370 was provided by University of Siena, Italy Wild type and mutant strains were grown at 37⁰C or 3O⁰C in Todd-Hewitt medium supplemented with 05% yeast extract (THY) (Difco), or THY agars supplemented with 5% defibπnated sheep blood L lactis subspecies cremons MG1363 was grown at 30°C in M17 (Difco) supplemented with 0,5% glucose (GM17) 20 μg/ml chloramphenicol was used in selective medium

Construction of GAS deletion mutants and complementation. In-frame deletion and complementation mutants of GAS strain SF370 were constructed as described before (Mora et al , 2005) Briefly, mutations were constructed by using splicing-by-overlap-extensionPCR (Horton, et al , 1990) The PCR deletion construct was cloned in the temperature-sensitive allelic exchange vector pJRS233, and transformation and allelic exchanges were performed as described in (Frameson et al , 1997, Caparon and Scott 1991 and Perez-Casal et al , 1993) Transformants were selected on THY plates with 1 μg/ml erythromycin (Sigma) at 30°C Drug sensitive colonies were screened and deletions were confirmed by PCR assay The complementation vectors pAM401 128 and pAM401 129 were constructed with the appropriate primers to amplify the fragment that includes the spyO128 or spyO129 gene, the predicted promoter and the P- independent terminator

L. lactis transformation with GAS pilus region. The complementation vector pAM401 pilMl was constructed with the appropriate primers to amplify the fragment that includes the genomic region comprised between spyO126 to spyO13O The fragment was cloned in the pAM vector containing the promoter and terminator regions of GBS adhesin island-2 (Buccato et al , 2006) The vector was then inserted in L lactis MG1363 competent cells by electroporation, and the transformants were selected on GM 17 plates with 20 μg/ml chloramphenicol Drug-resistant colonies were screened by PCR The expression of pilus subunits and their assembly into a covalently bound polymeric structure was confirmed by western blot analysis, using polyclonal sera obtained from mice immunized with the corresponding GAS pilus proteins Immunoblots on bacterial cell-wall fractions. Bacterial cell-wall fractions were prepared as described previously In particular, bacteria grown in THY to ODβoo = 04 at 37°C were pelletted, washed once in PBS, suspended in 1 ml of ice cold protoplasting buffer [40% sucrose, 0 1 M KPO4, pH 6 2, 10 mM MgC12, Complete EDTA free protease inhibitors (Roche), 2 mg/ml lysozime, 400 units of mutanolysin (Sigma)] and incubated at 37°C for 3 h After centπfuging at 13,000 x g for 15 min, the supernatants (cell- wall fractions) were frozen at -2O⁰C

Cell wall preparations were then separated by 3-8% gradient gels (NuPAGE Tπs-acetate gels, Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad) for immunoblot analysis with mouse polyclonal antisera at a 1 500 dilutuion obtained as described before (Mora et al , PNAS2005) and ECL enhanced chemilumiπescence detection (SuperSignal West Pico chemiluminescent substrate, Pierce) The secondary antibody (ECL, horseradish-peroxidase- hnked anti-mouse IgG, GE Healthcare) was used at a 1 5,000 dilution

Electron Microscopy GAS was grown on THY blood agar plates and resuspended in PBS Formvar-carbon- coated nickel grids were floated on drops of bacterial suspensions for 5 min, fixed in 2% PFA for 5 min, and placed in blocking solution (PBS containing 1% normal rabbit serum and 1% BSA) for 30 mm The grids were then floated on drops of primary antiserum diluted 1 20 in blocking solution for 30min at RT, washed, and floated on secondary antibody conjugated to 10 nm gold particles diluted 1 10 in 1% BSA for 30 min Bacteria were then fixed again for 10 min The grids were washed with PBS then distilled water and air dried and examined using a TEM GEOL 1200EX II transmission electron microscope Preimmune serum from the same animals were used as a negative control

Light microscopy. L lactis was grown in GM17 to mid-log phase 20 μl of bacterial suspension was placed on a glass slide, covered with a covershp and observed with a Bio-Rad confocal scanning microscope

Confocal microscopy GAS aggregation was observed by confocal laser scanning microscopy (CLSM). In particular, approximately 2xlO⁸ bacteria grown to OD_6Oo=O^ were seeded in 12-well plates containing sterile glass cover- slips coated with poly-lysine and were left growing upon the cover-slips up to the late exponential phase, when aggregation reaches a maximum Samples were then fixed with paraphormaldeyde 2,5% for 15 minutes, washed with PBS and blocked for 15 minutes Then samples were incubated with primary antibodies (rabbit-anti-GAS and mouse- anti-spyO128) for Ih at RT, washed in blocking solution and incubated for 30 minutes at RT with secondary antibodies Alexa Fluor dye 647 goat anti-rabbit and Alexa Fluor dye 568 goat anti-mouse (Molecular Probes) Cover-slips were then washed with blocking solution and mounted on glass slides with the Slow Fade reagent kit (Molecular Probes) containing 4_,6_-diamidino-2-phenylindole dihydrochloπde before they were viewed on a Bio-Rad confocal scanning microscope

For aggregation on eukaryotic cells surface, 2xlO⁵ Detroit-562 cells were seeded on glass cover-slips coated with polylysine in 12-well plates The day after 5xlO⁸ bacterial cfu of each strain from a logarithmic growth were extensively pipetted to break possible aggregates and used to infect mono-layers at 37°C in a 5%CO2 atmosphere After 15 minutes cells were washed 3 times with PBS to remove the unattached bacteπa, and infection was let continue to 30, 60 and 120 minutes Samples were then washed again, fixed, blocked and stained with rabbit-anti-GAS as a primary antibody and Alexa Fluor dye 488 goat anti-rabbit (Molecular Probes) as a secondary antibody Cells were stained with phalloidm conjugated with Alexa Fluor dye 647 (Molecular Probes) Mounting and viewing were performed as already described

For each strain of bio- film used in CLSM studies, a 1 10 dilution of an overnight culture in C-medium (Lyon et al , 1998) at 37°C was inoculated at RT on poly-lysine coated glass sterile cover-slips positioned in 50 ml falcon containing 10 ml of fresh C medium, as described elsewhere (Cho and Caparon, 2005) Five ml of C medium were replaced every 24 hours and preparations were collected after desired time points of growth (24, 48 and 72 hours) Samples were then fixed, blocked and stained with rabbit-anti-GAS and mouse-anti spyO128 as primary antibodies and Alexa Fluor dye 647 goat anti-rabbit and Alexa Fluor dye 568 goat ant-mouse as secondary antibodies (Molecular Probes) Exopolysacchaπdes (EPS) were stained by the FITC-conjugated lectin Concanavahn A (Sigma) Mounting and viewing were performed as already described Three-dimensional immunofluorescence images were reconstructed from 05-μm confocal optical sections by using VOLOCITY 3 5 (Improvision, Lexington, MA)

Bio-film formation assay. For each strain, a 1 10 dilution of an overnight culture in C-medium () at 37°C was inoculated in 1 ml of tresh medium in 24-well plates in triplicate Plates were incubated at room temperature for 16-24- 48-72 h, changing medium every 24 h At each time point, the medium was removed and adherent bacteria were stained with crystal violet (0,2% in distilled water) by incubating at room temperature for 10 minutes Crystal violet was then eluted with 1% SDS and bio-film formation was quantified by measuring the optical density at 540 nm

Eukaryotic cell cultures. The human pharynx carcinoma cell line Detroit-562 (ATCC CCL-138) was cultured in Dulbecco's modified Eagle's medium (EMEM, Life Technologies Gibco BRL) supplemented with 10% FCS (Life Technologies) and 5 mM glutamine (Life Technologies) at 37⁰C in an atmosphere containing 5% CO₂ For adherence assays, cells were resuspended at a concentration of approximately 3xlO⁵ cells/ml in EMEM, and seeded into 24-well tissue culture plates (Nunc), which were then incubated for 24 h For microscopic assays, approximately 6 x 10⁵ cells/ml were seeded onto 12-mm-diameter glass coverslips placed on the bottom of 24-well tissue culture plates

Adherence assay. Bacteria from exponential phase cultures were collected by centrifugation (3000xg, 5 min), resuspended in conditioned EMEM and used to infect Detroit 562 cells monolayers for 5, 15, 30 and 120 min at 37°C in a 5% CO₂ atmosphere A Multiplicity of Infection (MOI) of 100 1 (for GAS strains) or 10 1 (for L lactis strains) were used After infection, the wells were extensively washed with PBS to remove unattached bacteria, incubated with 1% saponin to lyse eukaryotic cells, and adherent bacteria were plated for enumeration Adherence results were expressed as the average number of bacteria recovered per ml for three independent determinations in a single assay and the percentage of adherence was calculated using the following equation bacteria recovered after infection (cfu/ml) /bacteria inoculated (cfu/ml) x 100 Tests were repeated at least three times and results are expressed as the averages + SD of three experiments performed in triplicate

Statistics. T student test was used to compare biofilm formation and cell adhesion of wild type and mutant strains. Data with p value <0,05 were reported as statistically significative Pilus dependent bacterial aggregation during in vitro growth

We previously showed that S pyogenes can display pilus-hke structures on their surface and that pili and their assembly machinery are encoded in a 11kb highly variable pathogenicity island known as the fibronectin binding, collagen binding, T antigen (FCT) region (Mora et al , 2005) In the transformable strain M1_SF37O, the genes for the three pihn components and the sortase enzyme involved in pilus assembly are located in the FCT-2 variant region In frame deletion of either the pilus backbone encoding gene (Ml_128) or the Cl sortase (Ml_129) resulted in abolished polymerization of all three pilin proteins, whereas the respective complemented strains produced again pili (FIG 248)

As a first step to investigate the phenotype of the two GAS derivatives unable to form pili, m vitro growth of the two mutants was compared to wild-type When SF370 was grown in liquid medium, it started forming large visible aggregates from the early exponential growth phase, which progressively precipitated to the bottom of the tube Although their growth rate was unaffected, the two mutant strains remained in solution for a longer period This observation led us to further investigate whether pili could be involved in self-aggregation of bacteria Using Confocal Laser Scanning Microscope (CLSM) we observed the vast aggregates formed by wild type SF370 grown to exponential phase and double labeled with sera raised against whole GAS bacteria and with Spyl28 purified recombinant protein (FIG 249A) Conversely, ΔSPyl28 and ΔSpyl29 mutants were not stained with anti-Spyl28 and they formed only the typical streptococcal chains without any or very low aggregation (FIG 249 B and C) Plasmid mediated complementation resulted in a partially restored capacity to self aggregate, even if not all the bacteria were labeled with anti-Spy 128, perhaps due to plasmid instability (FIG 249 D and E)

To further test whether pih could per se be responsible of the self-aggregating phenotype, we introduced the five genes involved in GAS SF370 pilus formation into L lactis, a non pathogenic Gram-positive microorganism which does not form aggregates during growth Lactococcal bacteria, already shown to correctly assemble pili from Streptococcus agalactiae (Buccato et al , 2006), expressed and assembled the GAS pilin proteins in a covalently bound polymerized structure, as could be inferred from the high molecular weight pattern visible in immunoblots (F]G 248) As hypothesized, light microscopy analysis (FIG 249 F-G) revealed that L lactis bearing the Ml pilus region acquired the capacity to form aggregates, whereas the strain transformed with the vector alone failed to show any aggregation phenotype, strongly suggesting an involvement of the pilus region in inter bacterial attachment GAS association to human pharyngeal epithelial cells is dependent on the presence of pili

To evaluate whether the in vitro observed aggregation phenomenon could be similar to the behavior of bacteria during adhesion to host cell epithelia, we co-cultured SF37O wild type, ΔSPyl28 and ΔSPyl29 strains with the human pharynx cell line Detroit-562 and observed bacteria adhering to cells by confocal microscopy In particular, 5xlO⁸ bacterial cfu from a logarithmic growth were extensively pipetted to break possible aggregates and used to infect monolayers of approximately 2xlO⁵ Detroit-562 cells After 15 minutes cells were thoroughly washed to eliminate loose- adherent bacteria and infection continued up to 30, 60 and 120 min As shown in FIG 250 A-D, after 30 minutes the wild type strain started forming aggregates which specifically adhered to the cell surface and became larger during longer incubation times Conversely the two mutant strains, although adhering to cells, started forming aggregates only after 1-2 hours of infection This suggests an involvement of GAS pili in concentrating large numbers of bacteria to the epithelial layers during the initial stages of colonization (FIG 250 E-H and I-N)

The results were confirmed by performing a classical adhesion assay in which a confluent cell monolayer was infected with 10⁸ thoroughly pipeted bacteria or its isogenic mutants ΔSPyl28 and ΔSpyl29 and the number of adhering bacteria after 5, 15, 30 and 120 minutes of infection was measured after extensive washing Bacterial growth was checked by counting the total number of cfu in parallel wells and was found to be equivalent in all strains As shown in FIG 251 A-B, the number of bacteria associated to cells was significantly lower for the two mutant strains compared to wild type after 30 and 120 minutes but also at shorter times, indicating a delay in initial adhesion as well as a slower formation of cell-associated aggregates as already described in confocal microscopy experiments These data suggest that, like Type IV pili in V cholerae, GAS pili contribute to cell adherence both by mediating micro-colony formation and by acting as a ligand for early binding to a surface exposed epithelial cell receptor

The role of pili in adherence to epithelial cells was confirmed in a new adhesion assay in which 10⁷ cfu of L lactis harboring either the GAS Ml pilus island or the recipient strain transformed with the plasmid vector as control, were co-cultured with Detroit-562 cells and adherent bacteria were counted after 15 and 120 minutes As shown in FIG 251 C, the acquisition of the pilus island strongly increased L lactis adhesion to epithelial cells both after 15 and 120 minutes, thus further confirming the involvement of GAS Ml pih in cell adhesion All these data strongly suggest that pili facilitate the early stages of GAS cell adhesion to pharyngeal eukaryotic cells, probably by means of the adhesin proteins of which they are composed GAS SF370 pili are involved in the cell surface interaction required for bio-film formation

Many bacteria, including S pyogenes aggregate during growth and form micro colonies which further develop into bio-film structures (reviewed in Hall-Stoodley et al , 2004) To investigate whether the described self-aggregation mediated by pili was instrumental to bio-film development, we performed a classical bio-film plate assay Bacteria were incubated at room temperature in C medium in 24-well plates, and stained with crystal violet A preliminary study indicated that GAS SF370 fully attached to polystyrene surfaces in 16 to 24 hours, whereas adhesion diminished after 48 and 72 hours Based on these data, the capacity of wild type and its mutants to form bio film was compared after 24 hour incubation

As shown in FIG 252, wild type bacteria formed significantly more bio-film than mutants unable to form pili (p val <0,025) Moreover, when Spyl28 and Spyl29 genes were again introduced in the mutant strains, we observed a partially restored bio-film forming ability (p val <005) The number of cfu after 24 hour growth was equivalent in all strains These data clearly demonstrate that a non polar deletion of pill considerably impairs bio-film formation Pili affect bioΩlm maturation

Bio-film assay on plates detects primarily the initial cell-surface interactions required for bio-film formation (O'Toole et al , 2000) To analyze subsequent stages of bio-film maturation wt, ΔSPyl28, ΔSPyl29 and their complemented strains ΔSPyl28(pAM128) and ΔSPyl29(pAM129) were grown on poly-lysine coated glass cover-slips, double labeled with anti GAS and anti Spyl28 sera and examined by confocal microscopy After 72 hours the bio-film formed by the wild-type strain showed an average thickness of 10 8 μm while the two mutants attached to the glass surface but failed to form a significant multilayered structure and thus a mature bio-film (three dimensionaland multilayered) (FIG 253) On the other hand the complemented strains ΔSPyl28(pAM128) and ΔSPyl29(pAM129) produced 11 8 μm and 4 5 μm thick bio-films respectively Furthermore, wt and complemented bacteria were able to produce the extra cellular saccharides that most bacterial cells secrete during bio-film development (EPS), stained in green by the lectin Concanavalin A labeled with FITC, whereas very low amounts of EPS could be detected in the mutant strains (FIG 253)

Example 27: Pili genomic islands are ubiquitous in clinical isolates of Streptococcus agalactiae: a basis for a broadly protective vaccine

In this example, we provide a thorough analysis of the distribution of the three pilus-like genomic islands among 289 clinical isolates of GBS collected at distant geographic sites Moreover, sequence variability of the PI genes coding for the three structural proteins of each pilus has been determined for 186 isolates This example has led to the definition of a combination of three antigens, one for each pilus island, that could form the basis for a broadly protective vaccine

Bacterial strains and growth conditions. Streptococcus agalactiae (GBS) isolates used in this work were collected from patients with invasive GBS infections and asymptomatic colonization The isolates came from three collections the Center for Disease Control and Prevention (CDC), Atlanta, Georgia (2000 to 2003), Baylor College of Medicine (BCM), Houston (2002 to 2005) and Istituto Supeπore di Sanita, Italy (1992 to 2006) Serotypmg of isolates at CDC and BCM used the capillary precipitin method of Lancefield GBS strains 2603 V/R (capsular serotype V), 515 (Ia), CJBl I l(V), H36B(Ib), COHl(III), used as source of DNA for amplification of pili genes, were a gift from Dr Dennis Kasper (Harvard Medical School, Boston, USA) Bacteria were grown at 37⁰C in Todd Hewitt Broth (THB, Difco Laboratories) or in trypticase soy agar supplemented with 5% sheep blood

DNA isolation. Genomic DNA was prepared by a standard protocol for gram-positive bacteria using a NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer's instructions In brief, GBS isolates were grown in 10ml of THB medium to OD600nm 05 The culture was centrifuged for 10 min at 3000rpm, the cell pellet was resuspended in 180 μl of lysis buffer containing 20 mM Tπs pH 8 0, 2 mM EDTA, 1% Triton X 100, 1 mg lysozyme (Sigma), 50 units of mutanolysin (Sigma) and incubated for 1 h at 37°C Then 25 μl of Proteinase K (20 mg/ml) was added and samples were incubated at 56⁰C for at least 1 h When a complete lysis was obtained, 10 μl of RNase A (20 mg/ml) were added and samples were incubated for an additional 10 min at 56°C The DNA from the bacterial clear lysates was isolated using NucleoSpin Tissue columns and eluted in sterile water

PCR amplification and DNA sequencing. Genes were amplified using primers external to the coding sequence The primers are listed in Table VII Each PCR reaction was performed in 100 μl containing 100 ng of GBS chromosomal DNA, 50 pM of each primer, 200 DM of each dNTP and 0 5 U of Pwo DNA polymerase (Roche) in Ix buffer with 1 5 mM MgCI2 The reaction conditions for denaturation were 94°C for 5 min , followed by 30 cycles (denaturation at 94°C for 30 sec primer annealing at 55°C for 45 sec and extension at 72°C for 1 2 mm ) The nucleotide sequences of PCR products were determined using a BigDye Terminator V3 1 Cycle Sequencing kit (Applied Biosystem) in an ABI PRISM 3700 DNA Analyzer (Applied Biosystem)

Sequence Alignments and Phylogenetic Analysis. The percentage of sequence identity was calculated by pair wise BLAST with the VECTOR NTI SUITE 9 for PC (Informax Bethesda) with gaps included Protein alignments were performed by using the program CLUSTAL W (1 83) included in the GCG Wisconsin Package version 11 1 Phylogenetic trees were inferred from the protein alignments by the neighbour joining distance-based method and bootstrapped 1,000 times The complete genome sequences of Streptococcus agalactiae strain 2603 V/R (V), A909 (Ia) and NEM316 (III) are available under accession numbers AE009948, CPOOOl 14, AL732656 The genome sequences m assembly of strains 18RS21 (II), 515 (Ia), CJBlI l (V), H36B (Ib) and COHl (III) are available under accession numbers AAJOOOOOOOOO, AAJPOOOOOOOO, AAJQOOOOOOOO, AAJSOOOOOOOO, AAJROOOOOOOO

Cloning, expression and purification of recombinant proteins. Recombinant proteins were expressed in E coli BL21DE3 cells (Novagen) as 6His-tagged fusion proteins by cloning the corresponding genes in pET24b+ (Novagen) and purified by affinity chromatography as previously reported (22) GBS strain 2603 V/R (serotype V) was used as source of DNA for cloning the sequences coding for the PM proteins (TIGR annotation SAG0645, SAG0646, SAG0649) and the PI-2a LPXTG proteins (TIGR annotation SAG1408, SAG1407, SAG1404) GBS strain 515 (Ia) and GBS strain CJB 111 (V) were used for cloning the sequences coding for the corresponding PI-2a backbone protein (TIGR annotation SAL1486, SAM1372) and GBS strain H36B (Ib) for the amplification of the gene coding for the PI-2a ancillary protein 1 (TIGR annotation SAI1512) GBS strain COHl (III) was used for cloning the genes coding for the PI- 2b proteins (TIGR annotation APl-2b, BP-2b and AP2-2b) Primers were designed to amplify the coding regions without the signal peptide sequence and the 3' terminal sequence starting from the region coding for the LPXTG motif

Mouse immunization. Purified recombinant GBS proteins were used for intraperitoneal immunization of groups of 6- to 8-week-old CD-I outbred mice (Charles River Laboratories, Calco, Italy) 20 μg of each protein was administered to mice on days 1 (emulsified in Complete Freund's adjuvant, CFA), 21 and 35 (in Incomplete Freund's adjuvant, IFA) Sera from each group of mice were collected on days 0 and 49, and the protein-specific immune response (total Ig) in pooled sera was monitored by ELISA

Flow cytometry. Exponential phase grown GBS strains were resuspended in PBS containing 005% paraformaldehyde, and incubated for 1 h at 37°C and then overnight at 4°C Fixed bacteria were then washed once with PBS, resuspended in Newborn Calf Serum (Sigma) and incubated for 20 mm at room temperature The cells were then incubated for 1 h at 4 °C in pre-immune or immune sera, diluted 1 200 in dilution buffer (PBS, 20% Newborn Calf Serum, 0 1% BSA) After centπfugation and washing in PBS/0 1% BSA, samples were incubated for 1 h at 4 ⁰C with R- Phycoerythπn conjugated F(ab)2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc ), diluted 1 100 After washing, cells were resuspended in PBS Samples were acquired by a FACS Calibur apparatus (Becton Dickinson, Mountain View, Ca ) and data were analyzed using the Cell Quest Software (Becton Dickinson) In the case of APl-I, to avoid cross-reactive binding of polyclonal sera, a pool of four monoclonal antibodies raised against the protein was used instead of mouse immune serum, a pool of two unrelated monoclonal antibodies was used as a control To analyze the surface exposure of the PI-2a backbone protein, antisera specific for the 2603, 515 and CJBl I l variants were used Data are expressed as the difference in fluorescence between cells stained with immune sera versus pre immune sera

Active mouse maternal immunization. A maternal immunization/neonatal pup challenge model of GBS infection was used to verify the protective efficacy of the antigens in mice, as previously described (11) In brief, CD-I female mice (6-8 weeks old) were immunized on days 1 (in CFA), 21 and 35 (IFA) with either PBS or 20 μg of protein per dose when immunized with a single antigen or 60 μg of protein per dose (20 μg of each antigen) when immunized with a combination of antigens Mice were bred 3 days after the last immunization Within 48 h of birth, pups were injected intraperitoneally with a dose of GBS bacteria calculated to cause 90% lethality Survival of pups was monitored for 2 days after challenge Protection was calculated as (percentage deadControl minus percentage deadVaccine) divided by percentage deadControl multiplied by 100. Statistical analysis was performed using Fisher's exact test.

Genomic islands coding for pilus-like structures are always present in clinical isolates of GBS. A total of 289 isolates of invasive and colonizing GBS collected at three centers (CDC, BCM and Istituto Superiore di Samta, Italy) (Table I) were analyzed for the presence, sequence variability and surface exposure of the three structural components of GBS pili. Two loci have been identified in the genome of GBS strains that can harbor pilus encoding islands Genes associated with the first locus, pilus island 1 (PI-I), are conserved and present in six of the eight GBS genomes sequenced (12).The second locus is occupied by either of two variants of pilus island 2, PI-2a and PI-2b, that show only limited similarity at the sequence and gene organization levels (13).

Screening by PCR for the presence of the genes coding for the structural components of GBS pill indicated that all 289 strains contained at least one of the pilus island regions (Table II). The PI-I locus was present in 208 (72%) strains and was always associated with the presence of a PI-2a or PI-2b allele at the second genomic region containing a pilus island. Therefore, while the genomic region at the first locus was empty in 28% of the strains, the second locus contained PI-2a /b in all GBS isolates. It should be noted that PI-2a was frequently present alone while PI-2b only rarely was not associated with PI-I. However, the most frequent combination was PI-l+PI-2a since it was present in over 45% of isolates.

Table II summarizes the distribution of pilus islands among the GBS isolates grouped by invasive disease manifestation or asymptomatic colonization. The data show no apparent association of disease or colonization with the presence of PI-I and/or PI-2a/b, albeit few colonizing strains were studied. Similarly, no significant difference in the distribution of the islands was found between these three groups of isolates collected from patients in different geographic areas (data not shown). Thus, all strains were combined in the additional analyses reported.

Pilus Islands distribution correlates with serotype. Extending the analysis of pilus island distribution to GBS strains grouped by serotype, a good correlation was observed between presence of a particular combination of PIs and CPS type. Most serotype IA isolates (91%) contained only the PI-2a island, while the large majority (85%) of type IB strains had PI-I inserted in their genome, as well as PI-2a (FIG. 256). Serotype II strains were always associated with PI- 2a, alone or together with PI-I in a nearly 50-50 ratio. In contrast, all serotype HI isolates except one contained PI-I in association with PI-2a (30%) or, more frequently, with PI-2b (69%) Indeed, 71 of 76 (94%) strains containing PI-l+PI- 2b were serotype III.

Only 10 serotype IV isolates were included in this study and the distribution of pilus islands among these few strains does not correlate with the presence of a specific PI, except that, of 289 isolates, the only four strains that contained PI-2b alone were serotype IV (FIG. 256). For serotype V strains, nearly all (96%) contained PI-l+PI-2a and the remaining 4% had only PI-2a Similarly, PI-I and PI-2a were both present in the majority of NT strains

Sequence conservation of PIs structural components. The PCR products obtained amplifying with specific primers the genomic regions coding for the three structural pili components of each island were sequenced for a total of 186 isolates, namely all the strains from the CDC and the Istituto Superiore di Sanita collections

A summary of this analysis is presented in FIG. 257 The three genes coding for the structural proteins of PI-I were extremely well conserved and their products differed by very few amino acids. In particular, the sequence of BP-I, the protein that is presumed to represent the backbone of pilus 1, showed a polymorphism at position 16 of the signal peptide sequence where a Met residue is substituted by He This was observed in 37 strains, mostly serotype V isolates (78%) Smaller groups of strains contained single amino acid variants at other positions such as the five isolates that had an Ala57Thr substitution or the eight strains, all serotype II isolates, that carried a point mutation resulting in a frameshift producing a termination codon after the Thr360 codon (FIG 257, panel A) Similarly, one of the alleles of API 1 , the major ancillary protein of pilus 1 , contained an Ilel90Asn polymorphism and all the strains that contained Asn at position 190 belonged to the serotype HI group

FIG 256, panel B displays a schematic representation of the distribution of alleles of pilus island 2a among the 137 GBS isolates that contained this island Since two of the structural proteins of PI-2a, BP-2a and APl-2a, are variable and variants corresponding to five of the strains whose genome has been fully sequenced already have been described ( 12), the sequences analyzed here have been assigned to the variants present in these reference strains Furthermore, two additional alleles have been identified among our isolates that are identical to those found in two laboratory strains, 090 (14) and DK21 Therefore, these were arbitrarily chosen as additional reference strains

The phylogenetic relationship of BP-2a and APl-2a variants m reference strains used in this study is shown in panel D of FIG 257 Variability among APl-2a alleles was limited and sequences from all reference strains fell into two groups that could be identified as the 2603 V/R and the H36B groups, which displayed 87% of amino acidic identity In contrast, BP-2a variants were clearly more distant and only two alleles, the CJBlI l and NEM316 variants that differ by 17 nucleotides or 11 amino acids, can be considered to be highly similar

All the isolates analyzed contained BP-2a and API -2a variants from the same reference strain Moreover, the distribution of PI-2a variants was strongly biased and correlated with strain serotype as well as with the presence/absence of PI-I in the same strain As shown in FIG 257, serotype IA strains were predominantly associated with the 515 variant for the backbone protein BP-2a and the ancillary protein API 2a Interestingly, all 31 strains containing the 515 variant belonged to the group of serotype IA isolates that had PI-2a alone, while the remaining 3 serotype IA strains, which were assigned to variant 090, contained PI-2a always in association with PI-I (FIG 256)

Similarly, the presence of PI-2a variant DK21 was restricted to serotype II strains devoid of PI 1 and, interestingly, this allele was found exclusively in serotype II strains from the CDC collection Serotype II strains carrying PI-I as well as PI-2a were associated with variants CJBl 11 or 2603 V/R The same was observed for serotype III isolates These nearly always contained PI-I together with variant 2603 V/R, since this allele was never found in strains with PI-2a alone This also was seen for variant CJBl I l which was found only in strains containing PI-I together with PI-2a, particularly in serotype IB and V strains

Sequence analysis of PI-2b in 40 isolates has shown that the structural components of this island are very conserved In particular, in 35 isolates, all of serotype III or IV, the sequences coding for the PI-2b pilus were 100% identical to those of strain COHl, whereas in the remaining 5 isolates, not belonging to serogroups III or IV, the sequence of the two genes coding for the ancillary proteins (APl-2b and AP2-2b) were 100% identical to the corresponding sequences of the A909 reference strain It is noteworthy that, as was found for the two reference strains, the gene coding for the pilus backbone (BP-2b) shared 100% identity in all 40 isolates (FIG 257, panel C)

Pilus Islands components are surface exposed. Surface exposure of pih components was assessed by flow cytometry (FACS) analysis using antisera specific for the backbone and the major ancillary protein of each PI in intact cells of all 289 GBS isolates The outcome of this analysis was instrumental in determining the relative amount of pilus component exposed on the bacterial surface and, more importantly, for assessing how suitable an antigen would be in protection against invasive GBS strains In fact, it has been established that a 5-fold or greater shift in fluorescence over that observed in the control, stained with preimmune sera, correlates well with protective immunity ( 1 1)

FIG 258 summarizes the results obtained after staining of all GBS isolates with antisera specific for BP-I and APl-I (PI-I), BP-2a and APl-2a (PI-2a), BP-2b and APl-2b (PI-2b) Data are presented showing shifts in fluorescence greater than 2-fold or 5-fold the negative control The lower threshold enables determination of the presence ot the antigen on the bacterial surface, while the higher threshold gives a good correlation with in vivo protection (10) Surface staining with sera specific for PI-I demonstrate that only 59% of the strains containing the island were FACS positive and only approximately half of these exposed the PI-I antigens at high levels (FIG 258) In contrast, the structural components of both PI-2a and PI-2b were surface exposed in over 90% of the strains and most of these showed a greater than 5-fold shift in fluorescence In fact, high surface exposure was observed in 82% of the strains containing PI-2a and 92 5% of those with PI-2b Since more than 70% of the isolates contained two pilus islands, it was important to establish if high surface exposure of components of two pilus types occurred simultaneously in the same strain The results, presented in Table III, highlight how PI-I antigens can be highly exposed on the surface in strains which contain, but do not expose, proteins of PI-2a or PI-2b It is noteworthy that serotype V isolates demonstrated high surface exposure exclusively of PI-I antigens in only 15 of 38 strains In conclusion, a total of 263 isolates, representing 91% of all strains analyzed in this work, expose on the bacterial surface at least one of the three PIs at high level

Each of the three types of pili contains two protective antigens. We have previously demonstrated that pilus components encoded by both PI-I (BP-I and APl-I) and PI-2a (BP-2a and APl-2a) are able to induce protective immunity in mice against GBS infection and that the levels of protection strongly correlate with antigen surface exposure ( 11) To investigate if structural components of PI-2b also elicit protection in vivo, we analyzed the recombinant proteins BP-2b and APl-2b, expressed in E coli as His-tagged fusions, by the active murine maternal immunization-neonatal pup challenge model previously described (11) CD-I female mice were immunized with three doses (days 1, 21, 35) of either 20μg of each antigen or PBS mixed with Freund's adjuvant, then mated and the resulting offspring were infected with a lethal dose of different GBS strains As reported in Table IV, both proteins conferred significant levels of protection against those challenge strains in which the antigens were present and highly exposed on the bacterial surface (> 5-fold shift in fluorescence)

APl-2a variants from Pilus Island 2a are cross-protective. Since antigens encoded from PI-2a (BP-2a and APl-2a, respectively the backbone and the ancillary protein 1) are the only protective pihn proteins showing gene variability, we investigated whether the allelic variants identified were protective not only against strains expressing a homologous protein but also against strains expressing a different variant

We overexpressed the APl-2a variants (2603 and H36B) and three of the six BP-2a variants (2603, 515 and CJBl 11) that together represent more than 80% of the sequenced genes coding for BP-2a Each soluble purified protein was assessed in the mouse model described above using as challenge strains expressing either a homologous or a heterologous variant As reported in Table V, all BP-2a proteins analyzed were able to protect only pups challenged with strains carrying the allelic variant used to immunize their mothers, while protection was not observed against strains expressing a heterologous allele We also tested the in vitro opsonophagocytic activity of sera from mice immunized with the single variants in the presence of human polymorphonuclear leukocytes (PMNs) and baby rabbit complement by using different GBS strains each expressing one allelic variant The results obtained uniformly correlated with the protection data reported above In fact, all sera promoted efficient, complement-dependent opsonophagocytosis and killing by PMNs of only those strains carrying the homologous allele (data not shown) Both GBS 67 variants were cross- protective (Table V), and able to protect the offspring of immunized mice against lethal challenge with strains expressing either homologous or heterologous variants and antisera specific for each allele were able to mediate killing of bacteria expressing both variants (data not shown)

A pilus-based vaccine against GBS infections. We previously have demonstrated that a combination of protective antigens not effective against all strains (either not present or not sufficiently exposed on the bacterial surface) can be useful to develop a broadly effective vaccine against GBS infections (11) Although the six pilus antigens identified so far (two for each pilus type, the backbone and the major ancillary protein) are not universally protective antigens, a combination of all three pill can confer broad protection as demonstrated by this example

In order to obtain the best minimal protein component vaccine formulation, we selected 3 antigens, one protein for each pilus type the backbone components from PI-I (BP-I) and PI 2b (BP-2b) and the ancillary protein 1 from Pl-2a (AP I -2a) In fact, as our antigens are co-expressed in pairs in the same strains the exclusion of one protein for each pilus should not impact the vaccine coverage, but would reduce vaccine complexity Selection criteria were based on gene variability results, on levels of protection in vivo compared with opsonophagocytic activity of each antigen in vitro and, finally, on difficulties of expression and purification Although BP-2a is the main component (the backbone) of pilus type 2a and a very high opsonophagocytic activity was observed in vitro with sera of mice immunized with this single protein, we excluded this antigen due to its high gene variability and because its variants were not cross-protective against each other For the pilus type 1 and type 2b, we excluded APl-I and APl-2b mainly on the basis of the lower levels of protection observed in mice with respect to the corresponding alternative protein The combination of the three selected antigens then was assessed in vivo in the same maternal/neonatal mouse model using a panel of GBS strains each expressing at high levels on the bacterial surface different combinations of pilus-like structures As reported in Table VI, we observed protection against all strains tested with levels ranging from 50% to 100% On the basis of the surface expression data of the three antigens in the collection of 289 isolates analyzed in this study and considering that at least one antigen was highly surface exposed (> 5-fold shift in fluorescence), we estimate the strain coverage of a potential pilus-based vaccine would exceed 91% of the circulating strains assuming that these strains are representative of all invasive GBS strains

Discussion. It has been shown that structural components of pill induce protective immunity in mouse models of GBS (11, 13) To date, three genomic islands coding for pilus-like structures have been identified in GBS (12, 13) However, as these islands are not conserved in all strains, a thorough study of their distribution was necessary to verify their potential as vaccine candidates In this Example, we have analyzed a large number of GBS clinical isolates, mainly from infants and adults invasive infections, in order to assess the distribution of the three pilus-like genomic islands

These represent regions of genomic diversity both in terms of presence/absence in the genome of a given GBS strain as well as for the sequence variability found between the same pilus components in different strains (12) An important finding in our analysis of 289 isolates collected in distant geographic areas was that all contained at least one of the three pilus genomic islands demonstrating that a vaccine with at least one antigen from each pilus island will provide broad protection Furthermore, the locus harboring the PI-2 alleles was never found empty, with PI-2a being the predominant allele (present in 72% of the strains) This indicates that attribution of the pilus genomic island PI-2 to the "dispensable genome" of GBS (12, 15) should be re-defined The finding that PI-2 alleles, different in structure but specifying similar functions (assembly of a pilus), are always present at the same locus, which as a consequence is never empty, suggests that this island represents a "variable" component of the core genome of GBS rather than a "dispensable" part of it Alternatively, the presence of either of the two PI-2 pilus structures is so critical to the pathogenesis of invasive GBS disease that we could not find a single clinical isolate devoid of PI-2 This further indicates that a vaccine with an antigen selected from each PI-2 variant will similarly have broad protective scope

As pili may be important for adhesion and host colonization, a first aim of our study was to verify if there was a correlation between the pilus islands genetic composition in clinical isolates and the type of invasive disease No apparent association was found between the presence/absence of a particular PI and type of disease or carriage This is in agreement with previous reports addressing the same question with regard to different genetic traits of GBS isolates, such as capsular serotypes and phylogeπetic lineages In general, reports in the literature indicate that there is no strong association between capsular loci and type of disease (16) However, there has been consistent and sustained epidemiologic evidence that serotype III strains are strongly associated with early- and late-onset meningitis as well as with late-onset infection irrespective of focus (5, 17) and, in particular, that specific lineages of serotype III GBS strains possibly correlate with early-onset disease (18, 19) Studies on distribution of several virulence factors (20) or pathogenicity islands (21) also failed to establish an absolute correlation between the presence in GBS isolates of a particular genetic determinant and the age at onset or clinical manifestation of disease

In this example, the presence of a particular pilus island allele in a clinical isolate correlates well only with the CPS serotype of the strain Generally, PI-I is rarely present in serotype Ia strains, which contain predominantly only PI- 2a, and is almost exclusively associated with PI-2a in serotype Ib and V strains The presence of PI-2b alleles is restricted mainly to serotype III and IV isolates Thus, in designing a vaccine, antigens from PI-2b are interchangeable with capsular polysaccharides from group II and IV Interestingly, the few cases that do not display this correlation always contain variants of the PI sequences not conserved with respect to those found in the other strains This is particularly true for the PI-2b genomic island, whereas for the PI-2a allele, which shows the broadest distribution and the highest degree of sequence divergence between the different strains, variants of the BP-2a and APl-2a pilus components correlate both with strain serotype and presence in the same strain of the PI-I pilus As an example, when PI-2a is present in conjunction with PI-I in serotype Ia strains, the sequences of BP-2a and APl-2a are identical to the 090 variant, while the presence of PI-2a alone is always associated with the 515 variants of these genes Thus, strains that differ in their PI composition with respect to the strains of the same serotype, invariably contain PI alleles different from those present in the other strains of that serotype

In the context of this work, the analysis of distribution and gene variability of pili in clinical isolates of GBS had the objective of defining the coverage that a pilus-based vaccine against GBS would give by using as antigens pili components that are highly conserved in a wide range of isolates A prerequisite of antigens used to induce protective immunity is that they should be well exposed on the bacterial surface In fact, the levels of protection against GBS infection in murine models strongly correlate with antigen surface exposure (11) Among the 289 clinical isolates analyzed, a relatively low percentage of strains harboring PM (31%) show high surface exposure of the PI-I pilus components, whereas most of the strains containing PI-2a (82%) or PI-2b (92 5%) expose high levels of pilus proteins on the surface The reason for this difference in behavior is unclear and more studies are needed to clarify this point Certainly, the low percentage of strains exposing the PM pilus on the surface cannot be ascribed to sequence diversity of PI-I components in different strains, since this island shows the highest sequence conservation It is more likely that, in our experimental conditions, expression and assembly of pili components are regulated in a different manner for the different types of pili Whatever the reason for this, it is very important to note that 15 serotype V clinical isolates, containing both PM and PI-2a, and 3 serotype III strains, which contain PM in conjunction with PI-2b, demonstrated high surface exposure only of PM components Thus, inclusion of the PM backbone protein in a GBS vaccine should induce protection against a significant number of serotype V GBS infections and therefore are interchangeable with capsular polysaccharides from serotype V The coverage by including the PI-2b backbone protein in a vaccine combination is, instead, substantial for serotype III GBS strains

References for Example 28

1 Telford, J L , M A Barocchi, I Margaπt, R Rappuoli, and G Grandi 2006 Pili in gram-positive pathogens Nat Rev Microbiol 4 509-519

2 Beckmann, C , J D Waggoner, T O Harris, G S Tamura, and C E Rubens 2002 Identification of novel adhesins from Group B streptococci by use of phage display reveals that C5a peptidase mediates fibronectin binding Infect Immun 70 2869-2876

3 Tamura, G S , and C E Rubens 1995 Group B streptococci adhere to a variant of fibronectin attached to a solid phase MoI Microbiol 15 581 589

4 Spellerberg B , E Rozdzinski, S Martin, J Weber-Heynemann, N Schnitzler, R Lutticken, and A Podbielski 1999 Lmb, a protein with similarities to the Lral adhesin family, mediates attachment of Streptococcus agalactiae to human laminin Infect Immun 67 871 878

5 Gibbs, R S S Schrag, and A Schuchat 2004 Perinatal infections due to group B streptococci Obstet Gynecol 104 1062-1076

6 Edwards, M S , and C J Baker 2005 Group B streptococcal infections in elderly adults Clin Infect Dis 41 839-847

7 Benson, J A ₁ A E Flores, C J Baker, S L Hillier, and P Ferπeπ 2002 Improved methods for typing nontypeable isolates of group B streptococci Int J Med Microbiol 292 37-42

8 Ramaswamy, S V , P Ferπeπ, A E Flores, and L C Paoletti 2006 Molecular characteπzation of nontypeable group B streptococcus J Clin Microbiol 44 2398-2403

9 Harrison, L H , J A Elliott, D M Dwyer, J P Libonati, P Ferrieri, L Billmann, and A Schuchat 1998 Serotype distribution of invasive group B streptococcal isolates in Maryland implications for vaccine formulation Maryland Emerging Infections Program J Infect Dis 177 998-1002

10 Baker, C J , D L Kasper, I Tager, A Paredes, S Alpert, W M McCormack, and D Goroff 1977 Quantitative determination of antibody to capsular polysaccharide in infection with type III strains of group B Streptococcus J Clin Invest 59 810-818

11 Maione, D , I Margaπt, C D Rinaudo, V Masignani, M Mora, M Scarselh, H Tettehn, C Brettoni, E T Iacobini, R Rosini, N D'Agostino, L Mioπn, S Buccato, M Mariani, G Galli, R Nogarotto, V N Dei, F Vegni, C Fraser, G Mancuso, G Teti, L C Madoff, L C Paoletti, R Rappuoh, D L Kasper, J L Telford, and G Grandi 2005 Identification of a Universal Group B Streptococcus Vaccine by Multiple Genome Screen Science 309 148-150

12 Tettelin, H , V Masignani, M J Cieslewicz, C Doπati, D Medim, N L Ward, S V Angiuoli, J Crabtree, A L Jones, A S Durkin, R T Deboy, T M Davidsen, M Mora, M Scarselh, Y R I Margaπt, J D Peterson, C R Hauser, J P Sundaram, W C Nelson, R Madupu, L M Bπnkac, R J Dodson, M J Rosovitz, S A Sullivan, S C Daugherty, D H Haft, J Selengut, M L Gwinn, L Zhou, N Zafar, H Khouπ, D Radune, G Dimitrov, K Watkms, J O'Connor K, S Smith, T R Utterback, O White, C E Rubens, G Grandi, L C Madoff, D L Kasper, J L Telford, M R Wessels, R Rappuoh, and C M Fraser 2005 Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae Implications for the microbial "pan-genome" Proc Natl Acad Sci U S A 102 13950-13955

13 Rosini, R , C D Rinaudo, M Soπani, P Lauer, M Mora, D Maione, A Taddei, I Santi, C Ghezzo, C Brettoni, S Buccato, I Margaπt, G Grandi, and J L Telford 2006 Identification of novel genomic islands coding for antigenic pilus-like structures in Streptococcus agalactiae MoI Microbiol 61 126-141

14 Lancefield, R C 1938 Two serological types of group B hemolytic streptococci with related, but not identical, type-specific substances J Exp Med 67 25-40

15 Medim, D , C Donati, H Tettelin, V Masignani, and R Rappuoh 2005 The microbial pan genome Curr Opm Genet Dev 15 589-594

16 Hauge, M , C Jespersgaard, K Poulsen, and M Kilian 1996 Population structure of Streptococcus agalactiae reveals an association between specific evolutionary lineages and putative virulence factors but not disease Infect Immun 64 919-925

17 Weisner, A M , A P Johnson T L Lamagni, E Arnold, M Warner P T Heath, and A Efstratiou 2004 Characterization of group B streptococci recovered from infants with invasive disease in England and Wales Clin Infect Dis 38 1203 1208

18 Jones, N , J F Bohnsack S Takahashi, K A Oliver, M S Chan, F Kunst, P Glaser, C Rusniok D W M Crook, R M Harding, N Bisharat and B G Spratt 2003 Multilocus Sequence Typing System for Group B Streptococcus J Clin Microbiol 41 2530-2536

19 Lin, F -Y C ₁ A Whiting, E Adderson, S Takahashi, D M Dunn, R Weiss, P H Azimi, J B Philips III, L E Weisman, J Regan, P Clark, G G Rhoads, C E Frasch, J Troendle, P Moyer, and J F Bohnsack 2006 Phylogenetic Lineages of Invasive and Colonizing Strains of Serotype III Group B Streptococci from Neonates a Multicenter Prospective Study J Clin Microbiol 44 1257-1261

20 Brochet, M , E Couve, M Zoume, T Vallaeys, C Rusniok, M C Lamy, C Buchπeser, P Tπeu-Cuot F Kunst, C Poyart, and P Glaser 2006 Genomic diversity and evolution within the species Streptococcus agalactiae Microbes Infect 8 1227-1243

21 Herbert, M A , C J Beveπdge, D McCormick, E Aten, N Jones, L A Snyder, and N J Saunders

2005 Genetic islands of Streptococcus agalactiae strains NEM316 and 2603 VR and their presence in other Group B streptococcal strains BMC Microbiol 5 31

22 Montigiani, S , F Falugi, M Scarselh, O Finco, R Petracca, G Galli, M Maπani, R Manetti, M Agnusdei, R Cevenini, M Donati, R Nogarotto, N Norais, I Garaguso, S Nuti, G Saletti, D Rosa, G Ratti, and G Grandi 2002 Genomic approach for analysis of surface proteins in Chlamydia pneumoniae Infect Immun 70 368-379

All publications mentioned in the above specification are herein incorporated by reference Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be covered by the present invention

Claims

Claims

We Claim

1 An immunogenic composition comprising a purified Gram positive bacteria adhesin island (AI) polypeptide in an oligomeric form

2 The immunogenic composition of embodiment 1 wherein the AI polypeptide comprises a sortase substrate motif

3 The immunogenic composition of embodiment 2 wherein the sortase substrate motif is an LPXTG motif 4 The immunogenic composition of embodiment 3 wherein the LPXTG motif is represented by the sequence XPXTG, wherein X at amino acid position 1 is L, I, or F and wherein X at amino acid position 3 is any amino acid residue

5 The immunogenic composition of embodiment 3 wherein the LPXTG motif is represented by XXXXG, wherein X at amino acid position 1 is L, V, E, I, F, or Q, wherein X at ammo acid position 2 is P if X at amino acid position 1 is L, I, or F, wherein X at ammo acid position 2 is V if X at amino acid position 1 is E or Q, wherein X at amino acid position 2 is V or P if X at amino acid position 1 is V, wherein X at amino acid position 3 is any amino acid residue, wherein X at amino acid position 4 is T if X at amino acid position 1 is V, E, I, F, or Q, and wherein X at amino acid position 4 is T, S, or A if X at amino acid position 1 is L

6 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria AI polypeptide affects the ability of Gram positive bacteria to adhere to epithelial cells

7 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria AI polypeptide affects the ability of Gram positive bacteria to invade epithelial cells

8 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria AI polypeptide affects the ability of Gram positive bacteria to translocate through an epithelial cell layer 9 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria AI polypeptide is capable of associating with an epithelial cell surface

10 The immunogenic composition of embodiment 9 wherein the associating with an epithelial cell surface is binding to the epithelial cell surface

11 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria AI polypeptide is a full-length protein

12 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria AI polypeptide is a fragment of a full-length protein

13 The immunogenic composition of embodiment 12 wherein the fragment comprises at least 7 contiguous amino acid residues of the Gram positive bacteria AI protein 14 The immunogenic composition of embodiment 1 wherein the Gram positive bacteria are of a genus selected from the group consisting of Streptococcus, Enterococcus, Staphylococcus, Clostridium, Corynebacterium, or Listeria

15 The immunogenic composition of embodiment 14 wherein the Gram positive bacteria are of the genus Streptococcus 16 The immunogenic composition of embodiment 15 wherein the bacteria are Group B Streptococcus

(GBS) 17 The immunogenic composition of embodiment 16 wherein the AI polypeptide is a GBS Adhesin Island 1 (AI-I) polypeptide

18 The immunogenic composition of embodiment 17 wherein the GBS AI 1 polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof 19 The immunogenic composition of embodiment 17 wherein the AI-I polypeptide is GBS 80

20 The immunogenic composition of embodiment 14 wherein the AI polypeptide is a GBS Adhesin Island 2 (AI-2) polypeptide

21 The immunogenic composition of embodiment 20 wherein the GBS AI-2 polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof 22 The immunogenic composition of embodiment 15 wherein the Gram positive bacteria are Group A

Streptococcus (GAS) polypeptide

23 The immunogenic composition of embodiment 22 wherein the AI polypeptide is a GAS Adhesin Island 1 (GAS Al-I) polypeptide

24 The immunogenic composition of embodiment 23 wherein the GAS AI-I polypeptide is selected from the group consisting of M6_SpyO157, M6_SpyO159, M6_SpyO16O, CDC SS 410_Fimbrιal, ISS3650_fimbπal,

DSM2071_fimbπal, and fragments thereof

25 The immunogenic composition of embodiment 22 wherein the polypeptide is a GAS Adhesin Island 2 (GAS AI-2) polypeptide

26 The immunogenic composition of embodiment 25 wherein the GAS AI-2 polypeptide is selected from the group consisting of GAS15, GAS16, GAS18, and fragments thereof

27 The immunogenic composition of embodiment 20 wherein the AI polypeptide is a GAS Adhesin Island 3 (GAS AI-3) polypeptide

28 The immunogenic composition of embodiment 27 wherein the GAS AI-3 polypeptide is selected from the group consisting of SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPsOlOo, orf78, orf80, orf82, orf84, spyM18_0126, spyM18_0128, spyM18_0130, spyM18_0132, SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, SpyoM01000149, ISS3040_fimbπal, ISS3776_fimbπal, ISS4959_fimbπal, and fragments thereof

29 The immunogenic composition of embodiment 22 wherein the AI polypeptide is a GAS Adhesin Island 4 (GAS AI 4) polypeptide 30 The immunogenic composition of embodiment 29 wherein the GAS AI-4 polypeptide is selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbπal, 20020069_fimbrial, CDC SS 635_fimbπal, ISS4883_fimbπal, ISS4538_fimbπal, and fragments thereof

31 The immunogenic composition of embodiment 22 wherein the AI polypeptide is a GAS Adhesin

Island 5 (AI-5) polypeptide 32 The immunogenic composition of embodiment 31 wherein the GAS AI 5 polypeptide is selected from the group consisting of MGAS10270_Spy0108, MGAS10270_Spy0109, MGAS 10270_Spy0110, MGAS10270_Spy0111, MGAS10270_Spy0112, MGAS10270_Spy0113, MGAS 10270_Spy0114,

MGAS10270_Spy0115, MGAS10270_Spy0116, and MGAS10270_Spy0117

33 The immunogenic composition of embodiment 22 wherein the AI polypeptide is a GAS Adhesin Island 6 (AI 6) polypeptide

34 The immunogenic composition of embodiment 33 wherein the GAS AI-6 polypeptide is selected from the group consisting of MGAS10750_Spy0113, MGAS 10750_Spy01 14, MGAS10750_Spy0115, MGAS10750_Spy01 16 MGAS10750_Spy01 17 MGAS10750_Spy01 18, MGAS 10750_Spy0119, and MGAS10750_SpyOI20

35 The immunogenic composition of embodiment 1 wherein the bacteria are Streptococcus pneumoniae (SP) 36 The immunogenic composition of embodiment 35 wherein the AI polypeptide is selected from the group consisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF3_19FTW, ORF4_19FTW, ORF5_19FTW, ORF3_23FP, ORF4_23FP, ORF5_23FP, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF3_6BF, ORF4_6BF, ORF5.6BF, ORF3_6BSP, ORF4_6BSP, ORF5_6BSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, and fragments thereof

37 The immunogenic composition of any of embodiments 1-36 wherein the oligomeπc form is a hyperoligomer

38 An immunogenic composition comprising a first and a second Gram positive bacteria adhesin island (AI) polypeptide 39 The immunogenic composition of embodiment 38 wherein the Gram positive bacteria are of a genus selected from the group consisting of Streptococcus, Enterococcus, Staphylococcus, Clostridium,

Corynebacterium, or Listeria

40 The immunogenic composition of embodiment 38 wherein the first AI polypeptide is a GBS AI 1 polypeptide 41 The immunogenic composition of embodiment 40 wherein the GBS AI-I polypeptide is selected from the group consisting of GBS 80, GBS 104, GBS 52, and fragments thereof

42 The immunogenic composition of embodiment 38 wherein the first AI polypeptide is a GBS Al-2 polypeptide

43 The immunogenic composition of embodiment 42 wherein the GBS AI 2 polypeptide is selected from the group consisting of GBS 59, GBS 67, GBS 150 01521, 01523, 01524, and fragments thereof

44 The immunogenic composition of embodiment 38 wherein the first AI polypeptide is GBS 80 and the second AI polypeptide is GBS 67

45 The immunogenic composition of embodiment 38 wherein the first AI polypeptide is a Group A Streptococcus (GAS) AI polypeptide 46 The immunogenic composition of embodiment 45 wherein the GAS AI polypeptide is a first GAS

AI-I polypeptide

47 The immunogenic composition of embodiment 46 wherein the first GAS AI-I polypeptide is selected from the group consisting of M6_SpyO157, M6_Spy0159, M6_Spy0160, CDC SS 410_fimbπal,

ISS3650_fimbπal, DSM2071_fimbπal, and fragments thereof 48 The immunogenic composition of embodiment 45 wherein the GAS AI polypeptide is a first GAS

AI-2 polypeptide

49 The immunogenic composition of embodiment 48 wherein the first GAS AI-2 polypeptide is selected from the group consisting of GAS15, GAS 16, GAS 18, and fragments thereof

50 The immunogenic composition of embodiment 45 wherein the GAS AI polypeptide is a first GAS AI 3 polypeptide

51 The immunogenic composition of embodiment 50 wherein the first GAS AI-3 polypeptide is selected from the group consisting of SpyM3_0098, SpyM3_0100, SpyM3_0102, SpyM3_0104, SPsOlOO, SPs0102, SPs0104, SPsOlOo, orf78, orf80, orf82, orf84, spyM18_0126 spyM18_0128, spyM 18_O13O, spyM 18_0132 SpyoM01000156, SpyoM01000155, SpyoMO 1000154, SpyoMO 1000153, SpyoM01000152, SpyoMOlOOOlS l , SpyoM01000150, SpyoM01000149, ISS3040_fimbπal, ISS3776_fimbπal, ISS4959_fimbπal, and fragments thereof

52 The immunogenic composition of embodiment 45 wherein the GAS AI polypeptide is a first GAS AI-4 polypeptide

53 The immunogenic composition of embodiment 52 wherein the First GAS AI-4 polypeptide is selected from the group consisting of 19224134, 19224135, 19224137, 19224139, 19224141, 20010296_fimbπal, 20020069_fimbπal, CDC SS 635_fimbπal, ISS4883_fimbπal, ISS4538_fimbπal, and fragments thereof

54 The immunogenic composition of embodiment 45 wherein the GAS AI polypeptide is a first GAS AI-5 polypeptide

55 The immunogenic composition of embodiment 54 wherein the first GAS AI-5 polypeptide is selected from the group consisting of MGAS 10270_Spy0108, MGAS 10270_Spy0109, MGAS10270_Spy0110, MGAS10270_Spy0111, MGAS10270_Spy0112, MGAS10270_Spy0113, MGAS10270_Spy0114, MGAS10270_Spy0115, MGAS10270_Spy0116, and MGAS10270_Spy0117 56 The immunogenic composition of embodiment 45 wherein the GAS AI polypeptide is a first GAS

AI-6 polypeptide

57 The immunogenic composition of embodiment 56 wherein the first GAS AI-6 polypeptide is selected from the group consisting of MGAS10750_Spy0113, MGAS10750_Spy0114, MGAS10750_Spy0115, MGAS10750_Spy0116, MGAS10750_Spy0117, MGAS10750_Spy0118, MGAS10750_Spy0119, and MGAS10750_Spy0120.

58 The immunogenic composition of any one of embodiments 45-57 wherein the second Gram positive bacteria AI polypeptide is selected from the group consisting of a second GAS AI-I polypeptide, a second GAS AI-2 polypeptide, a second GAS AJ-3 polypeptide, a second GAS AI-4 polypeptide, a second GAS AI-5 polypeptide, and a second GAS AI-6 polypeptide 59 The immunogenic composition of embodiment 38 comprising a first and a second S pneumoniae

AI polypeptide

60 The immunogenic composition of embodiment 59 wherein the first and the second S pneumoniae AI polypeptide are each selected from the group consisting of SP0462, SP0463, SP0464, orf3_670, orf4_670, orf5_670, ORF3_14CSR, ORF4_14CSR, ORF5_14CSR, ORF3_19AH, ORF4_19AH, ORF5_19AH, ORF3_19FTW, ORF4_19FTW, ORF5_19FTW, ORF3_23FP, ORF4_23FP, ORF5.23FP, ORF3_23FTW, ORF4_23FTW, ORF5_23FTW, ORF3_6BF, ORF4.6BF, ORF5.6BF, ORF3_6BSP, ORF4_6BSP, ORF5.6BSP, ORF3_9VSP, ORF4_9VSP, ORF5_9VSP, and fragments thereof

61 The immunogenic composition of embodiment 38 wherein a full length polynucleotide sequence encoding for the first Gram positive bacteria AI polypeptide is not present in a genome of a Gram positive bacteria comprising a full length polynucleotide sequence encoding for the second Gram positive bacteria AI polypeptide

62 The immunogenic composition of embodiment 38 wherein polynucleotides encoding the first and the second Gram positive bacteria AI polypeptide are each present in genomes of more than one Gram positive bacteria serotype and strain isolate

63 The immunogenic composition of embodiment 38 wherein the first and the second Gram positive bacteria AI polypeptides are of different Gram positive bacteria species

64 The immunogenic composition of embodiment 38 wherein the first and the second Gram positive bacteria AI polypeptides are of the same Gram positive bacteria species

65. The immunogenic composition of embodiment 38 wherein the first and the second Gram positive bacteria AI polypeptides are from different AI subtypes.

66. The immunogenic composition of embodiment 38 wherein the first and the second Gram positive bacteria AI polypeptides are from the same AI subtype. 67. The immunogenic composition of embodiment 38 wherein the first Gram positive bacteria AI polypeptide has detectable surface exposure on a first Gram positive bacteria strain or serotype but not a second Gram positive bacteria strain or subtype and the second Gram positive bacteria AI polypeptide has detectable surface exposure on the second Gram positive bacteria strain or serotype but not the first Gram positive bacteria strain or serotype. 68. The immunogenic composition of embodiment 38 wherein the Gram positive bacteria are S. pneumoniae, S. mutans, E. faecalis, E. faecium, C. difficile, L. monocytogenes, or C. diphtheriae.

69. An immunogenic composition comprising one or both of GBS59^DK21 and GBSSg⁰⁸¹¹⁰ polypeptides or fragments thereof.

70. The composition of embodiment 69 wherein the combination comprises GBS59^DK21 and GBS59^αBU0.

71. The immunogenic composition of any of embodiments 1-70 further comprising one or more GBS polypeptides selected from the group consisting of GBS80, GBS 104, GBS67²⁶⁰³, GBS67^H36B, GBS59²⁶⁰³, GBS59^αBI", GBS59⁵¹⁵, GBS59^H36B, 01524 and 01523.

72. The immunogenic composition of any of embodiments 1-71 further comprising one or more polypeptides not selected from an adhesin island.

73. The immunogenic composition of embodiment 72 wherein the one or more polypeptides are selected from the group consisting of: GBS293, GBS65, GBS97, GBS276, GBS84, GBS322, GBS 147 and GBS325.

74. The immunogenic composition of any of embodiments 1-73 wherein the composition further comprising one or more immunoregulatory agents. 75. The immunogenic composition of embodiment 74 wherein the one or more immunoregulatory agents include an adjuvant.

76. The immunogenic composition of any of embodiments 1-75 which is a vaccine.

77. A method for making the composition as defined in embodiment 69 or 70 comprising bringing into association: (a) an immunological effective amount of one or both GBS59^DK21 and GBS59^αB"° polypeptides; and (b) a pharmaceutically acceptable excipient.

78. A modified Gram positive bacterium adapted to produce increased levels of AI surface protein.

79. The modified Gram positive bacterium of embodiment 78 wherein the AI surface protein is in oligomeric form.

80. The modified Gram positive bacterium of embodiment 79 wherein the oligomeric form is a hyperoligomer.

81. The modified Gram positive bacterium of any one of embodiments 78-80 which is a nonpathogenic Gram positive bacterium.

82. The modified Gram positive bacterium of embodiment 81 wherein the non-pathogenic Gram positive bacterium is Lactococcus lactis or 5. gordonii. 83. A method for manufacturing an oligomeric adhesin island (AI) surface antigen comprising: culturing a Gram positive bacterium that expresses an oligomeric AI surface antigen; and isolating the expressed oligomeric AI surface antigen. 84 A method for manufacturing an oligomeric adhesin island (AI) surface antigen comprising cultuπng the Gram positive bacterium of any of embodiments 78-82, and isolating the expressed oligomeric AI surface antigen

85 A method of neutralizing a Streptococcal infection in a mammal comprising the step of administering to the mammal an effective amount of the immunogenic composition of any one of embodiments 1-76 or antibodies which recognize an immunogenic composition as defined in any one of embodiments 1 76

86 The method of embodiment 85 wherein the Streptococcal infection is a GBS infection

87 The method of embodiment 85 wherein the Streptococcal infection is a GAS infection

88 The method of embodiment 85 wherein the Streptococcal infection is a S pneumoniae infection 89 A method of raising an immune response in a mammal against a Streptococcal infection comprising administering to the mammal an effective amount of the immunogenic composition of any one of embodiments 1-76 or antibodies which recognize an immunogenic composition as defined in any one of embodiments 1-76

90 The method of embodiment 89 wherein the Streptococcal infection is a GBS infection 91 The method of embodiment 89 wherein the Streptococcal infection is a GAS infection

92 The method of embodiment 89 wherein the Streptococcal infection is a 5 pneumoniae infection

93 Use of the immunogenic composition according to any one of embodiments 1-76 in the preparation of a medicament for the prevention or treatment of a Streptococcal infection

94 The use of embodiment 93 wherein the Streptococcal infection is a GBS infection 95 The use of embodiment 93 wherein the Streptococcal infection is a GAS infection

The use of embodiment 93 wherein the Streptococcal infection is a S pneumoniae infection