Immunogenic compositions for streptococcus pyogenes

(Cross-reference to related applications for which priority is claimed) This application is incorporated by reference in its entirety, U.S. Patent Provisional Application No. 60 / 491,822 (filed July 31, 2003) and U.S. Patent Provisional Application No. 60 / 541,565 (filed February 3, 2004).

(Field of invention) The present invention belongs to the fields of immunology and vaccination. In particular, the present invention relates to multiple antigens obtained from Streptococcus pyogenes and their use in immunization. All documents cited herein are included herein by reference in their entirety.

(Background of invention) Group A streptococci ("GAS," S. pyogenes) are a common pathogen in humans and are estimated to be present in 5-15% of normal humans without signs of disease. On the other hand, if the host's defense mechanism is at stake, or if the microorganism is able to exert a malignant violence, or if the microorganism is introduced into a vulnerable tissue or host. Acute infections occur. Related diseases include puerperal fever, erythema, erysipelas, pharyngitis, impetigo, necrotizing fasciitis, myositis, and streptococcal toxic shock syndrome.

<p> GAS are Gram-positive, non-spore-forming spore-forming bacteria that usually appear as cells linked in chains or in pairs. Although S. pyogenes can be treated with antibiotics, a prophylactic vaccine is desirable to prevent the onset of the disease. Over the years, efforts have been devoted to the development of such vaccines. Various GAS vaccine methods have been proposed so far, and some of them are currently in clinical trials, but to date, no GAS vaccine is available to the general public.</p>

<p> An object of the present invention is to provide a new and improved composition for immunizing against GAS diseases and / or infectious diseases. The composition preferably contains one of the GAS toxic factors identified by Applicants and others, particularly GAS40 suitable for use as a vaccine. Furthermore, this composition is based on a combination of two or more (eg, three or more) GAS antigens.</p><p> (Outline of the invention) Applicants and others have discovered a GAS antigen group consisting of 30 types of antigens that are particularly suitable for immunization and particularly suitable for use in combination. Furthermore, Applicants and others have identified a GAS antigen (GAS40) that is particularly excellent in immunogenicity, either alone or in combination with another GAS antigen.</p><p> Therefore, the present invention provides an immunogenic composition comprising GAS40 (including a fragment thereof or a polypeptide having the same sequence as the fragment thereof). A preferred fragment of GAS40 comprises one or more coiled coil regions. The present invention further comprises a combination of GAS antigens. This combination consists of 2 to 10 GAS antigens and comprises GAS40, or a fragment thereof, or a polypeptide having the same sequence. The combination preferably consists of 3, 4, 5, 6, or 7 GAS antigens. The combination preferably consists of 3, 4, or 5 GAS antigens.</p><p> The present invention also provides an immunogenic composition comprising a composition comprising a combination of GAS antigens. The combination consists of 2 to 31 GAS antigens of the first antigen group, and the first antigen group is GAS117, GAS130, GAS277, GAS236, GAS40, GAS389, GAS504, GAS509, GAS366, GAS159, GAS217. , GAS309, GAS372, GAS039, GAS042, GAS058, GAS290, GAS511, GAS533, GAS527, GAS294, GAS253, GAS529, GAS045, GAS095, GAS193, GAS137, GAS084, GAS384, GAS202, and GAS057. These antigens are referred to herein as the "first antigen group". The combination of GAS antigens is preferably composed of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The GAS antigen combination preferably comprises 3, 4, or 5 GAS antigens selected from the first antigen group.</p><p> GAS39, GAS40, GAS57, GAS117, GAS202, GAS294, GAS527, GAS533, and GAS511 are particularly preferred GAS antigens. The GAS antigen combination preferably comprises either or both of GAS40 and GAS117. The combination preferably includes GAS40.</p><p> Some representative examples of the above antigen combinations will be discussed below.</p><p> The GAS antigen combination may consist of three GAS antigens selected from the first antigen group. Accordingly, in one embodiment, the GAS antigen combination consists of GAS40, GAS117, and another GAS antigen selected from the first antigen group. Preferred combinations include GAS40, GAS117, and a third GAS antigen selected from the group consisting of GAS39, GAS57, GAS202, GAS294, GAS527, GAS533, and GAS511.</p><p> In another embodiment, the GAS antigen combination comprises GAS40 and two additional GAS antigens selected from the first antigen group. Preferred combinations include GAS40 and two GAS antigens selected from the group consisting of GAS39, GAS57, GAS117, GAS202, GAS294, GAS527, GAS533, and GAS511. In another embodiment, the GAS antigen combination comprises GAS117 and two additional GAS antigens selected from the first antigen group.</p><p> The GAS antigen combination may consist of four GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS40, GAS117, and two additional GAS antigens selected from the first antigen group. A preferred combination consists of two GAS antigens selected from the group consisting of GAS40, GAS117, and GAS39, GAS57, GAS202, GAS294, GAS527, GAS533, and GAS511.</p><p> In another embodiment, the GAS antigen combination comprises GAS40 and three GAS antigens selected from the first antigen group. Preferred combinations include GAS40 and three additional GAS antigens selected from the group consisting of GAS39, GAS57, GAS117, GAS202, GAS294, GAS527, GAS533, and GAS511. In one embodiment, the GAS antigen combination comprises GAS117 and three additional antigens selected from the first antigen group.</p><p> The GAS antigen combination may consist of five GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS40, GAS117, and three additional antigens selected from the first antigen group. A preferred combination consists of GAS40, GAS117 and three additional GAS antigens selected from the group consisting of GAS39, GAS57, GAS202, GAS294, GAS527, GAS533, and GAS511.</p><p> In another embodiment, the GAS antigen combination comprises GAS40 and four additional GAS antigens selected from the first antigen group. Preferred combinations include GAS40 and four additional GAS antigens selected from the group consisting of GAS39, GAS57, GAS117, GAS202, GAS294, GAS527, GAS533, and GAS511. In one embodiment, the GAS antigen combination comprises GAS117 and four additional GAS antigens selected from the first antigen group.</p><p> The GAS antigen combination may consist of eight GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS40, GAS117, and six more GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS40 and an additional seven GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS117 and an additional seven GAS antigens selected from the first antigen group.</p><p> The GAS antigen combination may consist of 10 GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS40, GAS117, and an additional eight GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS40 and an additional nine GAS antigens selected from the first antigen group. In one embodiment, the GAS antigen combination comprises GAS117 and an additional nine GAS antigens selected from the first antigen group.</p><p> (Detailed description of the invention) As discussed above, the present invention provides compositions comprising a combination of GAS antigens. The combination may be selected from a group of antigens identified by Applicants and others as being particularly suitable for immunization, and particularly suitable when used in combination. In particular, the present invention includes compositions containing GAS40.</p><p> The first antigen group, GAS40, and other GAS antigens will be described in more detail below. Genome sequences of at least three GAS species are generally available. The genomic sequence of the M1GAS strain is reported in Ferretti et al., PNAS (2001) 98 (8): 4658-4663. The genomic sequence of the M3GAS strain is reported in Beres et al., PNAS (2002) 99 (15): 10078-10083. The genomic sequence of the M18GAS strain is reported in Smooth et al., PNAS (2002) 99 (7): 4668-4673. The GAS antigen of the present invention preferably contains the polypeptide or amino acid sequence of the M1, M3, or M18 GAS strain. It is more preferred that the GAS antigen of the present invention comprises the polynucleotide or amino acid sequence of the M1 strain.</p><p> Among the identified GAS antigens, GAS Due to variations between M-type and GAS isolates, the GAS amino acid or polynucleotide sequences described in the present invention preferably contain amino acids or polynucleotides having the same sequences. Preferred amino acid or polynucleotide sequences have 50% or more identity (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%). , 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more). Similarly, the GAS amino acid or polynucleotide sequences described in the present invention may contain fragments of those sequences (ie, fragments that retain or encode the immunological properties of the GAS antigen). preferable. Preferred amino acid fragments have n of 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, Contains at least n consecutive amino acids (200, 250 or more). Preferred polynucleotide fragments have n of 12 or more (eg, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 30, 35, 40, 45, 50). , 60, 70, 80, 90, 100, 150, 200, 250 or more) contains at least n consecutive polynucleotides. In one embodiment, the amino acid or polynucleotide fragment of the invention is not identical to an amino acid or polynucleotide derived from another (non-GAS) bacterium (eg, the fragment is not identical to the sequence of another streptococcus). Absent).</p><p> ((1) GAS40) GAS40 matches M1 GenBank registration number GI: 13621545 and GI: 15674449, M3 GenBank registration number GI: 21909733, M18 GenBank registration number GI: 1945402, "Spy0269" (M1), "SpyM3_0197" (M3), "SpyM18_0256" "(M18), and also called" prgA ". GAS40 has also been identified as a putative surface exclusion protein. The amino acid and polynucleotide sequences of GAS40 of M1 species are listed in SEQ ID NOs: 1 and 2 below.<u style="single">As</u>Listed in the sequence list.</p><p><chemistry num="1"><img file="JP4875490B2_D0001.tif" /></chemistry></p><p><chemistry num="2"><img file="JP4875490B2_D0002.tif" /></chemistry> Preferred GAS40 proteins used in accordance with the present invention are (a) 50% or more (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91) relative to SEQ ID NO: 1. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more) of the amino acid sequence and / or (b) SEQ ID NO: 1 In amino acids, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, Contains an amino acid sequence containing fragments consisting of at least n consecutive amino acids (200, 250 or more). These GAS40 proteins include variants of SEQ ID NO: 1 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 1. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 1. One or more amino acids to be lost and / or from the N-terminus of SEQ ID NO: 1 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) To be deleted.</p><p> For example, in one embodiment, the underlined amino acid sequence (leader sequence) in the N-terminal sequence of SEQ ID NO: 1 is removed (the amino acid and polynucleotide sequences of this N-terminal leader sequence are sequenced as SEQ ID NOs: 3 and 4). Listed. The amino acid and polynucleotide sequences of the remaining GAS40 fragments are listed as SEQ ID NOs: 5 and 6).</p><p> As another example, in one embodiment, the C-terminal underlined amino acid sequence (transmembrane region) of SEQ ID NO: 1 is removed (the amino acid and polynucleotide sequences in this transmembrane region are SEQ ID NOs: 7 and 8). The amino acid and polynucleotide sequences of the remaining GAS40 fragments are listed as SEQ ID NOs: 9 and 10. ).</p><p> In other fragments, one or more domains of the protein may be omitted (eg, signal peptide, plasma domain, transmembrane domain, extracellular domain omitted).</p><p> More detailed illustrations of the domains inside GAS40 are shown in Figures 1 and 2. As shown in these figures, the amino acid of GAS40 (SEQ ID NO: 1) is the leader peptide sequence within amino acid 1-26 (eg, SEQ ID NO: 3) and the first coiled coil within amino acids 58-261 (SEQ ID NO: 12). , Contains a second coiled coil within amino acids 556-733 (SEQ ID NO: 13), a leucine zipper region within amino acids 673-701 (SEQ ID NO: 14), and a transmembrane region within amino acids 855-866 (SEQ ID NO: 11). .. FIG. 1 shows these regions in the amino acid sequence of GAS40, while FIG. 2 schematically shows the regions along the long axis of the GAS40 protein.</p><p> The coiled coil region identified inside the GAS40 appears to form an alpha helix coil. Such structures are often involved in oligomerization interactions, for example, between different regions of a protein or between regions of two separate proteins. The leucine zipper motif in the second coiled coil region contains a series of leucine (or isoleucine) amino acid residues separated from each other so that a special oligomeric interaction can be formed between the two alpha helices. In the leucine zipper motif, it is preferable that six amino acid residues are sandwiched between the leucine residues that repeatedly appear. In the leucine zipper oligomer structure, the alpha helix is believed to be linked by a hydrophobic interaction between leucine residues located on one side of each helix. Leucine zipper motifs are often involved in dimer-forming interactions. The position of the leucine zipper motif inside the coiled coil region indicates that the region of the GAS40 protein is likely to be involved in the oligomerization interaction.</p><p> Figure 2 also shows that the identified regions of GAS40 have regions with low homology to other streptococcal proteins with known or predicted two-dimensional structures or surface arrangements. This low level of homology may indicate some similar secondary structure, or function. For example, amino acids 33-324 of GAS40, including the first coiled coil region, are regions with proteins derived from Streptococcus gordonii (amino acids 112-392), called streptococcal surface protein A (SpA) precursors (see Genbank GI25990270). , It has almost 22% sequence homology with SEQ ID NO: 15). This protein is considered to be a surface protein adhesive involved in the adhesion of the streptococcus to the mammalian host cell membrane. This S. gordonii SpA is a member of the streptococcal antigen I / II family of protein deposits and recognizes salivary aglutinin glycoprotein (gp-340) and type I collagen. Amino acids 33-258 of GAS40 also show low levels of sequence homology (23%) with another S. gordonii protein, a streptococcal surface protein B precursor (Genbank reference GI25055226, SEQ ID NO: 16).</p><p> A similar region of GAS40 (amino acid 43-238) that also overlaps the first coiled coil region is a region of protein derived from Streptococcus pneumoniae (amino acid 43-238) (Genbank reference GI282335, SEQ ID NO:) called the surface protein pspA precursor. It shows about 23% homology to 17). The amino-terminal domain of pspA is believed to be essential for streptococcus to be sufficiently virulent, whereas monoclonal antibodies protect mice from streptococcal infection. The pspA domain has an axial monomeric form with a ratio of about 1:12, typical of fibrous proteins. Sequence analysis showed that this monomer molecule had an alpha-helix type coiled coil structure with only a few loop seams in the helix.</p><p> The second coiled coil region of GAS40 is a region with a protein derived from Streptococcus equi called immunoreactive protein Se89.9 (amino acid 509-717) (see Genbank GI2330384, SEQ ID NO: 18) (The full length sequence of Se89.9 is , Also available from http://pedant.gsf.de) and has approximately 46% sequence homology. This Streptococcus equi protein is predicted to be surface-exposed. Figure 3 shows an alignment diagram of each of these streptococcal sequences and GAS40 by BLAST.</p><p> Further, an explanatory diagram of the two-dimensional structure of GAS40 is shown in FIG. First, FIG. 4 (a) shows a predictive analysis of secondary structure aligned with the amino acid sequence of GAS40. In FIG. 4, the predicted alpha helix region almost coincides with the coiled coil region described above. In FIG. 4 (b), the position of the coiled coil candidate was predicted using the pair coil prediction. In this figure, two coils are identified that roughly correspond to the first and second coiled coil regions. FIG. 4 (c) shows the leucine zipper region extracted and shows the regular repetition of leucine (or isoleucine) amino acid residues that are thought to be involved in the leucine zipper.</p><p> From the above, the first coiled coil region of GAS40 is at least 10 selected from the N-terminal half of the full length array of GAS40 (for example, at least 10, 13, 15, 18, 20, 25, 30, 35, It contains 40, 50, 70, 90, 100 or more) contiguous amino acid residues, and contains an amino acid sequence that is predicted to form an alpha-helix complex based on the functional characteristics of the amino acid residues in the sequence. SEQ ID NO: 12 shows the preferred first coiled coil region of GAS40.</p><p> The second coiled coil region of GAS40 is at least 10 selected from the C-terminal half of the full length array of GAS40 (eg, at least 10, 13, 15, 18, 20, 25, 30, 35, 40, 50, 50, Contains 70, 90, 100 or more) contiguous amino acid residues and contains an amino acid sequence that is predicted to form an alpha-helix complex based on the functional characteristics of the amino acid residues in the sequence. The second coiled coil region preferably contains a leucine zipper motif. SEQ ID NO: 13 shows the preferred second coiled coil region of GAS40.</p><p> The coiled coil region of GAS40 appears to be involved in the formation of oligomers such as dimers or trimmers. This oligomer can be a homomer (containing two or more GAS40 proteins involved in oligomerization) or a heteromer (containing one or more additional GAS proteins oligomerized with GAS40). Good. Alternatively, the first coiled coil region and the second coiled coil region may interact within the GAS40 protein to form an oligomeric reaction between the first coiled coil region and the second coiled coil region.</p><p> Thus, in one embodiment, the composition of the invention comprises a GAS40 antigen in the form of an oligomer. This oligomer may contain two or more GAS40 antigens or fragments thereof, or may contain GAS40 or fragments thereof that oligomerize with a second GAS antigen. A preferred GAS40 fragment comprises an amino acid sequence selected from the group consisting of a first coiled coil region and a second coiled coil region. This preferred GAS40 fragment may be used alone or in combination as in the present invention.</p><p> The GAS polynucleotides and amino acid sequences of the present invention may be engineered to promote or optimize recombinant expression. For example, the N-terminal leader sequence may be replaced with a sequence encoding a tag protein, such as polyhistidine (HIS), or glutathione S-transferase (GST). Such tag proteins can be used to facilitate purification, detection, and stabilization of expressed proteins. Various variants of such modifications for GAS40 are discussed below. Such modifications may be applied to any GAS protein of the invention.</p><p> An example of a GAS40 sequence tagged with two tags, GST and HIS, is referred to herein as "GST40HIS". This construct is GAS40 with the leader sequence removed, the GST tag coding sequence added to the N-terminus (eg, using the pGEXNNH vector containing the NdeI and NotI restriction sites), and the HIS tag coding sequence added to the C-terminus. Contains an array. The fusion region of the GST tag, the GAS40 sequence, and the polynucleotide and amino acid sequences of the C-terminal HIS tag of GST40HIS are shown in SEQ ID NOs: 19 and 20.</p><p> Alternatively, a single tag sequence may be used. An example of a GAS40 sequence with only HIS tags is displayed as "40a-HIS". This construct contains an N-terminal leader sequence and a GAS40 sequence with the C-terminus removed, including a transmembrane sequence. In this construct, the HIS tag sequence is added to the C-terminus (eg, using a cloning vector such as pET21b + (Novagen) containing NdeI and NotI restriction sites). The polynucleotide and amino acid sequences of 40a-HIS are shown in SEQ ID NOs: 21 and 22.</p><p> In addition to the addition of the purified tag, the recombinant expression may be facilitated by further optimizing such as increasing the coding sequence in a larger amount or improving access to the recombinant host. For example, the polynucleotide sequence AGA encodes an arginine amino acid residue, whereas arginine can also be encoded by the polynucleotide sequence CTG. This CTG codon is preferred by the E. coli transcription enzyme. In SEQ ID NO: 21 of the 40a-HIS polynucleotide sequence, the C-terminal CTG encoding arginine is replaced with CGT.</p><p> The following codons, namely AGA, AGG and CGA, are generally less reproducible in E. coli. If these codons appear in the GAS polynucleotide sequence, they may be replaced by one of the other two alternative codons encoding the same amino acid residue.</p><p> In the "40a-RR-HIS" construct, SEQ ID NOs: 23 and 24, a total of 3 ATG codons have been replaced with CTG for optimization. Also, SEQ ID NO: 23 with the optimized codons underlined is shown below (40a-RR-HIS is the same as 40a-HIS, except for additional codon optimization).</p><p><chemistry num="3-1"><img file="JP4875490B2_D0003.tif" /></chemistry></p><p><chemistry num="3-2"><img file="JP4875490B2_D0004.tif" /></chemistry> Codon optimization may be used without a purification tag. Constructs "40a-RR-Nat", SEQ ID NOs: 25 and 26 are such examples. This construct does not contain the N-terminal leader sequence and the C-terminal transmembrane sequence, but contains GAS40 (and does not contain the HIS tag sequence), which contains three codon optimizations.</p><p> Various cloning vectors can be used to optimize expression in different host cells under different culture conditions. For the above-mentioned construct, pET21b + (Novagen) containing an IPTG-induced promoter was used. Alternatively, an E. coli / B. subtilis expression shuttle vector, such as pSM214gNH, may be used. This vector uses a constitutive promoter instead of the IPTG-induced promoter. An example of a GAS40 construct using this vector is labeled "HIS-40a-NH" and its sequence is shown in SEQ ID NOs: 27 and 28. In this construct, the N-terminal leader sequence and the C-terminal transmembrane sequence are removed and a HIS tag is added to the N-terminus. Yet another N-terminal amino acid is introduced by cloning. In addition, two nucleotide changes, probably occurring during PCR, are shown, neither of which results in amino acid changes.</p><p> As yet another embodiment, the pSM214gCH shuttle vector may be used. An example of a GAS40 construct using this vector is displayed as "HIS-40a-CH" and SEQ ID NOs: 29 and 30. In this construct, the N-terminal leader sequence and the C-terminal transmembrane sequence are removed and the HIS tag is<u style="single">C</u>Added to the end. Two more amino acids are introduced at the amino terminus. The three nucleotide changes introduced by cloning are shown in the DNA sequence, and the resulting amino acid changes are shown in the protein sequence (amino acids F to S).</p><p> Codon optimization can also be used for these different cloning vectors. The GAS40 construct "HIS-40a-RR-NH" contains "HIS-40a-NH" which contains three codon optimization sites. The sequence of HIS-40a-RR-NH is set forth in SEQ ID NOs: 31 and 32.</p><p> From the above, the GAS antigen used in the present invention may be produced by a recombinant technique using an expression construct that promotes recombinant production. Preferred sequence modifications to promote expression may be selected from the group consisting of (1) addition of purified tag sequences and (2) codon optimization.</p><p> As mentioned above, Applicants and others have identified GAS40 as particularly suitable for use as an immunogenic composition, whether alone or in combination. The use of GAS40 as a particularly effective GAS antigen is supported by its toxicity association, surface localization, and its effectiveness in bacterial opsonin phagocytosis assays and immunostimulatory experiments. In addition, the antigen is likely to be specific to GAS (compared to other streptococci), probably because the virulence factor was acquired horizontally. Yet another evidence supporting the antigenic properties of GAS40 is that coiled-coil regions have been identified within the two-dimensional structure of GAS40, and that these regions are found in other streptococci, including some adherent proteins. It has a low level of homology with surface proteins.</p><p> When the position of GAS40 in the Streptococcus pyogenes genome was analyzed by Applicants and others, this toxic factor was found to be horizontal gene transfer during evolution.<u style="single">propagation</u>As a result, it seems that it was acquired by GAS. FIG. 5A shows GAS40 within the GAS genome. GAS40 is a "purine operon repressor" or "pur" on the 5'end side.<u style="single">R</u>Comes next to the array displayed. On the 3'end side, next to GAS40 are the two sequences encoding the ribosomal protein, namely the sequences labeled "ribosome protein S12" or "rpsL" and "ribosome protein S7" or "rpsG". Is. (Amino acid and polynucleotide sequences for these flanking genes are available from GenBank publications. (PurR sequences can be found, for example, in Genbank reference GI: 15674250.<u style="single">The RpsL sequence can be found, for example, at Genbank reference GI: 15674250.</u>The RpsG sequence can be found, for example, at Genbank reference GI: 15674250. Of note, there are two expected promoter sequences at the origin of the rpsL sequence. FIG. 5B shows a GAS mutation in which most of GAS40 has been deleted. The only remaining portion of the GAS40 sequence corresponds to polynucleotide 1-97 of SEQ ID NO: 2. The deletion also included one of the rpsL promoters, leaving the second P * intact. (The horizontal arrow below the schematic diagram indicates the deletion region).</p><p> Figure 5C shows yet another detail of the wild-type GAS sequence. In this figure, the direct repetitive sequence labeled "DR" is shown adjacent to the 5'end and 3'end of GAS40. (The corresponding sequence of the deletion mutation in GAS40 is identified in Figure 5D). These direct repeats are about 8 base pairs. An example of such a base pair direct repeat sequence is shown in SEQ ID NO: 136. Such sequence motifs in the bacterial genome are often horizontal gene transfer.<u style="single">propagation</u>Is shown. In vivo infection experiments have shown that GAS40 deletion mutations are several logs less toxic than wild-type strains.<u style="single">(Details of this experiment are provided in Example 2.)</u></p><p> The combination of flanking direct repeats with GAS40-related toxicity suggests that the GAS40 sequence was horizontally acquired by Streptococcus pyogenes during evolution. It should be noted that the associated purR and rpsL are present in the similar streptococcus Streptococcus agalactiae and Streptococcus mutants, but none of these bacteria have a GAS40 homology (Fig. 5E, S. a. It schematically shows the location of purR, rpsL, and rpsG homologues in agalactiae (Group B streptococcus, GBS), and shows the percent homology between GBS homologues and the corresponding homologues of GAS. , GBS does not contain side-adjacent direct repeat sequence pairs of the GAS40 sequence by having only one of the direct repeat sequences.)</p><p> The surface position of GAS40 is indicated by the FACS diagram shown in FIG. (Discussion on protocols related to FACS analysis is presented in Example 1). FIG. 6 contains FACS diagrams of both wild-type GAS (denoted as DSM2071, M23 type of GAS) and deletion mutation (DSM2071 40). Absorption shifts in wild-type strains indicate that GAS40 was recognized on the bacterial surface by anti-GAS40 antibodies (GAS40 is not recognized on the surface of deletion mutations).</p><p> Surface exposure of GAS40 is further demonstrated by the bacterial opsonin phagocytosis assay shown in Figure 7 and Example 3. In this assay, GAS strains were incubated with pre-immune and immune sera, polynuclear cells, and complement. (Immune serum was produced by immunizing mice with the indicated GAS protein). Bacterial phagocytosis or growth was measured logarithmically. Positive histogram bars represent phagocytosis (ie cell death). Negative histogram bars represent cell proliferation. As shown in FIG. 7, the immune sera produced by each GAS40-expressing protein leads to bacterial depletion (positive histogram bar).</p><p> Immunization induction experiments with GAS40 are discussed in detail in Example 4. As seen in this example, GAS40 obtained with various constructs provides substantial protection in adult mice. Notably, many GAS40s provide about the same degree of protection as the GAS M protein. (GAS M protein was used for comparison as it is known to be highly immunogenic. However, M protein is generally not considered a suitable GAS vaccine candidate, because this protein. Is highly variable in GAS strains and may have epitopes that are cross-reactive with human tissues.) In addition, the N-terminal cut of GAS40<u style="single">Piece</u>Also gave significant protection in this model. This N-terminal fragment contains approximately 292 amino acids from the overlap between the N-terminus of GAS40 and the first coiled coil region. "40N-HIS" (SEQ ID NOs: 33 and 34) is an example of this GAS40 fragment, which includes the coiled coil region of GAS40 and the C-terminal HIS tag.</p><p> ((2) GAS117) GAS117 corresponds to M1, GenBank registration number GI: 13621679 and GI: 15674571, M3, GenBank registration number GI: 21909852, M18, GenBank registration number GI: 19745578, and also "Spy0448" (M1), "SpyM3_0316" ( Also called M3), and "Spy M18_0491" (M18). Examples of the amino acid and polynucleotide sequences of GAS117 of the M1 strain are set forth in SEQ ID NOs: 35 and 36.</p><p> Preferred GAS117 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 35 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 35, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids.<u style="single">These GAS117 proteins include variants of SEQ ID NO: 35 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes).</u>The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 1. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 35. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 35 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 35 (shown below) is removed. (SEQ ID NO: 37 contains the removed N-terminal amino acid sequence). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="4"><img file="JP4875490B2_D0005.tif" /></chemistry> ((3) GAS130) GAS130 supports M1, GenBank registration number GI: 13621794 and GI: 15674677, M3, GenBank registration number GI: 21909954, M18, GenBank registration number GI: 19745704, and also "Spy0591" (M1), "SpyM3_0418" ( Also called M3), and "Spy M18_0660" (M18). GAS130 may be identified as a protease. Examples of the amino acid and polynucleotide sequences of GAS130 of the M1 strain are set forth in SEQ ID NOs: 39 and 40.</p><p> The preferred GAS130 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 39 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) SEQ ID NO: 3<u style="single">9</u>N is 7 or more in the amino acids of (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100).<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS130 proteins include variants of SEQ ID NO: 39 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 39. Other preferred fragments are SEQ ID NO: 3<u style="single">9</u>One or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from the C-terminus of, and / or SEQ ID NO: 3<u style="single">9</u>Delete one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((4) GAS277) GAS277 corresponds to M1, GenBank registration number GI: 13622962 and GI: 15675742, M3, GenBank registration number GI: 21911206, M18, GenBank registration number GI: 19746852, and also "Spy1939" (M1), "SpyM3_1670" ( Also called M3), and "SpyM18_2006" (M18). Examples of the amino acid and polynucleotide sequences of GAS277 of the M1 strain are set forth in SEQ ID NOs: 41 and 42.</p><p> Preferred GAS277 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 41 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 41, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS277 proteins include variants of SEQ ID NO: 41 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 41. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 41. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 41 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 41 (shown below) is removed. (SEQ ID NO: 43 contains the removed N-terminal amino acid sequence; SEQ ID NO: 44 contains a fragment of GAS277 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="5"><img file="JP4875490B2_D0006.tif" /></chemistry> ((5) GAS236) GAS236 corresponds to M1, GenBank registration number GI: 13622264 and GI: 15675106, M3, GenBank registration number GI: 21910321, M18, GenBank registration number GI: 1946075, and also "Spy1126" (M1), "SpyM3_0785" ( Also called M3), and "Spy M18_1087" (M18). Examples of the amino acid and polynucleotide sequences of GAS236 of the M1 strain are set forth in SEQ ID NOs: 45 and 46.</p><p> Preferred GAS236 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 45 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 45, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS236 proteins include variants of SEQ ID NO: 45 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 45. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 45. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 45 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 45 (shown below) is removed. (SEQ ID NO: 47 contains the removed N-terminal amino acid sequence; SEQ ID NO: 48 contains a fragment of GAS236 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="6"><img file="JP4875490B2_D0007.tif" /></chemistry> ((6) GAS389) GAS389 supports M1, GenBank registration number GI: 13622996 and GI: 15675772, M3, GenBank registration number GI: 21911237, M18, GenBank registration number GI: 19746884, and also "Spy1981" (M1), "SpyM3_1701" ( Also called M3), and "Spy M18_2045" (M18) and "relA". GAS389 has also been identified as (p) ppGpp synthase. Examples of the amino acid and polynucleotide sequences of GAS389 of the M1 strain are set forth in SEQ ID NOs: 49 and 50.</p><p> Preferred GAS389 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 49 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 49, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200、250</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids.<u style="single">These GAS389 proteins include variants of SEQ ID NO: 49 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes).</u>The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 49. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 49. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 35 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((7) GAS504) GAS504 corresponds to M1, GenBank registration number GI: 13622806 and GI: 15675600, M3, GenBank registration number GI: 21911061, M18, GenBank registration number GI: 19476708, and also "Spy1751" (M1), "SpyM3_1525", Also known as "Spy M18_1823" (M18) and "fabK". GAS504 has also been identified as the expected trans-2-enoyl-ACP reductase II. Examples of the amino acid and polynucleotide sequences of GAS504 of the M1 strain are set forth in SEQ ID NOs: 51 and 52.</p><p> Preferred GAS504 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 51 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 51, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS504 proteins include variants of SEQ ID NO: 51 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 51. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 51. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 51 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((8) GAS509) GAS509 supports M1, GenBank registration number GI: 13622692 and GI: 15675496, M3, GenBank registration number GI: 21910899, M18, GenBank registration number GI: 19746544, and also "Spy1618" (M1), "SpyM3_1363".<u style="single">(M3)</u>, Also called "SpyM18_1627" (M18) and "cysM". GAS509 has also been identified as the expected O-acetylserine lyase. Examples of the amino acid and polynucleotide sequences of GAS509 of the M1 strain are set forth in SEQ ID NOs: 53 and 54.</p><p> Preferred GAS509 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 53 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 53, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS509 proteins include variants of SEQ ID NO: 53 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 53. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 53. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 53 To be deleted. For example, in one embodiment, SEQ ID NO: 53<u style="single">C</u>The terminal underlined amino acid sequence (shown below) is removed. (SEQ ID NO: 55 has been removed<u style="single">C</u>Contains the terminal amino acid sequence. SEQ ID NO: 56<u style="single">C</u>Contains a fragment of GAS509 with the terminal amino acid sequence removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="7"><img file="JP4875490B2_D0008.tif" /></chemistry> ((9) GAS366) GAS366 supports M1, GenBank registration number GI: 13622612, GI: 15675424 and GI: 30315979, M3, GenBank registration number GI: 21910712, M18, GenBank registration number GI: 19746474, and also "Spy1525" (M1), Also called "SpyM3_1176" (M3), "SpyM18_1542" (M18) and "murD". GAS366 has also been identified as UDP-N-acetylmuramoylalanine-D-glutamate ligase, or D glutamate deductase. Examples of the amino acid and polynucleotide sequences of GAS366 of the M1 strain are set forth in SEQ ID NOs: 57 and 58.</p><p> The preferred GAS366 used in accordance with the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 57 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 57, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS366 proteins include variants of SEQ ID NO: 57 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 57. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 57. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 57 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 57 (shown below) is removed. (SEQ ID NO: 59 contains the removed N-terminal amino acid sequence; SEQ ID NO: 60 contains a fragment of GAS366 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="8"><img file="JP4875490B2_D0009.tif" /></chemistry> ((10) GAS159) GAS159 supports M1, GenBank registration number GI: 13622244, and GI: 15675588, M3, GenBank registration number GI: 21910303, M18, GenBank registration number GI: 1946056, and also "Spy1105" (M1), "SpyM3_0767". Also called (M3), "SpyM18_1067" (M18) and "potD". GAS159 has also been identified as the expected spellidine / putrescine ABC transporter (protoplasmic peritransport protein). Examples of the amino acid and polynucleotide sequences of GAS159 of the M1 strain are set forth in SEQ ID NOs: 61 and 62.</p><p><chemistry num="9"><img file="JP4875490B2_D0010.tif" /></chemistry></p><p><chemistry num="10"><img file="JP4875490B2_D0011.tif" /></chemistry> The preferred GAS159 used in accordance with the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 61 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 61, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS159 proteins include variants of SEQ ID NO: 61 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 61. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 61. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 61 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 61 (shown below) is removed. (SEQ ID NO: 63 contains the removed N-terminal amino acid sequence; SEQ ID NO: 64 contains a fragment of GAS159 from which the N-terminal amino acid sequence has been removed). In another embodiment, of SEQ ID NO: 61<u style="single">C</u>The terminal underlined amino acid sequence is removed. (SEQ ID NO: 65 contains a C-terminal hydrophobic region. SEQ ID NO: 66 contains a fragment of GAS159 with the C-terminal hydrophobic region removed. SEQ ID NO: 67 is an N-terminal leader sequence and a C-terminal hydrophobic region. Includes a fragment of GAS159 from which Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="11"><img file="JP4875490B2_D0012.tif" /></chemistry> ((11) GAS217) GAS217 supports M1, GenBank registration number GI: 13622089 and GI: 15674945, M3, GenBank registration number GI: 21910174, M18, GenBank registration number GI: 19745987, and also "Spy0925" (M1), "SpyM3_0638" ( Also called M3), "Spy M18_0982" (M18). GAS217 has also been identified as a expected redox enzyme. Examples of the amino acid and polynucleotide sequences of GAS217 of the M1 strain are set forth in SEQ ID NOs: 68 and 69.</p><p> Preferred GAS217 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 68 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 68, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS217 proteins include variants of SEQ ID NO: 68 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 68. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 68. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 68 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((12) GAS309) GAS309 corresponds to M1, GenBank registration number GI: 13621426 and GI: 15674341, M3, GenBank registration number GI: 21909633, M18, GenBank registration number GI: 19745363, and also "Spy0124" (M1), "SpyM3_0097" ( Also called M3), "SpyM18_0205" (M18), "nra" and "rofA". GAS309 has also been identified as a regulatory protein and a negative transcriptional regulator. Examples of the amino acid and polynucleotide sequences of GAS309 of the M1 strain are set forth in SEQ ID NOs: 70 and 71.</p><p> Preferred GAS309 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 70 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 70, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS309 proteins include variants of SEQ ID NO: 70 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 70. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 70. Lose and / or one or more amino acids from the N-terminus of SEQ ID NO: 70 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((13) GAS372) GAS372 supports M1, GenBank registration number GI: 13622698 and GI: 15675501, M3, GenBank registration number GI: 21910905, M18, GenBank registration number GI: 19476500, and also "Spy1625" (M1), "SpyM3_1369" ( Also called M3), "Spy M18_1634" (M18). GAS372 has also been identified as a predicted protein kinase, or a predicted eukaryotic serine / threonine kinase. The amino acid and polynucleotide sequences of GAS372 of the M1 strain are set forth in SEQ ID NOs: 72 and 73.</p><p> Preferred GAS372 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 72 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 72, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200、250</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS372 proteins include variants of SEQ ID NO: 72 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 72. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 72. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 72 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((14) GAS039) GAS039 corresponds to M1, GenBank registration number GI: 13621542 and GI: 15674446, M3, GenBank registration number GI: 21909730, M18, GenBank registration number GI: 19745398, and also "Spy0266" (M1), "SpyM3_0194" ( Also called M3), "Spy M18_0250" (M18). The amino acid and polynucleotide sequences of GAS039 of the M1 strain are set forth in SEQ ID NOs: 74 and 75.</p><p> Preferred GAS039 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 74 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 74, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS039 proteins include variants of SEQ ID NO: 74 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 74. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 74. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 74 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((15) GAS042) GAS042 supports M1, GenBank registration number GI: 13621559 and GI: 15674461, M3, GenBank registration number GI: 21909745, M18, GenBank registration number GI: 19745415, and also "Spy0287" (M1), "SpyM3_0209" ( Also called M3), "Spy M18_0275" (M18). The amino acid and polynucleotide sequences of GAS042 of the M1 strain are set forth in SEQ ID NOs: 76 and 77.</p><p> Preferred GAS042 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 76 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 76, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS042 proteins include variants of SEQ ID NO: 76 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 76. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 76. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 76 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((16) GAS058) GAS058 corresponds to M1, GenBank registration number GI: 13621663, GI: 15674556, M3, GenBank registration number GI: 21909841, M18, GenBank registration number GI: 19745567, and also "Spy0430" (M1), "SpyM3_0305" ( Also called M3), and "Spy M18_0477" (M18). Examples of the amino acid and polynucleotide sequences of GAS058 of the M1 strain are set forth in SEQ ID NOs: 78 and 79.</p><p> The preferred GAS058 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 78 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 78, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS058 proteins include variants of SEQ ID NO: 78 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 78. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 78. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 78 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 78 (shown below) is removed. (SEQ ID NO: 80 contains the removed N-terminal amino acid sequence; SEQ ID NO: 81 contains a fragment of GAS058 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="12"><img file="JP4875490B2_D0013.tif" /></chemistry> ((17) GAS290) GAS290 supports M1, GenBank registration number GI: 13622978 and GI: 15675757, M3, GenBank registration number GI: 21911221, M18, GenBank registration number GI: 19746869, and also "Spy1959" (M1), "SpyM3_1685" ( Also called M3), "Spy M18_2026" (M18). The amino acid and polynucleotide sequences of GAS290 of the M1 strain are set forth in SEQ ID NOs: 82 and 83.</p><p> The preferred GAS290 used in accordance with the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 82 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 82, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS290 proteins include variants of SEQ ID NO: 82 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 82. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 82. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 82 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((18) GAS511) GAS511 supports M1, GenBank registration number GI: 13622798 and GI: 15675592, M3, GenBank registration number GI: 21911053, M18, GenBank registration number GI: 19476700, and also "Spy1743" (M1), "SpyM3_1517" ( Also called M3), "Spy M18_1815" (M18) and "accA". The amino acid and polynucleotide sequences of GAS511 of the M1 strain are set forth in SEQ ID NOs: 84 and 85.</p><p> Preferred GAS511 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 84 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 84, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS511 proteins include variants of SEQ ID NO: 84 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 84. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 84. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 84 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((19) GAS533) GAS533 corresponds to M1, GenBank registration number GI: 13622912 and GI: 15675696, M3, GenBank registration number GI: 21911157, M18, GenBank registration number GI: 19746804, and also "Spy1877" (M1), "SpyM3_1621" ( Also called M3), "Spy M18_1942" (M18) and "glnA". GAS533 is also identified as the expected glutamine synthetase. The amino acid and polynucleotide sequences of GAS533 of the M1 strain are set forth in SEQ ID NOs: 86 and 87.</p><p> Preferred GAS533 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 86 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 86, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS533 proteins include variants of SEQ ID NO: 86 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 86. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 86. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 86 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((20) GAS527) GAS527 supports M1, GenBank registration numbers GI: 13622332, GI: 15675169, and GI: 24217164, M3, GenBank registration numbers GI: 21910381, M18, GenBank registration numbers GI: 19476136, and also "Spy1204" (M1). , Also called "SpyM3_0845" (M3), "SpyM18_1155" (M18) and "guaA". GAS527 is also identified as GMP synthase (glutamate hydrolyzable) (glutamate amide transferase). The amino acid and polynucleotide sequences of GAS527 of the M1 strain are set forth in SEQ ID NOs: 88 and 89.</p><p> Preferred GAS527 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 88 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 88, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS527 proteins include variants of SEQ ID NO: 88 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 88. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 88. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 88 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((21) GAS294) GAS294 corresponds to M1, GenBank registration number GI: 13622306, GI: 15675145, and GI: 26006773, M3, GenBank registration number GI: 21910357, M18, GenBank registration number GI: 19746111, and also "Spy1173" (M1). , Also called "SpyM3_0821" (M3), "SpyM18_1125" (M18) and "gid". GAS294 is also identified as a glucose-suppressing compartment protein. The amino acid and polynucleotide sequences of GAS294 of the M1 strain are set forth in SEQ ID NOs: 90 and 91.</p><p> Preferred GAS294 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 90 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 90, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS294 proteins include variants of SEQ ID NO: 90 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 90. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 90. Lose and / or one or more amino acids from the N-terminus of SEQ ID NO: 90 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((22) GAS253) GAS253 supports M1, GenBank registration numbers GI: 13622611, GI: 15675423, and GI: 21362716, M3, GenBank registration numbers GI: 21910711, M18, GenBank registration numbers GI: 19746473, and also "Spy1524" (M1). , Also called "SpyM3_1175" (M3), "SpyM18_1541" (M18) and "murG". GAS253 is also identified as the expected undecaprenyl-PP-MurNAc-pentapeptide-UDPGlcNAcGlcNAc transferase. The amino acid and polynucleotide sequences of GAS253 of the M1 strain are set forth in SEQ ID NOs: 92 and 93.</p><p> Preferred GAS253 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 92 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 92, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS253 proteins include variants of SEQ ID NO: 92 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 92. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 92. Lose and / or one or more amino acids from the N-terminus of SEQ ID NO: 92 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((23) GAS529) GAS529 supports M1, GenBank registration number GI: 13622403, GI: 15675233, and GI: 21759132, M3, GenBank registration number GI: 21910446, M18, GenBank registration number GI: 19476203, and also "Spy1280" (M1). , Also called "SpyM3_0910" (M3), "SpyM18_1228" (M18) and "glmS". GAS529 is also identified as the expected L-glutamine-D-fructose-6-phosphate transaminase (glucosamine-6-phosphate synthase). The amino acid and polynucleotide sequences of GAS529 of the M1 strain are set forth in SEQ ID NOs: 94 and 95.</p><p> The preferred GAS529 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 94 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 94, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS529 proteins include variants of SEQ ID NO: 94 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 94. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 94. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 94 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((24) GAS045) GAS045 corresponds to M3, GenBank registration numbers GI: 21909751, M18, GenBank registration numbers GI: 19745421, and is also called "SpyM3_0215" (M3), "SpyM18_oppA" (M18), and "oppA".<u style="single">GAS045 is identified as an oligopeptide permease.</u>Examples of the amino acid and polynucleotide sequences of GAS045 of the M1 strain are set forth in SEQ ID NOs: 96 and 97.</p><p> The preferred GAS045 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 96 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 96, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS045 proteins include variants of SEQ ID NO: 96 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 96. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 96. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 96 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 96 (shown below) is removed. (SEQ ID NO: 98 contains the removed N-terminal amino acid sequence; SEQ ID NO: 99 contains a fragment of GAS45 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="13"><img file="JP4875490B2_D0014.tif" /></chemistry> ((25) GAS095) GAS095 corresponds to M1, GenBank registration number GI: 13622787 and GI: 15675582, M3, GenBank registration number GI: 21911042, M18, GenBank registration number GI: 19746634, and also "Spy1733" (M1), "SpyM3_1506" ( Also called M3), "Spy M18_1741" (M18).<u style="single">GAS095 is also identified as a predictive transcriptional regulator.</u>Examples of the amino acid and polynucleotide sequences of GAS095 of the M1 strain are set forth in SEQ ID NOs: 100 and 101.</p><p> Preferred GAS095 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 100 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 100, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS095 proteins include variants of SEQ ID NO: 100 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 100. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 100. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 100 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 100 (shown below) is removed. (SEQ ID NO: 102 contains the removed N-terminal amino acid sequence; SEQ ID NO: 103 contains a fragment of GAS95 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="14"><img file="JP4875490B2_D0015.tif" /></chemistry> ((26) GAS193) GAS193 supports M1, GenBank registration number GI: 13623029, and GI: 15675802, M3, GenBank registration number GI: 21911267, M18, GenBank registration number GI: 19746914, and also "Spy2025" (M1), "SpyM3_1731". Also called (M3), "SpyM18_2082" (M18) and "isp". GAS193 is also identified as an immunogenic secretory protein precursor. The amino acid and polynucleotide sequences of GAS193 of the M1 strain are set forth in SEQ ID NOs: 104 and 105.</p><p> Preferred GAS193 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 104 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 104, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS193 proteins include variants of SEQ ID NO: 104 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 104. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 104. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 104 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((27) GAS137) GAS137 corresponds to M1, GenBank registration number GI: 13621842, GI: 15674720, and GI: 30173748, M3, GenBank registration number GI: 21909998, M18, GenBank registration number GI: 19745749, and also "Spy0652" (M1). , Also called "SpyM3_0462", "SpyM18_0713" (M18). The amino acid and polynucleotide sequences of GAS137 of the M1 strain are set forth in SEQ ID NOs: 106 and 107.</p><p> Preferred GAS137 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 106 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 106, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS137 proteins include variants of SEQ ID NO: 106 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 106. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 106. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 106 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((28) GAS084) GAS084 corresponds to M1, GenBank registration number GI: 13622398 and GI: 15675229, M3, GenBank registration number GI: 21910442, M18, GenBank registration number GI: 19476199, and also "Spy1274" (M1), "SpyM3_0906", Also called "Spy M18_1223" (M18). GAS084 has also been identified as an expected amino acid ABC transporter / protoplasmic peripheral amino acid binding protein. The amino acid and polynucleotide sequences of GAS084 of the M1 strain are set forth in SEQ ID NOs: 108 and 109.</p><p> Preferred GAS084 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 108 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 108, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS084 proteins include variants of SEQ ID NO: 108 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 108. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 108. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 108 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 108 (shown below) is removed. (SEQ ID NO: 110 contains the removed N-terminal amino acid sequence; SEQ ID NO: 111 contains a fragment of GAS84 from which the N-terminal amino acid sequence has been removed). Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="15"><img file="JP4875490B2_D0016.tif" /></chemistry> ((29) GAS384) GAS384 supports M1, GenBank registration number GI: 13622908, and GI: 15675693, M3, GenBank registration number GI: 21911154, M18, GenBank registration number GI: 19746801, and also "Spy1874" (M1), "SpyM3_1618". Also called (M3), "Spy M18_1939" (M18). GAS384 is also identified as the expected glycoprotein endopeptidase. The amino acid and polynucleotide sequences of GAS384 of the M1 strain are set forth in SEQ ID NOs: 112 and 113.</p><p> Preferred GAS384 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 112 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 112, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS384 proteins include variants of SEQ ID NO: 112 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 112. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 112. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 112 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((30) GAS202) GAS202 supports M1, GenBank registration number GI: 13622431, and GI: 15675258, M3, GenBank registration number GI: 21910527, M18, GenBank registration number GI: 19476290, and also "Spy13094" (M1), "SpyM3_0991". Also called (M3), "SpyM18_1321" (M18) and "dltD". GAS202 is also<u style="single">Expected</u>It is also identified as an extramembrane protein. The amino acid and polynucleotide sequences of GAS202 of the M1 strain are set forth in SEQ ID NOs: 114 and 115.</p><p> Preferred GAS202 used according to the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 114 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 114, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS202 proteins include variants of SEQ ID NO: 114 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 114. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 114. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 114 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((31) GAS057) GAS057 corresponds to M1, GenBank registration number GI: 13621655 and GI: 15674549, M3, GenBank registration number GI: 21909834, M18, GenBank registration number GI: 19745560, and also "Spy0416" (M1), "SpyM3_0298" ( Also called M3), "SpyM18_0464" (M18) and "prtS". GAS057 has also been identified as a predicted cellular envelope proteinase. The amino acid and polynucleotide sequences of GAS057 of the M1 strain are set forth in SEQ ID NOs: 116 and 117.</p><p> Preferred GAS057 used according to the present invention<u style="single">(a)</u>50% or more for SEQ ID NO: 116 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) in the amino acid of SEQ ID NO: 116, n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150、200</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS057 proteins include variants of SEQ ID NO: 116 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 116. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 116. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 116 To be deleted. For example, in one embodiment, the N-terminal underlined amino acid sequence of SEQ ID NO: 116 (shown below) is removed. (SEQ ID NO: 118 contains the removed N-terminal amino acid sequence; SEQ ID NO: 119 contains a fragment of GAS57 from which the N-terminal amino acid sequence has been removed). In another embodiment, the C-terminal underlined amino acid sequence of SEQ ID NO: 116 is removed. (SEQ ID NO: 120 contains a C-terminal hydrophobic region. SEQ ID NO: 121 contains a fragment of GAS57 from which the C-terminal hydrophobic region has been removed. SEQ ID NO: 122 contains an N-terminal leader sequence and a C-terminal hydrophobic region. Includes a fragment of GAS57 from which Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p><chemistry num="16"><img file="JP4875490B2_D0017.tif" /></chemistry> A typical example of immunization with the GAS antigen of the present invention in the rodent mouse model described above is summarized in FIG. The first column identifies the GAS antigen used in the experiment. In some cases, this list refers to the purification phase. Also, modifications to the polynucleotide sequence made to promote recombinant expression of the antigen are shown on the chart with the notes below. That is, "a" indicates that the hydrophobic region at the N- or C-terminus has been removed. RR indicates codon optimization. "NH" and "CH" correspond to the same expression vectors shown in the GAS40 construct example. Given a p-value, it is calculated based on the HIS termination value of the control at the bottom of the chart.</p><p> Mice immunized with GAS40 had a substantially improved viability against challenge (antigen injection). In a population of more than 100 mice, immunization with GAS40 resulted in survival rates of&gt; 50%. Other GAS antigens found on the chart also provide some protection<u style="single">did</u>However, this may improve protection, for example when combined with GAS40.</p><p> The immunogenicity of other known GAS antigens may also be improved by combining with two or more GAS species in the first antigen group. Such other known GAS antigens are (1) one or more variants or fragments of M surface proteins, (2) fibronectin binding proteins, (3) streptococcal heme-related proteins, or (4). Contains a second group of antigens consisting of SagA. These antigens are referred to herein as the "second antigen group".</p><p> Accordingly, the present invention includes immunogenic compositions comprising a combination of GAS antigens. However, the combination consists of 2 to 31 GAS antigens in the first antigen group and 1, 2, 3, or 4 GAS antigens in the second antigen group.<u style="single">Preferably this</u>The combination consists of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The combination is more preferably composed of 3, 4, or 5 GAS antigens selected from the first antigen group. The GAS antigen combination preferably comprises either or both of GAS40 and GAS117. The GAS antigen combination preferably comprises one or more variants of the M surface protein.</p><p> Each GAS antigen in the second antigen group will be described in more detail later.</p><p> ((1) M surface protein) M protein is a GAS toxic factor associated with colony formation and resistance to phagocytosis. For M protein, over 100 different types of variants have been identified based on antigen specificity, and M protein is believed to be a major contributor to antigenic shifts and drifts in GAS. The M protein also binds to serum fibrinogen, blocking complement's binding to internal peptidoglycan. This activity is thought to increase GAS survival within the mammalian host by suppressing phagocytosis.</p><p> Unfortunately, the GAS M protein contains several epitopes that mimic those of mammalian muscle and connective tissue. Some GAS M proteins cause the development of rheumatism. This protein contains epitopes similar to the myocardium and can lead to autoimmune rheumatic carditis (rheumatic fever) after acute infection.</p><p> Multiple epitopes that increase bacterial activity but are less likely to cross-react with human tissue have been identified in the amino-terminal region, and these are associated with each other and about 6, 7, or 8 linked in series. It is a fusion protein containing an M protein fragment. Hu et al., Infection &amp; Immunity (2002) 70 (4): 2171-2177; Dale, Vaccine (1999) 17: 193-200; Dale et al., Vaccine 14 (10): 944-948; WO 02/094851, And WO94 / 06465. (Each of the M protein variants, fragments, and fusion proteins described in these references is specifically included herein by reference.) Based on the above, the composition of the present invention may further contain a GAS M surface protein, or a fragment or derivative thereof. One or more GAS M surface protein fragments may be bound in one fusion protein. Separately, one or more GAS M surface protein fragments are combined with the GAS antigen of the first antigen group, or a fragment thereof. GAS One example of M protein is SEQ ID NO: 123<u style="single">As</u>Listed in the sequence list.</p><p> The GAS M protein used in the present invention is<u style="single">(a)</u>50% or more of the known M protein of SEQ ID NO: 123 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, An amino acid sequence having 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity) and / or (b) a known M protein of SEQ ID NO: 123 with n greater than or equal to 7. Yes (eg 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100<u style="single">、150</u>Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These GAS M proteins include variants of SEQ ID NO: 123 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from a known M protein, such as SEQ ID NO: 123. The fragment is preferably one of those described in the above literature. Fragments are preferably constructed in fusion proteins with one or more additional M protein fragments. Other preferred fragments are one or more from the C-terminus of a known M protein, such as SEQ ID NO: 123 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 20). Deletion of 25 or more amino acids and / or one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20) from the N-terminus of the M protein , 25 or more) amino acids are deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((2) Fibronectin binding protein) GAS fibronectin-binding protein (SfbI) mediates bacterial attachment to host cells, promotes bacterial uptake into cells, binds to Fc fragments of human IgG, and binds to Fc receptor-mediated phagocytosis and antibodies. It is a multifunctional bacterial protein that interferes with dependent cellular cytotoxicity. Immunization with mouse SfbI and "H12 fragments" (encoded by position 1240-1854 of the SfbI gene) is Schulze et al., Vaccine (2003) 21: 1958-1964; Schulze et al., Infection and Immunity (2001) ) 69 (1): 622-625, and Guzman et al., Journal of Infectious Diseases (1999) 179: 901-906. An example of the amino acid sequence of GAS SfbI is SEQ ID NO: 124.<u style="single">As</u>Shown in the sequence list.</p><p> The preferred SfbI protein used in the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 124 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) SEQ ID NO: 124, where n is 7 or greater (eg, 8, 10, 12, (14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These SfbI proteins include variants of SEQ ID NO: 124 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 124. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 124. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 124 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((3) Streptococcal heme-related protein) The GAS streptococcal heme-related protein (Shp) has been identified as a GAS cell surface protein. It is believed to be transcribed with genes encoding homologues of ABC transporters involved in ion uptake in Gram-negative bacteria. The Shp protein is more refined and is described in Lei et al., "Identification and characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes," Infection and Immunity (2002) 70 (8): 4494-4500. There is. One example of the Shp protein is SEQ ID NO: 125<u style="single">As</u>Array list<u style="single">To</u>Shown.</p><p> The preferred Shp protein used in the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 125 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) SEQ ID NO: 125, where n is 7 or greater (eg, 8, 10, 12, (14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more) Includes an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These Shp proteins include variants of SEQ ID NO: 125 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes). The preferred fragment of (b) comprises an epitope derived from SEQ ID NO: 125. Other preferred fragments lack one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus of SEQ ID NO: 125. Lose and / or one or more amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 125 To be deleted. Other fragments delete one or more domains of the protein (eg, deletion of signal peptide, plasma domain, transmembrane domain, or extracellular domain).</p><p> ((4) SagA) Streptolysin S (SLS), also known as "SagA", is believed to be produced in almost all GAS colonies. This cytolytic toxin causes beta-hemolysis surrounding GAS colonies cultured on blood agar and is thought to be associated with toxicity. Full-length SagA peptides have not been shown to be immunogenic, but fragments of amino acids 10-30 (SagA10-30) have been used to produce neutralized antibodies. Dale et See al., "Antibodies against a Synthetic Peptide of SagA Neutralize the Cytolytic Activity of Streptolysin S from Group A Streptococci," Infection and Immunicty (2002) 70 (4): 2166-2170. The amino acid sequence of SagA10-30, SEQ ID NO: 126<u style="single">As</u>Shown in the sequence list.</p><p> The preferred SagA10-30 protein used in the present invention is<u style="single">(a)</u>50% or more for SEQ ID NO: 126 (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 99.5% or more) and / or (b) SEQ ID NO: 126, where n is 7 or greater (eg, 8, 10, 12, 14, 16, 18,<u style="single">Or</u>2<u style="single">0)</u>Contains an amino acid sequence containing a fragment consisting of at least n consecutive amino acids. These SagA10-30 proteins include variants of SEQ ID NO: 126 (eg, allelic variants, homologous genes, ausologas genes, paralogus genes, mutated genes).</p><p> There is an upper limit to the number of GAS antigens that may be present in the compositions of the present invention. The number of GAS antigens in the compositions 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. More preferably, the number of GAS antigens in the composition of the invention is less than 6, less than 5, or less than 4. It is even more preferable that the number of GAS antigens in the composition of the present invention is three. The GAS antigen used in the present invention is preferably isolated, i.e. isolated, from an organism in which the molecule is innately found. Alternatively, if the polynucleotide or polypeptide is not naturally found, it may be sufficiently released from other biological macromolecules that the polynucleotide or polypeptide can be used for its intended purpose. preferable.</p><p> (Fusion protein) The GAS antigens used in the present invention may be present as individual polypeptides in the composition, but at least two of those antigens (ie, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) are expressed as a single polypeptide chain (hybrid polypeptide). Hybrid polypeptides offer two major advantages. First, polypeptides that are unstable or underexpressed by themselves are assisted by the addition of suitable partner hybrids that overcome the problem. Second, commercial production is simplified because only one expression / purification step is required to produce two polypeptides that are both antigenically effective.</p><p> The hybrid polypeptide contains two or more polypeptide sequences selected from the first antigen group. Therefore, the present invention is a composition containing a first amino acid sequence and a second amino acid sequence, and the first and second amino acid sequences are selected from the GAS antigen of the first antigen group or a fragment thereof. Contains the characteristic composition. The first and second amino acid sequences in the hybrid polypeptide preferably contain different epitopes.</p><p> The hybrid polypeptide may contain one or more polypeptide sequences selected from the first antigen group and one or more polypeptide sequences selected from the second antigen group. Therefore, the present invention is a composition containing a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence is selected from the GAS antigen of the first antigen group or a fragment thereof, and the second amino acid sequence. Contains a composition characterized by being selected from the GAS antigen of a second group of antigens or fragments thereof. The first and second amino acid sequences in the hybrid polypeptide preferably contain different epitopes.</p><p> Hybrids consisting of amino acid sequences obtained from 2, 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens are preferred. In particular, hybrids consisting of amino acid sequences obtained from 2, 3, 4, or 5 GAS antigens are preferred.</p><p> Various hybrid polypeptides may be mixed in a single formulation. In such a combination, one GAS antigen may be present in more than one hybrid polypeptide and / or non-hybrid polypeptide. However, the antigen preferably exists as either hybrid or non-hybrid, and is not preferably present as both.</p><p> The hybrid peptide is of formula NH₂-A- {-XL-}_nIt can be represented by -B-COOH. In the formula, X is the amino acid sequence of the GAS antigen or fragment thereof of the first or second antigen group; L is the optionally used linker amino acid sequence; A is required. Is an optionally used N-terminal amino acid sequence; B is an optionally used C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, or 15.</p><p> If the -X- component is wild-type and has a leader polypeptide sequence, it may be included or omitted in the hybrid protein. In certain embodiments, the leader peptide is deleted. However, in the -X- component located at the N-terminus of the hybrid protein, the leader peptide X₁Is retained, but the leader peptide X₂... X_nIs omitted. This removes all leader peptides and leader peptide X₁Is equivalent to using as component -A-.</p><p> {-XL-} n For each of the examples<u style="single">I</u>The linker amino acid -L- may or may not be present. For example, if n = 2, the hybrid is NH₂-X₁-L₁-X₂-L₂-COOH, NH₂-X₁-X₂-COOH, NH₂-X₁-L1-X₂-COOH, NH₂-X₁-X₂-L₂-It may be COOH or the like. Linker amino acid sequence (single or multiple)<u style="single">-L-</u>Are usually short (eg, 20 or less amino acids, ie 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). An example is a short polypeptide sequence that facilitates cloning, a polyglycine linker (ie, Gly)._nIncluding, here n = 2,3,4,5,6,7,8,9,10 and above), and histidine tags (ie His)_nAnd includes n = 3, 4, 5, 6, 7, 8, 9, 10 and above). Other suitable linker amino acid sequences will be apparent to those of skill in the art. A useful linker is GSGGGG, which forms a Gly-Ser dipeptide from the BamHI restriction site, which aids in cloning and manipulation. Also, (Gly)₄Tetrapeptides are typical polyglycine linkers.</p><p> -A- is an optionally used N-terminal amino acid sequence. This is usually short (eg, 40 or less amino acids, ie 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). As an example, of protein<u style="single">Transport</u>A leader sequence that directs delivery, or a short peptide sequence that facilitates cloning or purification (eg, histidine tag, ie His)._nAnd n = 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those of skill in the art. X₁-A- is an oligopeptide that provides N-terminal methionine (eg, has 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) if it lacks its own N-terminal methionine. Is preferable.</p><p> -B- is a C-terminal amino acid sequence that is optionally used. This is usually short (eg, 40 or less amino acids, ie 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). As an example, of protein<u style="single">Transport</u>A leader sequence that directs delivery, or a short peptide sequence that facilitates cloning or purification (eg, histidine tag, ie His)._nAnd n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences that improve protein stability. Other suitable<u style="single">C</u>The terminal amino acid sequence is apparent to those of skill in the art.</p><p> Most preferably n is 2 or 3.</p><p> The fusion construct of the present invention comprises a combination of two or more GAS antigens, which combination may comprise GAS40, or a fragment thereof, or a polypeptide having the same sequence relative to it.</p><p> The fusion construct of the present invention is a combination of GAS antigens, the combination consisting of 2 to 31 GAS antigens belonging to the first antigen group, and the first antigen group is GAS117, GAS130, GAS277. , GAS236, GAS40, GAS389, GAS504, GAS509, GAS366, GAS159, GAS217, GAS309, GAS372, GAS039, GAS042, GAS058, GAS290, GAS511, GAS533, GAS527, GAS294, GAS253, GAS529, GAS045, GAS095 , GAS384, GAS202, and GAS057. The combination of GAS antigens is preferably composed of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The GAS antigen combination preferably comprises 3, 4, or 5 GAS antigens selected from the first antigen group.</p><p> GAS39, GAS40, GAS57, GAS117, GAS202, GAS294, GAS527, GAS533, and GAS511 are particularly preferred GAS antigens for use in the fusion constructs of the present invention. The GAS antigen combination preferably comprises either or both of GAS40 and GAS117. The combination preferably includes GAS40.</p><p> Recombinant expression of the fusion construct of the present invention may be improved or optimized by the same method described above for expression of GAS antigen alone. The fusion construct of GAS40 and GAS117 is illustrated below.</p><p> In the first embodiment, GAS117 is bound to GAS40a-RR. (As mentioned above, GAS40a-RR is a codon-optimized GAS40 sequence with the N-terminal leader sequence and C-terminal transmembrane sequence removed). In this construct, the GAS117 fragment (with the N-terminal leader sequence removed) is placed at the N-terminus of the GAS40 sequence and a HIS tag is added to the C-terminus of the GAS40 sequence. This construct is labeled "117-40a-RR". The amino acid and polynucleotide sequences of this construct are SEQ ID NOs: 127 and 128.<u style="single">As</u>Shown in the sequence list.</p><p> The GAS117 and GAS40 sequences are preferably linked by a linker sequence containing multiple glycine residues. For example, the linker sequence of SEQ ID NO: 129 (YASGGGS) is used as the linker used in the 117-40a-RR fusion construct.</p><p> In the second embodiment, the relative positions of the GAS40 and GAS117 sequences are interchangeable. In this construct labeled "40a-RR-117", the GAS40a-RR sequence is placed at the N-terminus of GAS117 and the HIS tag is added to the C-terminus of the GAS117 sequence. The amino acid and polynucleotide sequences of this fusion construct are SEQ ID NOs: 130 and 131.<u style="single">As</u>Shown in the sequence list.</p><p> Apart from that, the fusion construct may be designed without codon optimization. For example, the amino acid and polynucleotide sequences of fusion construct "117-40a" are SEQ ID NOs: 132 and 133.<u style="single">As</u>Shown in the sequence list. (Since no codon optimization is used, three point mutations are likely to occur during cloning, one of which contained a conservative amino acid change (glucin to glycine). Rodent immunization model. So (as mentioned above), immunization with "117-40a" resulted in up to 80% survival for antigen infusion.</p><p> A preferred GAS40 fusion sequence is a GAS40 fragment containing one or more coiled coil regions. For example, the fusion construct may include a GAS40 sequence that includes a first coiled coil region. "117-40N" is an example of this type of construct. The amino acid and polynucleotide sequences of this construct are SEQ ID NOs: 132 and 133.<u style="single">As</u>Shown in the sequence list.</p><p> The present invention also provides nucleic acids encoding the hybrid polypeptides of the present invention. Furthermore, the present invention provides nucleic acids capable of hybridizing to this nucleic acid under "high rigor" conditions (eg, at 65 ° C, 0.1xSSC, 0.5% SDS solution).</p><p> The GAS antigens of the present invention may also be used to prepare antibodies specific for the GAS antigen. The antibody is preferably specific for the first or second coiled coil region of GAS40. The present invention also includes GBS80, GAS117, GAS130, GAS277, GAS236, GAS40, GAS389, GAS504, GAS509, GAS366, GAS159, GAS217, GAS309, GAS372, GAS039, GAS042, GAS058, GAS290, GAS511, GAS533, GAS527, GAS294, GAS253. , GAS529, GAS045, GAS095, GAS193, GAS137, GAS084, GAS384, GAS202, and the use of a combination of two or more antibodies selected from the group consisting of antibodies specific for GAS057. The combination preferably comprises an antibody specific for GAS40, or a fragment thereof.</p><p> The GAS-specific antibodies of the invention contain one or more biological components capable of binding or associating with an epitope of a GAS polypeptide by chemical or physical means. Antibodies of the invention include antibodies that specifically bind to the GAS antigen, preferably GAS80. The present invention includes the following, including antibodies obtained from polyclonal and monoclonal preparations. That is, see hybrid (chimeric) antibody molecules (see, eg, Winter et al. (1991) Nature 349: 293-299, and US Pat. No. 4,816,567; F (ab').₂And F (ab) fragment; F_vMolecules (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-strand Fv molecules (see sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85: 5897-5883); 2<u style="single">Dimer</u>O<u style="single">Yo</u>And 3<u style="single">Dimer</u>Antibody fragment constructs; minibodies (see, eg, Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (eg, Riechmann et al). (1988) Nature 332: 323-327; Verhoeyan et al. (1988) Science 239: 1534-1536; and UK Patent Publication No. GB2,276,169 published September 21, 1994); and obtained from the above antibodies. Any functional fragment of an antibody that retains the immunological binding properties of the original antibody molecule. The present invention further comprises antibodies obtained by unusual processes such as phage display.</p><p> The GAS-specific antibody of the present invention is preferably a monoclonal antibody. The monoclonal antibody of the present invention comprises an antibody composition having a uniform antibody population. The monoclonal antibody of the present invention may be obtained from a human monoclonal antibody obtained by using a human hybridoma instead of a rodent, including a rodent hybridoma. See, for example, Cote et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p77.</p><p> The polypeptides of the invention are prepared by various methods (eg, recombinant expression, purification of cell culture, chemical synthesis, etc.) and in various forms (eg, natural, fusion, non-glycosylation, lipidation, etc.). It is possible to do. The polypeptide is preferably prepared in a substantially pure form (ie, substantially free of other GAS or host cell proteins).</p><p> Nucleic acids according to the invention can be prepared in many ways (eg, from chemical synthesis, genome or cDNA libraries, from the organism itself, etc.) and can take various forms (eg, from the organism itself). , Single-stranded, double-stranded, vector, probe, etc.). This nucleic acid is preferably prepared in a substantially pure form (ie, substantially free of nucleic acids from other GAS or host cells).</p><p> The term "nucleic acid" includes DNA and RNA and their analogs, such as those with a modified backbone (eg, phosphorothioate, etc.), and further peptide nucleic acids (PNA), and the like. The present invention also includes sequences complementary to the aforementioned nucleic acids (eg, for antisense or probe purposes, etc.).</p><p> The present invention is also a method for producing the polypeptide of the present invention, which is characterized by the nucleic acid of the present invention.<u style="single">Turn</u>Provided is a method comprising culturing a converted host cell under conditions that induce polypeptide expression.</p><p> The present invention also provides a method of producing the polypeptide of the invention, comprising the step of synthesizing at least a portion of the polypeptide by chemical means.</p><p> The present invention also provides a method for producing the nucleic acid of the present invention, which comprises a step of amplifying the nucleic acid using a primer-based amplification method (eg, PCR).</p><p> The present invention also provides a method for producing the nucleic acid of the present invention, which comprises the step of synthesizing at least a part of the nucleic acid by chemical means.</p><p> (Strain) Preferred polypeptides of the invention include the amino acid sequences found in the M1, M3, or M18 strains of GAS. The genomic sequence of the M1 GAS strain is reported in Ferretti et al, PNAS (2001) 98 (8): 4658-4663. The genomic sequence of the M3 GAS strain is reported in Berres et al, PNAS (2002) 99 (15): 10078-10083. The genome sequence of the M18 GAS strain is Smooth. It is reported in et al, PNAS (2002) 99 (7): 4668-4673.</p><p> When using a hybrid polypeptide, the individual antigens in the hybrid (ie, the individual -X-components) may be from one or more strains. When n = 2, for example, X₂Is X₁It may be obtained from the same strain as, or it may be obtained from a different strain. If n = 3, the stock is (i) X₁= X₂= X₃, (Ii) X₁= X₂ X₃, (Iii) X₁ X₂= X₃, (Iv) X₁ X₂ X₃, Or (v) X₁= X₃ X₂It may be.</p><p> (Purification and recombinant expression) The GAS antigen of the present invention may be isolated from Streptococcus pyogenes or may be produced, for example, by recombinant techniques in a heterologous host. The GAS antigen is preferably prepared using a heterologous host. The heterologous host may be pronuclear (eg, bacterial) or eukaryotic. E. coli is preferred, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (eg, M. tuberculosis), yeast and the like.</p><p> Recombinant production of polypepritide is facilitated by adding a tag protein to the GAS antigen so that it is expressed as a fusion protein containing the tag protein and the GAS antigen. Such tag proteins improve the purification, detection, and stability of expressed proteins. Suitable tag proteins for use in the present invention include polyarginine tags (Arg-tags), polyhistidine tags (His-tags), FLAG-tags, Strep-tags, c-myc-tags, S-tags, etc. Carmodulin-binding tag, cellulose-binding domain, SBP-tag, chitin-binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination antitrantrolytic factor (NusA), E.coli thioredoxin (TrxA) , And protein disulfide isomerase I (DsbA). Preferred tag proteins include His-tag and GST. A detailed discussion of the use of tag proteins can be found in Terpe et al., Appl Microbiol Biotechnol (2003) 60: 523-533.</p><p> After purification, the tag protein may be optionally removed from the expression fusion protein, i.e., by a specifically tailored enzyme treatment known in the art. Commonly used proteases include enterokinase, tobacco etch virus (TEV), thrombin, and factor Xa.</p><p> (Immunogenic composition and drug) The composition of the present invention is preferably an immunogenic composition, more preferably a vaccine composition. The pH of the composition is preferably between 6 and 8, about 7. The pH may be maintained with buffer. The composition may be sterile and / or pyrogen-free. The composition may be isotonic to humans.</p><p> Vaccines according to the invention may be prophylactic (ie, prevent infection) or therapeutic (ie, treat infection), but are typically prophylactic. Accordingly, the present invention includes methods for therapeutic or prophylactic treatment of Streptococcus pyogenes in animals susceptible to streptococcal infection, which methods are therapeutically or prophylactically effective amounts of the immunogenic compositions of the present invention. Includes administration to the animal. The immunogenic composition is a combination of GAS antigens, preferably containing a combination of 2 to 31 GAS antigens in the first antigen group. The combination of GAS antigens is preferably composed of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The GAS antigen combination preferably comprises 3, 4, or 5 GAS antigens selected from the first antigen group. The GAS antigen combination preferably comprises either or both of GAS40 and GAS117.</p><p> Separately, the present invention is a combination of GAS antigens, which comprises 2 to 31 GAS antigens in the first antigen group and 1, 2, 3, or 4 GAS antigens in the second antigen group. Includes an immunogen composition that contains. The GAS antigen combination preferably consists of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The GAS antigen combination is more preferably composed of 3, 4, or 5 GAS antigens selected from the first antigen group. The GAS antigen combination preferably comprises either or both of GAS40 and GAS117. The GAS antigen combination preferably comprises one or more variants of the M surface protein.</p><p> The present invention also provides the compositions of the present invention as agents. The agent is preferably capable of inducing an immune response in a mammal (ie, an immunogenic composition), more preferably a vaccine.</p><p> The present invention also provides the use of the compositions of the present invention in the manufacture of agents that elicit an immune response in mammals. The drug is preferably a vaccine. The present invention also provides a kit comprising a first composition comprising a combination of GAS antigens. In one embodiment, the GAS antigen combination consists of a mixture of 2 to 31 GAS antigens selected from the first antigen group. The combination preferably consists of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The combination preferably comprises 3, 4, or 5 GAS antigens selected from the first antigen group. The combination preferably comprises either or both of GAS40 and GAS117.</p><p> In another embodiment, the kit is a combination of GAS antigens from 2 to 31 GAS antigens in the first antigen group and 1, 2, 3, or 4 GAS antigens in the second antigen group. Contains the first component, including the combination consisting of. The combination preferably consists of 3, 4, 5, 6, 7, 8, 9, or 10 GAS antigens selected from the first antigen group. The combination is more preferably composed of 3, 4, or 5 GAS antigens selected from the first antigen group.<u style="single">Preferably of the GAS antigen</u>The combination preferably comprises either or both of GAS40 and GAS117. The GAS antigen combination preferably comprises one or more variants of the M surface protein.</p><p> The present invention is also prefilled with the immunogenic compositions of the present invention.<u style="single">Delivery</u>Provide the device.</p><p> The present invention also provides a method of inducing an immune response in a mammal, comprising the step of administering an effective amount of the composition of the present invention. The immune response is protective and preferably comprises antibodies and / or cell-mediated immunity. The method may elicit a booster reaction.</p><p> The mammal is preferably human. If the vaccine is prophylactic, the human is preferably a child (eg, infant or toddler) or a teenager. If the vaccine is therapeutic, humans are preferably teenagers or adults. Vaccines for children may be administered to adults, for example, to assess safety, dose, immunogenicity, etc.</p><p> These uses and methods include diseases caused by Streptococcus pyogenes (eg, pharyngitis (eg, streptococcal throat), rheumatic fever, impetigo, erysipelas, honeycombitis, septicemia, toxic shock syndrome, necrotizing myelitis (muscle invasion). Disease), and for the prevention and / or treatment of sequelae (eg, rheumatic fever and acute streptococcal nephritis). The composition may also be effective against other streptococci.</p><p> One way to check the effectiveness of a therapeutic procedure is to monitor GAS infection after administration of the compositions of the invention. One way to check the effectiveness of prophylactic treatment is to monitor the immune response to the GAS antigen in the compositions of the invention after administration of the composition.</p><p> The compositions of the present invention are generally administered directly to a patient. direct<u style="single">Delivery</u>By parenteral injection (eg, subcutaneous, intraperitoneal, intravenous, intramuscular, or tissue interstitial space), or rectal, oral (eg, tablets, sprays), vaginal, topical application, trans. Intrathecal (see, eg, WO99 / 27961), or transdermal (see, eg, WO02 / 074444 and WO02 / 064162), intranasal (see, eg, WO03 / 028760), intraocular, intraoural, lung or other. It is realized by mucosal administration of. The present invention may be used to induce systemic and / or mucosal immunity.</p><p> The medication procedure can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in the primary immunization schedule and / or in the booster immunization schedule. In a multi-dose schedule, different doses may be given from the same or different routes, eg, parenteral preparatory, mucosal boost, or mucosal preparatory and parenterally. Boost may be administered.</p><p> The compositions of the present invention can be prepared in various forms. For example, the composition may be prepared as a solution or suspension for injection. Liquid before injection<u style="single">Bi</u>Solids suitable for making a solution or suspension in a hicle can also be prepared (eg, lyophilized compositions). The composition may be prepared for topical administration, eg, as an ointment, cream, or powder. The composition may be prepared for oral administration, eg, as tablets or capsules, as a spray, as a syrup (with optional flavors if desired). The composition may be prepared for pulmonary administration, eg, as an inhalant with a fine powder or spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, ear cavity, or eyeball administration, eg, as nasal drops, ear drops, eye drops. The composition may take the form of a kit designed to reconstitute the combined composition immediately prior to administration to the patient. The kit may include one or more antigens in the form of a solution and one or more lyophilized antigens. The immunogenic composition used as a vaccine contains an immunologically effective amount of the antigen (s) and other components as needed. "Immunologically effective amount" means that the amount is effective for treatment or prevention when administered to an individual either alone or as part of a series of administrations. This amount is the health and physical condition of the individual being treated, the age, the taxonomic category of the individual being treated (eg, non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, and the desired protection. Depends on the degree of vaccine, the evaluation of the treating physician's medical condition, and other related factors. This amount is expected to be relatively widespread, as determined by conventional attempts.</p><p> (Other components of the composition) The compositions of the present invention typically include one or more "pharmaceutically acceptable carriers" in addition to the components described above. The carrier can be any carrier as long as it does not induce the production of antibodies that are harmful to the individual receiving the composition. Suitable carriers are usually large, hard-to-metabolize macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, and lipid aggregates (eg, oil droplets or liposomes). Such carriers are well known to those of skill in the art. Vaccines may also contain diluents such as water, saline, glycerol and the like. In addition, auxiliary substances such as lubricants or emulsifiers, pH buffers and the like may be present. A detailed description of pharmaceutically acceptable excipients is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th ed., ISBN: 0683306472.</p><p> The vaccine of the present invention may be administered in combination with other immunomodulators. In particular, the composition usually comprises an adjuvant.</p><p> More preferred adjuvants include, but are not limited to, one or more selected from those listed below.</p><p> (A. Mineral-containing composition) Mineral-containing compositions suitable for use as adjuvants in the present invention include mineral salts such as aluminum and calcium salts. The present invention uses mineral salts such as hydroxides (eg, oxyhydroxydos), phosphates (eg, hydroxyphosphates, orthophosphates), sulfates and the like (eg, chapters 8 &amp; 9 of Vaccine design: the subunit and). adjuvant approach (1995) Powell &amp; Newman. ISBN0-306-44867-X), or a mixture of different mineral compounds, the compound of which is of any suitable form (eg, gel, crystalline, amorphous, etc.). Contains a mixture that is preferably adsorbed. Mineral-containing compositions may also be formulated as particles of metal salts. See WO 00/23105.</p><p> (B. Oil emulsion) As an oil emulsion suitable for use as an adjuvant in the present invention, a squalene aqueous emulsion, for example, MF59 (5% squalene, 0.5% Tween80, and 0.5% Span85, which are made into submicron particles using a microfluidizer). Can be mentioned. See WO 90/14837. 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, "see Vaccine (2003) 21: 4234-4237. MF59 was used as an adjuvant for the FLUAD influenza virus trivalent subunit vaccine.</p><p> A particularly suitable adjuvant for use in the composition is a submicron oil droplet emulsion of submicron, a submicron oil droplet emulsion suitable for use in the present invention is optionally in varying amounts of MTP-. Squalene / water emulsions containing PE, such as 4-5% w / v squalene, 0.25-1.0% w / vTween80 (polyoxyethylene sorbitan monooleate), and / or 0.25-1.0% Span85 . (Sorbitan trioleate), and optionally N-acetylmuramil-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxy) Phosphoryloxy) -ethylamine (MTP-PE), eg, "MF59" (International Publication WO 90/14837; US Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entirety; 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, MF and Newman, MJeds.) Plenum Press, New York,<u style="single">1955</u> pp.277-296). MF59 is 4-5% w / v squalene (eg 4.3%),<u style="single">0.25~</u>0.5% w / vTween80 and 0.5%<u style="single">w / v</u>It contains Span85 , and optionally varying amounts of MTP-PE, which can be made into submicron particles using a microfluidic device, such as the Microfluidics Model 110Y (Microfluidics, Newton, Mass.). Including those that have been For example, MTP-PE may be present in an amount of about 0-500 μg / dose, more preferably 0-250 μg / dose, most preferably 0-100 μg / dose. As used herein, "MF59-0" refers to the aforementioned submicron oil-in-water emulsion that does not contain MTP-PE, while the term MF59-MTP refers to formulations that contain MTP-PE. For example, "MF59-100" contains 100 μg of MTP-PE per dose. Another submicron oil droplet emulsion used in the present invention, MF69, is 4.3% w / v squalene, 0.25% w / vTween80 , and 0.75%.<u style="single">w / v</u>Includes Span85 and optionally MTP-PE. Yet another submicron oil droplet emulsion, also known as SAF, contains 10% w / v squalene, 0.4% w / vTween80 , 5% pluronic block polymer L121, and thr-MDP. , Including those made into submicron particles using a microfluidic device. MF75-MTP refers to an MF75 formulation containing MTP, eg, a formulation containing 100-400 μg of MTP-PE per dose. Formulations of the submicron oil-in-water agents used in the compositions, the agents thereof and immunostimulatory agents, such as muramyl peptide, are described in detail in International Publication WO 90/14837 and US Pat. Nos. 6,299,884 and 6,451,325. ..</p><p> Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA) can also be used as the adjuvants of the present invention.</p><p> (C. Saponin prescription) Saponin formulations may also be used as an adjuvant in the present invention. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, trunks, roots, and sometimes flowers of a wide range of plant species. Quillaia saponaria Molina Saponins obtained from the bark of trees have been widely studied as adjuvants. Saponins are also Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria. Industrially collected from officialis (soap root). Saponin adjuvant formulations include purified formulations such as QS21 and lipid formulations such as ISCOM. The saponin composition is purified using high performance thin layer chromatography (HP-LC) and reverse phase high performance liquid chromatography (RP-HPLC). Specific purified fractions were identified using these techniques. Such fractions include QS7, QS17, QS18, QS21, QH-A, QH-B, and QH-C. The saponin is preferably QS21. The recipe for QS21 is disclosed in US Pat. No. 5,057,540. Saponin formulations may also include sterols, such as cholesterol (see WO 96/33739).</p><p> The combination of saponin and cholesterol can be used to form unique particles called immunostimulatory complexes (ISCOM). ISCOM also typically comprises phospholipids such as phosphatidylethanolamine or phosphatidylcholine. It is possible to use any known saponin in ISCOM. ISCOM preferably contains one or more of Quil A, QHA, and QHC. ISCOM is described in more detail in EP0109942, WO96 / 11711, and WO96 / 33739.<u style="single">If desired, the ISCOM may lack an additional surfactant.</u>See WO 00/07621.</p><p> A comprehensive overview of the development of saponin-based adjuvants, Barr, et al., Advanced Found in Drug Delivery Reviews (1998) 32: 247-271. See also Sjolander, et al., Advanced Drug Delivery Reviews (1998) 32: 321-338.</p><p> (C. Willosome and Virus-like Particles (VLP)) Willosomes and virus-like particles (VLPs) can also be used as adjuvants of the present invention. These structures generally include one or more proteins derived from the virus, optionally combined or formulated with phospholipids. This structure is generally non-pathogenic, non-replicating, and generally completely free of the natural viral genome . Viral proteins are either recombinantly produced or isolated from the whole virus. We<u style="single">B</u>Suitable viral proteins for use in som or VLP are influenza virus (eg HA or NA), hepatitis B virus (eg core or capsid protein), hepatitis E virus, measles virus, sinobis virus, rotavirus. , Orthopedic virus, retrovirus, nowalk virus, human papillomavirus, HIV, RNA-phage, Qβ-phage (eg coat protein), GA-phage, fr-phage, AP205 phage, and Ty (eg retrotransposon Ty protein) It is a protein obtained from p1). VLPs are WO03 / 024480, WO03 / 024481, and Niikura et al., Virology (2002) 293: 273-280; Lenz et al., Journal of Immunology (2001) 5246-5355; Pinto et al., Journal of Infectious Diseases (2003) 188: 327-338; Gerber et al., More details are given in the Journal of Virology (2001) 75 (10): 4752-4760. Willosomes are further discussed, for example, in Gluck et al., Vaccine (2002) 20: B10-B16.</p><p> (D. Bacterial or microbial derivative) Suitable adjuvants for use in the present invention include bacterial or microbial derivatives such as:</p><p> ((1) Non-toxic derivative of enterobacteria lipopolysaccharide (LPS)) Such derivatives include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3De-O-acylated monophosphoryl lipid A with 4, 5, or 6 acylated chains. Preferred "small particles" of 3De-O-acylated monophosphoryl lipid A are disclosed in EP0689454. Such "small particle" 3dMPLs are small enough to be filtered through a 0.22 micron membrane and sterilized (see EP0689454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics, such as aminoalkyl glucosamide phosphate derivatives, such as RC-529. See Johnson et al. (1999) Bioorg Med Chem Lett 9: 2273-2278.</p><p> ((2) Lipid A derivative) Lipid A derivatives include derivatives of Lipid A obtained from E. coli, such as OM-174. OM-174 is described, for example, in Meraldi et al., Vaccine (2003) 21: 2485-2491 and Pajak, et al., Vaccine (2003) 21: 836-842.</p><p> ((3) Immunostimulatory oligonucleotide) Immunostimulatory oligonucleotides preferably used as adjuvants of the present invention include nucleic acid sequences containing a CpG motif (a sequence containing unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double-stranded RNA or oligonucleotides, including palindromes or poly (dG) sequences, have also been found to be immunostimulatory.</p><p> CpG can include nucleotide modified / analogs, such as phosphorothioate modified, which may be double-stranded or single-stranded. Optionally, guanosine may be replaced by an analog, such as 2'-deoxy-7-deazaguanosine. See Kandi mala, et al., Nucleic Acids Research (2003) 31 (9): 2393-2400; WO 02/26757 and WO 99/62923 for examples of similar possible substitutions. For the adjuvant action of CpG oligonucleotides, see Krieg, Nature Medicine (2003) 9 (7): 831-835; McCluskie, et al., FEMS Immunology and Medical Microbiology (2002) 32: 179-185; WO 98/40100; USA It is discussed in Patent No. 6,207,646; US Pat. No. 6,239,116; and US Pat. No. 6,429,199.</p><p> The CpG sequence may be directed to TLR9, eg, motif GTCGTT or TTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003) 31 (part 3); 654-658. The CpG sequence may be specific for inducing a Th1 immune response, for example CpG-A ODN, or specific for inducing a B cell response, for example CpG-B ODN. It may be a target. For CpG-A and CpG-B ODN, Blackwell, et al., J.Immunol. (2003) 170 (8): 4061-4068; Krieg, TRENDS in Immunology (2002) 23 (2): 64-65, And WO 01/95935. CpG is preferably CpG-A ODN.</p><p> CpG oligonucleotides are preferably constructed so that the 5'end is accessible for receptor recognition. If necessary, two CpG oligonucleotide sequences may be optionally attached to the 3'end to form an "immunomer". For example, Kandimalla, et al., BBRC (2003) 306: 948-953; Kandimalla, et al., Biochemical Society Transactions (2003) 31 (part3); 654-658; Bhagat et al., BBRC (2003) 300: See 853-861 and WO 03/035836.</p><p> ((4) ADP-ribosylated toxin and its detoxified derivative) Bacterial ADP-ribosylated toxin and its detoxified derivatives may be used as an adjuvant in the present invention. The protein is preferably derived from E. coli (ie, E. coli heat-sensitive endotoxin "LT"), Vibrio cholerae ("CT"), or Bordetella pertussis ("PT"). .. The use of detoxified ADP-ribosylated toxin as a mucosal adjuvant is described in WO95 / 17211 and the use as a parenteral adjuvant is described in WO98 / 42375. The adjuvant is preferably a detoxified LT mutation, eg LT-K63.</p><p> (E. Human immunomodulatory factor) Human immunomodulators suitable for use as adjuvants in the present invention include cytokines such as interleukins (eg IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL). -12 etc.), interferon (eg, interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.</p><p> (F. Bio-adhesives and mucosal adhesives) Bioadhesives and mucosal adhesives can also be used as the adjuvants of the present invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al. (2001) J.Cont.Rele. 70: 267-276), mucosal adhesives, such as crosslinked derivatives of poly (acrylic acid). Polyvinyl alcohols, polyvinyl pyrrolidones, polysaccharides, and carboxymethyl cellulose can be mentioned. Chitosan and its derivatives can also be used as an adjuvant in the present invention. For example, WO99 / 27960.</p><p> (G. Fine particles) Fine particles may also be used as an adjuvant in the present invention. Fine particles (ie, diameters from about 100 nm to about 150 μm, more preferably from about 200 nm to about 30 μm, most preferably from about 500 nm to about 10 μm), biodegradable and non-toxic materials (eg, poly (α-hydroxy acids), poly). It is formed from (hydroxyl butyric acid, polyorthoester, polyanhydride, polocaprolactone, etc.), but is preferably made of poly (lactide-co-glycolide), preferably with a negatively charged surface (optionally). It is treated (eg, by SDS) or to have a positively charged surface (eg, by a cationic surfactant, eg, CTAB).</p><p> (H. Liposome) Examples of liposome formulations suitable for use as an adjuvant are described in US Pat. No. 6,090,406, US Pat. No. 5,916,588, and EP0626169.</p><p> (I. Polyoxyethylene ether and polyoxyethylene ester formulation) Suitable adjuvants for use in the present invention include polyoxyethylene ethers and polyoxyethylene esters.<u style="single">WO 99/52549.</u>The formulation further includes a polyoxyethylene sorbitan ester surfactant (WO01 / 21207) in combination with octoxinol and at least one additional nonionic surfactant, eg, polyoxy in combination with octoxinol. Includes ethylene alkyl ether, or ester surfactant (WO01 / 21152).</p><p> Preferred polyoxyethylene ethers are selected from the following groups. That is, polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steroyl ether, polyoxyethylene-8-steroyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether. , And polyoxyethylene-23-lauryl ether.</p><p> (J. Polyphosfazen (PCPP)) For PCPP formulations, Andrianov et al., "Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazene solutions," Biomaterials (1998) 19 (1-3): 109-115, and Payne et al., "Protein Release from Polyphosphazene Matrices". , Adv. Drug Delivery Review (1998) 31 (3): 185-196.</p><p> (K. Muramyl peptide) Examples of muramyl peptides preferably used as adjuvants in the present invention are N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramil-L-alanyl-D-. Isoglutamine (nor-MDP), and N-Acetyl Muramyl-L-Alanyl-D-Isoglutaminer-L-Alanine-2- (1'-2'-Dipalmityl-sn-Glycero-3-Hydroxyphosphoryloxy) )-Ethylamine MTP-PE).</p><p> (L. Imidazoquinolone compound) Examples of the imidazoquinolone compound preferably used as an adjuvant in the present invention include imikimod and its homologues. For these, Stanley, "Imiquimod and imidazoquinolones: mechanism of action and therapeutic potential" Clin Exp Dermatol (2002) 27 (7): 571-577, and Jones, "Resiquimod 3M," Curr Opin Investig Drugs (2003) 4 ( 2): Described in more detail at 214-218.</p><p> The present invention may include a combination of one or more aspects of the adjuvant identified above. For example, the following adjuvant compositions may be used in the present invention: (1) Saponin and oil drop emulsion in water (WO99 / 11241) (2) Saponin (eg QS21) + non-toxic LPS derivative (eg 3dMPL) (see WO94 / 00153); (3) Saponin (eg QS21) + non-toxic LPS derivative (eg 3dMPL) + cholesterol; (4) Saponin (eg QS21) + 3dMPL + IL-12 (optionally + sterols) (WO98 / 57659); (5) 3dMPL and, for example, QS21 and / or oil drop emulsion in water (European patent applications 0835318, 0735898, and 0761231); (6) SAF containing 10% squalene, 0.4% Tween80, 5% pluronic block polymer L121, and thr-MDP, which were microfluidically treated into a submicron emulsion, or relatively swirled and agitated. Emulsion with a large particle size; (7) Ribi adjuvant system (RAS) (Ribi Immunochem) with 2% squalene, 0.2% Tween80, and monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton ( An adjuvant system containing one or more cell wall components selected from the group consisting of CWS) and MPL + CWS (Detox ); (8) One or more mineral salts (eg aluminum salts) + non-toxic derivatives of LPS (eg 3dPML); (9) One or more mineral salts (eg, aluminum salts) + immunostimulatory oligonucleotides (eg, nucleotide sequences containing CpG motifs).</p><p> For parenteral administration, aluminum salts and MF59 are the preferred adjuvants. A preferred mucosal adjuvant is a mutant bacterial toxin.</p><p> The composition may include antibiotics.</p><p> (Additional antigen) The compositions of the invention may further comprise one or more additional non-GAS antigens, including antigens including additional bacterial, viral, or parasitic antigens.</p><p> In one embodiment, the GAS antigen mixture of the present invention may be combined with one or more additional, non-GAS antigens for use as a pediatric vaccine. For example, the GAS antigen mixture is N. meningitidis (serum groups A, B, C, W135, and / or Y), Streptococcus pneumoniae, Bordella pertussis, Moraxella catarrhalis, Tetanus, Diphtheria, RS virus (RSV), polio, It may be combined with one or more antigens obtained from a bacterium or virus selected from the group consisting of hashika, mumps cold, rubella, and rotavirus.</p><p> In another embodiment, the GAS antigen mixture of the present invention may be combined with one or more additional non-GAS antigens that are suitably used in vaccines designed to protect the elderly or immunocompromised individuals. Good. For example, the GAS antigen mixture is obtained from a bacterium or virus selected from the group consisting of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Psudomonas aeruginosa, Legionella penumonphila, Listeria monocytogenes, influenza, and Palinefuenza virus (PIV). It may be combined with more than one species of antigen.</p><p> When using saccharide or carbohydrate antigens, the antigen is preferably conjugated to a carrier protein to enhance immunogenicity (eg, Ramsay et al. (2001) Lancet 357 (9251): 195-196; Lindberg (1999). Vaccine17Suppl2: S28-36; Buttery &amp; Moxon (2000) JR Coll Physicans Lond34: 163-168; Ahmad &amp; Chapnick (1999) Infect Dis Clin North Am 13: 113-133, vii.Goldblatt (1998) J.Med.Microbiol.47 : 563-567; European Patent No. 0477508; US Pat. No. 5,306,492; WO98 / 42721; Conjugate Vaccines (eds.Cruse et al.) ISBN3805549326, especially vol.10: 48-114; Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X). Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoid. CRM₁₉₇Diphtheria toxoids are particularly preferred (Research Disclosure, 453077 (Jan 2002). Other carrier proteins include N. meningitidis external membrane proteins (EP-A-0372501), synthetic peptides (EP-A-0378881 and EP-A-). 0427347), heat shock protein (WO93 / 17712 and WO94 / 03208), pertussis protein (WO98 / 58668 and EP-A-0471177), protein derived from H. influenza<u style="single">D</u>Toxins A or B from (WO00 / 56360), cytokines (WO91 / 01146), phosphokines, hormones, growth factors, C. difficile<u style="single">(WO00 / 61761)</u>, Iron absorbing protein (WO01 / 72337) and the like. If the mixture contains capsule (capsule) saccharides from both serum groups A and C, the MenA saccharide: MenC saccharide ratio (w / w) is greater than 1 (eg 2: 1, 3: 1, 4). It is preferably 1, 1, 5: 1, 10: 1 or more). It is possible to attach various saccharides to the same or different types of carrier proteins. Any bonding reaction may be used as long as a suitable linker is available, if necessary.</p><p> Toxic protein antigens may be detoxified, if desired, such that pertussis toxin is detoxified by, for example, chemical and / or genetic means.</p><p> If the diphtheria antigen is included in the composition, it is preferred to also include the tetanus antigen and the whooping cough antigen. Similarly, if the tetanus antigen is included in the composition, it is preferred to also include the diphtheria antigen and the whooping cough antigen. If the whooping cough antigen is included in the composition, it is preferable to also include the diphtheria antigen and the tetanus antigen.</p><p> The antigens in the composition are typically present at a concentration of at least 1 μg / ml each. In general, the concentration of antigen is sufficient to elicit an immune response against that antigen.</p><p> As an alternative to using protein antigens in the compositions of the present invention, nucleic acids encoding the antigens may be used (eg Robinson &amp; Torres (1997) Seminars in Immunology 9: 271-283; Donnely et al. (1997). ) Annu Rev Immunol 15: 617-648; Scott-Taylor &amp; Dalgleich (2000) Expert Opin Investig Drugs 9: 471-480; Apostolopoulos &amp; Plebanski (2000) Curr Opin Mol Ther 2: 441-447; Ilan (1999) Curr Opin Mol Ther 1: 116-120; Dubensky et al. (2000) Mol Med 6: 723-732; Robinson &amp; Pertner (2000) Adv Virus Res 55: 1-74; Donnelly et al. (2000) Am J Respir Crit Care Med 162 (4Pt2): S190-S193; Davis (1999) Mt.Sinai J.Med.66: 84-90). The protein component of the composition of the invention may be replaced by a nucleic acid (in DNA, eg, in the form of a plasmid) encoding the protein.</p><p> (Definition) The term "contains" means "consisting of", including "contains," for example, a composition that "contains" X may contain only X, or something else, such as X. It may contain + Y.</p><p> The term "about" with respect to the number x means, for example, x ± 10%.</p><p> When referring to the percent sequence identity between two amino acid sequences, it is the percentage of amino acids that, when aligned, are the same when comparing the two sequences. For this alignment and percent homology or sequence identity, use software programs known in the art, such as those described in Section 7.7.18 of Current Protocols in Molecular Biology (FM Ausubel et al., Eds., 1987). It is possible to confirm. The preferred alignment is defined by the Smith-Waterman homology search algorithm. This algorithm has a gap opening penalty of 12 and a gap expansion penalty of 2, and uses an affine gap search using the BLOSUM matrix of 62. Smith-Waterman Homology Search Program is Smith &amp; Disclosed in Waterman (1981) Adv. Appl. Math.2: 482-489. Similar sequence identity methods can be used to determine homology between two polynucleotide sequences.</p><p> The examples below illustrate one way to prepare the recombinant GAS antigens of the invention and test their efficacy in a rodent model.</p>

(Example 1: Preparation of recombinant GAS antigen of the present invention and proof of efficacy in rodent model) Recombinant GAS proteins corresponding to two or more GAS antigens in the first antigen group are expressed as follows.

(1. Cloning of GAS antigen for E. coli expression) The GAS antigens selected were cloned to give two different recombinant proteins. That is, (1) a protein having a 6-histidine tag at the carboxy terminus (Gas-His), (2) a protein having a 6-histidine tag at the carboxy terminus and Gst at the amino terminus (Gst-Gas-His). Type (1) protein was obtained by cloning into a pET21b + vector (obtained from Novagen). Type (2) protein was obtained by cloning into a pGEX-NNH vector. This cloning strategy allows GAS genomic DNA to be used to amplify selected genes by PCR, perform single-enzyme digestion of the PCR product, and then clone the digested product simultaneously in both vectors. became.

((a) Construction of pGEX-NNH expression vector) DNA synthesizer<u style="single">ABI394</u>(Perkin Elmer) and Cruachem<u style="single">(Glasgow, Scotland)</u>Two couples of complementary oligodeoxyribonucleotides were synthesized using reagents obtained from. Equal moles of oligo pairs (50 ng for each oligo) were annealed in T4 DNA ligase buffer (New England Biolabs) for 10 minutes with a final volume of 50 μl and allowed to slowly cool to room temperature. The following DNA linker is obtained by the treatment step described below.

<chemistry num="17"><img file="JP4875490B2_D0018.tif" /></chemistry> The plasmid pGEX-KG (KLGuan and JEDixon, Anal.Biochem.192,262 (1991)) was digested with BamHI and HindIII, and 100 ng was subjected to linker / plasmid molar ratio of 3 using 200 units of T4 DNA ligase (New England Biolabs). Ligate to linker gexNN at 16 ° C at 1: 1. E. coli ligation product<u style="single">DH5</u>Traits<u style="single">Turn</u>After conversion, clones containing the pGEX-NN plasmid with the appropriate linker were selected by restriction enzyme analysis and DNA sequencing.

This novel plasmid pGEX-NN was digested with SalI and HindIII and ligated to the linker gexNNH. E. coli ligation product<u style="single">DH5</u>Traits<u style="single">Turn</u>After conversion, clones containing the pGEX-NN plasmid with the appropriate linker were selected by restriction enzyme analysis and DNA sequencing.

((B) Preparation of chromosomal DNA) GAS SF370, OD₆₀₀In THY culture until<u style="single">Proliferate</u>The bacteria were then centrifuged, suspended in a TES buffer containing lysozyme (10 mg / ml) and mutanolicin (10 U / μl), and incubated at 37 ° C for 1 hour. Bacterial suspensions are treated with RNase, proteinase K and 10% sarcosyl / EDTA, followed by protein extraction with saturated phenol and phenol / chloroform. The resulting supernatant is precipitated with sodium acetate / ethanol, the extracted DNA is pelleted by centrifugation, suspended in Tris buffer and stored at -20 ° C.

((C) Oligonucleotide design) Synthetic oligonucleotides based on the coding sequence of each GAS antigen using the sequence of the Streptococcus pyogenes SF370 M1 strain<u style="single">Primer</u>To design. Predicted by deriving the 5'end amplification primer sequence immediately downstream of the predicted leader sequence<u style="single">Signal peptide</u>Was removed altogether. For many GAS antigens<u style="single">I</u>And the 5'tail of the primer (table below)<u style="single">1</u>See), it contains only one restriction enzyme recognition site (NdeI, or NheI, or SpeI, depending on the restriction pattern of the gene itself). 3'primer tail (see Table 1) contains XhoI, or NotI, or HindIII restriction sites<u style="single">。</u>

<chemistry num="18"><img file="JP4875490B2_D0019.tif" /></chemistry> (Table 1: Oligonucleotide tail of the primer used to amplify the gene encoding the selected GAS antigen) Primers not only contain restriction enzyme recognition sites, but also nucleotides that hybridize to the amplified sequence. The number of nucleotides to hybridize depends on the melting temperature of the primers, which can be determined as previously described (Breslauer et al., Proc. Nat. Acad. Sci. 83,3746-50 (1986)). ). The average melting temperature of the selected oligos is 50-55 ° C for the hybridized region alone and 65-75 ° C for the whole oligo. Oligo was purchased from MWG-Biotech SpA (Florence, Italy).

((d) PCR amplification) The standard PCR protocol is as follows. Using 50 ng of genomic DNA as a template, each primer 0.2 μM, each dNTP 200 μM, 1.5 mM MgCl₂, 1xPCR buffer minus Mg (Gibco-BRL), and 2 units of Taq DNA polymerase (Platinum Taq, Gibco-BRL) to a final volume of 100 μl. Each sample is amplified in two steps. The first 5 cycles are performed at the hybridization temperature of the oligo excluding the restriction enzyme tail, and the next 25 cycles are performed according to the hybridization temperature of the full length of the primer. The standard cycle is as follows. 1 Cycle Degeneration: 94 ° C, 2 minutes 5 cycles Degeneration: 94 ° C, 30 seconds Hybridization: 51 ° C, 50 seconds Elongation: 72 ° C, 1 minute or 2 minutes and 40 seconds 25 cycles Degeneration: 94 ° C, 30 seconds Hybridization: 70 ° C, 50 seconds Elongation: 72 ° C, 1 minute or 2 minutes and 40 seconds 72 ° C, 7 minutes 4 ° C.

The extension time for GAS antigens encoded by ORFs shorter than 2000 bp is 1 minute, and for ORFs longer than 2000 bp it is 40 seconds. Amplification was performed using the Gene Amp PCR system 9600 (Perkin Elmer).

To check the amplification results, load 4 μl of each PCR product on a 1-1.5 agarose gel and determine the size of the amplified fragment to the DNA molecular weight standard (DNA marker III or I).<u style="single">X</u>, Roche). The PCR product was loaded on an agarose gel, and after electrophoresis, a band of the desired size was excised from the gel. DNA was purified from agarose using a gel extraction kit (Qiagen) according to the manufacturer's instructions. The final elution volume of DNA is 50 μl TE (10 mM Tris-HCl, 1 m).<u style="single">M</u> EDTA, pH 8). 1 μl of each purified DNA was loaded on an agarose gel and its product was evaluated.

((e) Digestion of PCR fragments) Purified PCR product<u style="single">1~</u>2 μg was double digested overnight at 37 ° C with a suitable restriction enzyme (60 units of each enzyme) in a final volume of 100 μl with a suitable restriction buffer. Restriction enzymes and digestion buffers were obtained from the New England Buffer. After purification of the digested DNA (PCR purification kit, Qiagen) and elution with 30 μl TE, 1 μl was subjected to agarose gel electrophoresis and the product was evaluated by comparison with the molecular weight standard of simultaneous electrophoresis (DNA marker III or IX, Roche).

((f) Digestion of cloning vector (pET21b + and pGEX-NNH)) Incubate 10 μg of plasmid overnight at 37 ° C with 100 units of each restriction enzyme in 400 μl of reaction in the presence of suitable buffer.<u style="single">Double digested by</u>.. After electrophoresis with 1% agarose, the band corresponding to the digested vector was purified using Qiagen's Qiaex II gel extraction kit and the DNA was eluted with 50 μl TE. DNA concentration, sample OD₂₆₀Was evaluated by measuring.

((g) Cloning of PCR product) 75 ng of appropriately digested and purified vector and each selected<u style="single">GAS</u>The digested and purified fragment corresponding to the antigen is in a manufacturer-supplied buffer using 400 units of T4 DNA ligase (New England Biolabs) in a final volume of 10-20 μl containing a fragment / vector molar ratio of 1: 1. Ligated in the presence. The reaction was incubated overnight at 16 ° C.

Escherichia coli BL21 (Novagen) and Escherichia coli BL21-DE3 (Novagen) electroreactive cell traits<u style="single">Turn</u>The conversion is performed using the pGEX-NHH conjugate and the pET21b + conjugate, respectively. Traits<u style="single">Turn</u>The conversion process is as follows. 1-2 μl of ligation reaction was mixed with 50 μl of ice-cold competent cells, and then the cells were poured into a 0.1 cm electrode cuvette (Biorad) of the gene pulsar. After applying a pulsed current to the cells in a micropulsar electroporator (Biorad) according to the manufacturer's instructions, the cells were suspended in 0.95 ml SOC culture medium.<u style="single">Shake</u>Incubate at 37 ° C for 45 minutes. Sprinkle 100 and 900 μl of cell suspension on separate plates of 100 μg / ml ampicillin agar LB and incubate this plate overnight at 37 ° C. Traits<u style="single">Turn</u>Screening for replacement cells is performed by PCR. Randomly selected traits<u style="single">Turn</u>Take up the replacement cells, PCR buffer, 4dNTP, 1.5 mM MgCl₂, Taq polymerase, and 30 μl PCR reaction mixture containing the appropriate antegrade and retrograde oligonucleotide primers. The above primers are pET21b + or pGEX-NNH vectors<u style="single">Polylinker</u>It is capable of hybridizing from upstream to downstream. After 30 cycles of PCR, 5 μl of product was subjected to agarose gel electrophoresis analysis to select positive clones with the expected PCR band. PCR-positive clones were selected based on the appropriate size of the PCR product according to evaluation by comparison with a suitable molecular weight marker (DNA marker III or IX, Roche).

(2. Protein expression) PCR-positive clones were inoculated into 3 ml LB 100 μg / ml ampicillin and overnight at 37 ° C.<u style="single">Proliferated</u>.. 70 μl of this overnight culture was inoculated into 2 ml of LB / Amp and the OD of the pET clone.₆₀₀Until is 0.4-0.8 value, or OD of pGEX clone₆₀₀At 37 ° C until is 0.8-1<u style="single">Proliferate</u>Ta. Protein expression is then induced by adding 1 mM IPTG (isopropyl β-D thio-galacto-pyranoside) to this miniculture. Final OD after incubating at 37 ° C for 3 hours₆₀₀Check and cool the culture on ice. After centrifugation of 0.5 ml of culture, cell pellet is subjected to 50 μl protein-loaded sample buffer (60 mM TRIS-HCl, pH 6.8, 5% w / v SDS, 10% v / v glycerin, 0.1% w / v bromophenol blue. , 100 mM DTT) and incubate at 100 ° C for 5 minutes. One volume of boiled sample corresponding to 0.1 OD600 culture was analyzed by SDS-PAGE and the presence of induced protein bands was confirmed by Coomassie blue staining.

(3. Purification of recombinant protein) A single colony is inoculated into 25 ml LB 100 μg / ml ampicillin and overnight at 37 ° C.<u style="single">Proliferate</u>Ta. Inoculate 500 ml of LB / Amp with this overnight culture and OD₆₀₀Until it reaches a value of 0.4-0.7<u style="single">Shake</u>While at 25 ° C<u style="single">Proliferate</u>Ta. Protein expression is then induced by adding 1 mM IPTG to this culture. Final OD after incubating at 25 ° C for 3.5 hours₆₀₀Check and cool the culture on ice. After centrifugation at 6000 rpm (JA10 rotor, Beckman), cell pellet was treated for purification or frozen at -20 ° C.

((a) Step for purifying soluble His-tagged protein from E. coli) (1) Transfer the pellets from -20 ° C to an ice bath and add 10 ml of 50 mM NaHPO.₄Reconstitute in buffer, 300 mM NaCl, pH 8.0, transfer to a 40-50 ml centrifuge tube and destroy cells as outlined below. (2) Destroy cells with a French press while passing them through serial washing 3 times. (3) Centrifuge at about 30-40000g for 15-20 minutes. Use rotor JA25.50 (21000rpm, 15 minutes) or JA-20 (18000rpm, 15 minutes) (4) Equilibrate the polyprep column with 1 ml of fast chelated sepharose resin containing 50 mM phosphate buffer, 300 mM NaCl, pH 8.0. (5) Store the centrifugal pellet at -20 ° C and load the supernatant on the column. (6) Collect spillage (7) Add 5 of 10ml (2ml + 2ml + 4ml) to the column.<u style="single">0</u>Wash with mM phosphate buffer, 300 mM NaCl, pH 8.0 (8) Wash again with 10 ml of 20 mM imidazole buffer, 50 mM phosphoric acid, 300 mM NaCl, pH 8.0. (9) The protein bound to the column is eluted with 4.5 ml (1.5 ml + 1.5 ml + 1.5 ml) of 250 mM imidazole buffer, 50 mM phosphoric acid, 300 mM NaCl, and pH 8.0, and each corresponds to about 1.5 ml. Collect fractions. Add 15 μl DTT 200 mM (final concentration 2 mM) to each tube (10) Measure the protein concentration of the first 2 fractions by the Bradford method, collect 10 μg of each sample and analyze by SDS-PAGE (Note: 21 μl + if the sample is overdiluted) Load 7 μl load buffer) (11) Save the collected fraction at + 4 ° C while waiting for the results of SDS-PAGE analysis. (12) For immunization, prepare 4-5 separate solutions in which 100 μg of each is dissolved in 0.5 ml of 40% glycerol. The dilution buffer is the above-mentioned elution buffer, plus 2 mM. DDT. Store the separatory funnel at -20 ° C until immunization.

((B) Purification of His-tagged protein from inclusion bodies) Purification was effectively performed according to the protocol below: (1) Bacteria are collected by centrifugation from a 500 ml culture. If desired, store bacterial pellets at -20 ° C. After extraction, each bacterial pellet is resuspended in 10 ml 50 mM TRIS-HCl, pH 8.5 buffer in an ice bath. (2) Destroy the resuspended bacteria while passing them twice with a French press. (3) Centrifuge at 35000xg for 15 minutes and collect pellets. Use Beckman rotor JA25.50 (21000rpm, 15 minutes) or JA-20 (18000rpm, 15 minutes) (4) Dissolve the centrifugal pellet in 50 mM TRIS-HCl, 1 mM TCEP (Tris (2-carboxyethyl) -phosphine hydrochloride, Pierce), 6M guanidine chloride, pH 8.5. Stir with a magnet bar for about 10 minutes (5) Centrifuge as described above and collect the supernatant. (6) Prepare a sufficient number of Polyprep (Bio-Rad) columns containing 1 ml of fast chelated Pharmacia saturated with Nitchell according to the manufacturer's instructions. Column, 5 ml H₂Wash twice with O and equilibrate with 50 mM TRIS-HCl, 1 mM TCEP, 6M guanidine chloride, pH 8.5 (7) Load the supernatant from step (5) onto a column and wash with 5 ml of 50 mM TRIS-HCl, 1 mM TCEP, 6M urea, pH 8.5. (8) Wash the column with 10 ml of 20 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5 and set the first 5 ml aside for use as a control in some cases. (9) Add 4.5 ml of 250 mM imidazole, 50 mM to the protein bound to the column. Elute at TRIS-HCl, 6M urea, 1mM TCEP, pH 8.5. Add elution buffer to 3 1.5 ml separators and collect 3 corresponding fractions. Add 15 μl DTT 200 mM (final concentration 2 mM) to each fraction (10) Measure the eluted protein concentration by the Bradford method, and analyze about 10 μg of protein separator by SDS-PAGE. (11) Protein, 40% (v / v) glycerol, 50mM TRIS-HCl, 2M urea, 0.5M arginine, 2mM Dissolve in DTT, 0.3 mM TCEP, 83.3 mM imidazole, pH 8.5 and store at -20 ° C.

((C) Step for purifying GST-fusion protein from E. coli) (1) Bacterial pellets were transferred from -20 ° C to an ice bath and suspended in 7.5 ml PBS, pH 7.4, in which a mixture of protease inhibitors (COMPLETE -Boehringer Mannheim, 1 in 25 ml buffer). Lock) (2) Transfer to a 40-50 ml centrifuge tube and sonicate according to the following steps. Place the probe approximately 0.5 cm from the bottom of the tube b. Clamp the tube c. Immerse the tube in an ice bath d. Set the ultrasonic device as follows. Timer-&gt; Hold, Work Cycle-&gt; 55, Output Control-&gt; 6 e. Perform 5 cycles of 10 pulses per minute (ie 1 cycle = 10 pulses + about 45''hold; b.10 pulses + about 45''hold; c.10 pulses + about 45''hold; d.10 Pulse + approx. 45''hold; e.10 pulse + approx. 45''hold) (3) Centrifuge at 30-40000xg for 15-20 minutes. For example, use Beckman rotor JA25.50 at 21000 rpm for 15 minutes. (4) Store the centrifugal pellet at -20 ° C and load the supernatant on the chromatography column as shown below. (5) The polyprep (Bio-Rad) column was equilibrated with 0.5 ml (about 1 ml suspension) of glutathione-sepharose 4B resin and 2 ml (1 + 1) H.₂Wash with O, then with 10 ml (2 + 4 + 4) PBS, pH 7.4 (6) Load the supernatant on the column and discard the effluent. (7) Wash the column with 10 ml (2 + 4 + 4) PBS, pH 7.4. (8) Elute the protein bound to the column with 4.5 ml of 50 mM TRIS buffer, 10 mM reduced glutathione, pH 8.0, add 1.5 ml + 1.5 ml + 1.5 ml, and add about 1.5 ml of each of the three fractions. To collect (9) Measure the protein concentration of the first 2 fractions by the Bradford method, collect 10 μg protein fraction from each sample, and analyze by SDS-PAGE (Note: 21 μl if the sample is overdiluted). Load +7 μl load buffer) (10) Save the collected fraction at + 4 ° C while waiting for the results of SDS-PAGE analysis. (11) For each protein scheduled to be used for immunization, prepare a 4-5 fraction in which 100 μg of each protein is dissolved in 0.5 ml of 40% glycerol. The dilution buffer is 50 mM TRIS HCl, 2 mM DDT, pH 8.0. Store the separatory funnel at -20 ° C until immunization.

(4. Rodent model protected from GAS infection) ((a) Immunization protocol) A group of 10 CD1 female mice aged 6 to 7 weeks is immunized with two or more GAS antigens of the invention (appropriate solution).<u style="single">In 100 μl</u>20 μg of each recombinant GAS antigen suspended in. Administer 3 times on days 0, 21, and 45 for each group. Immunization is by intraperitoneally injecting protein with an equal dose of Freund's complete adjuvant (CFA) in the first dose and with an equal dose of Freund's incomplete adjuvant (IFA) in subsequent two doses. went. Negative and positive control groups are used in each immunization scheme.

In the negative control group, mice were treated with an E. coli protein that was eluted from a purified column after treatment with a total bacterial extract of an E. coli strain and was either a pET21b vector or a pGEX-NNH vector (thus expressing only GST). Immunized with E. coli protein containing or but without cloned GAS ORF (groups labeled as His stop or GST stop, respectively).

In the positive control group, mice are immunized with purified GAS M cloned from either the GAS SF370 or GAS DSM2071 strain (groups are labeled 192SF and 192DSM, respectively).

Serum pooled from each group is collected before the first immunization and 2 weeks after the last immunization. Mice are infected with GAS about 1 week later.

Immunized mice are infected with a different GAS strain than that used to clone the selected protein. For example, the GAS strain may be DSM2071M23, available from the German Microbial and Cell Culture Preservation and Collection Facility (DSMZ).

DSM2071 for infection experiments, OD₆₀₀In THY liquid medium at 37 ° C until 0.4<u style="single">Proliferate</u>To. Bacteria are pelleted by centrifugation, washed once with PBS, suspended in PBS to obtain an appropriate concentration of bacteria per ml, and administered to mice by intraperitoneal injection. From the results of quantifying the separated liquid of the bacterial suspension by sprinkling it on 5 THY plates, 50 to 100 bacteria were administered to each mouse. Observe animals daily to check survival.

(5. Analysis of immune serum) ((a) Preparation of GAS whole protein extract) Whole protein extract, bacterial culture, OD₆₀₀Incubate in Tris 50 mM, pH 6.8 / mutanolicin (20 units / ml) for 2 hours at 37 ° C until 0.4-0.5, then incubate on ice for 10 minutes in 0.24N NaOH and 0.96% β-mercaptoethanol. .. The extracted protein is precipitated by the addition of trichloroacetic acid, washed with ice-cold acetone and suspended in a protein loading buffer.

((B) Western blot analysis) Whole mixed with SDS-loaded buffer (1x: 60 mM TRIS-HCl, pH6.8, 5% w / vSDS, 10% v / v glycerin, 0.1% bromophenol blue, 100 mM DDT) and boiled at 95 ° C for 5 minutes. Separations of protein extracts were loaded on a 12.5% SDS-PAGE precast gel (Biorad). Gel, 250 mM Transfer using an SDS-PAGE working buffer containing TRIS, 2.5 mM glycine, and 0.1% SDS. The gel is electroblasted onto a nitrocellulose membrane at 200 mA for 60 minutes. Membranes were blocked with PBS / 0.05% Tween-20 (Sigma), 10% scum milk powder in 60 minutes and at 4 ° C with PBS / 0.05% Tween-20, 1% scum milk powder and a suitable dilution of serum. Incubate with. After washing twice with PBS / 0.05% Tween, the membrane is incubated with 1: 4000 diluted peroxidase-conjugated anti-mouse secondary antibody (Amersham) for 2 hours. The nitrocellulose is washed 3 times with PBS / 0.05% Tween for 10 minutes, 1 time with PBS, and then developed with the Opti-4CN Substrate Kit (Biorad).

((C) Preparation of paraformaldehyde-treated GAS culture) OD₆₀₀Until 0.4-0.5<u style="single">Proliferate</u>Bacterial cultures are washed once with PBS and concentrated 4-fold in PBS / 0.05% paraformaldehyde. After incubation at 37 ° C for 1 hour with shaking, the treated cultures are left overnight at 4 ° C, then complete inactivation of the bacteria is controlled by sprinkling the separator on a THY blood agar plate. To do.

((d) FACS analysis of paraformaldehyde-treated GAS cultures with mouse immune sera) About 10⁵The paraformaldehyde-inactivated bacteria are washed on a 96-well U-bottom plate with 200 μl PBS and centrifuged at 4 ° C for 10 minutes at 3000 g. Discard the supernatant and suspend the bacteria in 20 μl PBS-0.1% BSA. 80 μl of either pre-immune or immune serum diluted with PBS-0.1% BSA with a final dilution of 1: 100, 1: 250, or 1: 500<u style="single">Any of</u>Add to bacterial suspension and incubate on ice for 30 minutes. Bacteria were washed once by adding 100 μl PBS-0.1% BSA, centrifuged at 4 ° C for 10 minutes at 3000 g, suspended in 200 μl PBS-0.1% BSA, centrifuged again and 10 μl goat anti-mouse. Suspend in PBS-0.1% BSA solution of IgG so that it is finally diluted 1: 100. This antibody is F (ab')₂Fragment, specific-R-phycoerythrin-conjugate (Jackson Immunoresearch Laboratories Inc., Catalog No. 115-116-072). Darken this suspension<u style="single">At the place</u>Incubate on ice for 30 minutes. Bacteria are washed once with 180 μl PBS-0.1% BSA and centrifuged at 4 ° C for 10 minutes at 3000 g. Discard the supernatant and suspend the bacteria in 200 μl PBS. Bacterial suspensions are passed through a cytometric chamber in FACS Calibur (Becton Dikinson, Mountain View, CA, USA) to obtain 10,000 data. Data are analyzed using Cell Quest Software (Becton Dikinson, Mountain View, CA, USA) by drawing morphological dot plots against bacterial signals (using forward and side scattering parameters). Next, a histogram plot is created for the FL2 intensity on the logarithmic scale of fluorescence, recalling the morphological region of the bacterium.

(Example 2: Comparison of toxicity between wild-type GAS strain (including GAS40) and GAS40 deletion mutation) The example below shows a toxicity comparison between a wild-type GAS strain and a GAS40 deletion mutation. GAS mutants from which most of GAS40 was removed were prepared by standard methods. Several immunized groups, consisting of 10 mice per group, were injected with either wild GAS strains or mutant GAS strains. As shown below, infusion of wild-type isolates in a certain concentration range results in lethality of mice, whereas infusion of GAS Δ40 mutant strains is not lethal.

<chemistry num="19"><img file="JP4875490B2_D0020.tif" /></chemistry> (Example 3: Bacterial opsonin phagocytosis assay of GAS40 construct) The example below demonstrates surface exposure of GAS40 using a bacterial opsonin phagocytosis assay. The following GAS constructs were used in the assay. Each of them is described in detail above. That is, 40a-CH, 40a-RR-NH, 40a-RR, GST-40, 40a, 40a and 40a-NH. (There are two references to "40a" in Figure 7, but these refer to sera prepared on different days).

The assay was performed as follows.

(1. Preparation of bacterial inoculum) Central log phase (OD) of GAS bacteria in THY culture₆₀₀At 37 ° C until 0.4) is reached<u style="single">Proliferate</u>To. Bacteria are washed twice with cold saline and suspended in HBSS culture. At that time, the volume of each strain is adjusted according to the amount of bacteria used. Bacterial cells are kept on ice until use.

(2. Preparation of PMN) PMN is a heparinized blood of healthy volunteers<u style="single">Buffy coat</u>Prepare from.<u style="single">Buffy coat</u>Incubate in a solution containing dextran, NaCl and heparin (1: 1 ratio) for 30 minutes. After incubation, the leukocyte-rich supernatant is removed, transferred to a clean tube and centrifuged at 700 g for 20 minutes. A short wash with water is performed to destroy the red blood cells, then a NaCl solution is added to restore the appropriate salt concentration. After this step, the cells are centrifuged and suspended in MEM at an appropriate concentration.

(3. Opson Phagocytosis Assay) Heat-inactivated immune mouse serum, human PMN, and baby rabbit complement obtained from immunization of the GAS strain (prepared as described above) with the indicated GAS antigen (or pre-immunization in the control). Incubate with. 1 hour incubation at 37 ° C. Inoculate THY blood agar plates with samples taken immediately before and after incubation. Phagocytosis is evaluated for pre-immune and immune sera by comparing the difference in colony numbers at two time points. The data is expressed as the logarithm of the number of growing colonies at t = 0 and the logarithm of the number of growing colonies at t = 60.

The results of the assay are shown in Figure 7. The Y-axis represents the difference between the logarithm of the number of colonies at 0 and the logarithm of the number of colonies after 60 seconds: log (CFU @ T0)-log (CFU @ T60'). If there is proliferation (ie, if the bacteria are not actively killed), a negative number (negative bar) is obtained. If the bacteria are killed, a positive number (positive histogram bar) is obtained. As shown in FIG. 7, a positive histogram bar is recorded for each GAS construct. The last four yellow bars in Figure 7 represent controls. That is, B = bacteria only, B PMN = bacteria + polymorphonuclear cells, BC = bacteria + complement, P PMN C = bacteria + polymorphonuclear cells + complement (without serum).

(Example 4: In a rodent mouse model for protection<u style="single">GAS40</u>Immunization Challenge Experiment) Below is a sample of the survival rate results obtained from a number of rodent mouse model experiments performed using the GAS40 antigen. The note indicates where in the construct used to express the recombinant GAS40 antigen, it was modified to promote expression.

<chemistry num="20"><img file="JP4875490B2_D0021.tif" /></chemistry> It will be appreciated that the present invention has been described for illustration purposes only and that modifications can be made while remaining within the scope and spirit of the invention.<maths num="1"><img file="JP4875490B2_D0022.tif" /></maths><maths num="2"><img file="JP4875490B2_D0023.tif" /></maths><maths num="3"><img file="JP4875490B2_D0024.tif" /></maths><maths num="4"><img file="JP4875490B2_D0025.tif" /></maths><maths num="5"><img file="JP4875490B2_D0026.tif" /></maths><maths num="6"><img file="JP4875490B2_D0027.tif" /></maths><maths num="7"><img file="JP4875490B2_D0028.tif" /></maths><maths num="8"><img file="JP4875490B2_D0029.tif" /></maths><maths num="9"><img file="JP4875490B2_D0030.tif" /></maths><maths num="10"><img file="JP4875490B2_D0031.tif" /></maths><maths num="11"><img file="JP4875490B2_D0032.tif" /></maths><maths num="12"><img file="JP4875490B2_D0033.tif" /></maths><maths num="13"><img file="JP4875490B2_D0034.tif" /></maths><maths num="14"><img file="JP4875490B2_D0035.tif" /></maths><maths num="15"><img file="JP4875490B2_D0036.tif" /></maths><maths num="16"><img file="JP4875490B2_D0037.tif" /></maths><maths num="17"><img file="JP4875490B2_D0038.tif" /></maths><maths num="18"><img file="JP4875490B2_D0039.tif" /></maths><maths num="19"><img file="JP4875490B2_D0040.tif" /></maths><maths num="20"><img file="JP4875490B2_D0041.tif" /></maths><maths num="21"><img file="JP4875490B2_D0042.tif" /></maths><maths num="22"><img file="JP4875490B2_D0043.tif" /></maths><maths num="23"><img file="JP4875490B2_D0044.tif" /></maths><maths num="24"><img file="JP4875490B2_D0045.tif" /></maths><maths num="25"><img file="JP4875490B2_D0046.tif" /></maths><maths num="26"><img file="JP4875490B2_D0047.tif" /></maths><maths num="27"><img file="JP4875490B2_D0048.tif" /></maths><maths num="28"><img file="JP4875490B2_D0049.tif" /></maths><maths num="29"><img file="JP4875490B2_D0050.tif" /></maths><maths num="30"><img file="JP4875490B2_D0051.tif" /></maths><maths num="31"><img file="JP4875490B2_D0052.tif" /></maths><maths num="32"><img file="JP4875490B2_D0053.tif" /></maths><maths num="33"><img file="JP4875490B2_D0054.tif" /></maths><maths num="34"><img file="JP4875490B2_D0055.tif" /></maths><maths num="35"><img file="JP4875490B2_D0056.tif" /></maths><maths num="36"><img file="JP4875490B2_D0057.tif" /></maths><maths num="37"><img file="JP4875490B2_D0058.tif" /></maths><maths num="38"><img file="JP4875490B2_D0059.tif" /></maths>

<figref num="1">FIG. 1 identifies a leader peptide sequence, two coiled coil sequences, a leucine zipper sequence, and a transmembrane sequence in the GAS40 amino acid sequence.</figref><figref num="2">FIG. 2 shows a schematic representation of GAS40 identifying a leader peptide sequence, two coiled coil sequences, a leucine zipper sequence, and a transmembrane sequence, as well as other known or predicted functions. It shows the coiled-coil region of GAS40, which has only a low degree of homology with the streptococcal protein.</figref><figref num="3-1">FIG. 3 includes BLAST alignment analysis of the identified GAS40 coiled coil region with other streptococci.</figref><figref num="3-2">FIG. 3 includes BLAST alignment analysis of the identified GAS40 coiled coil region with other streptococci.</figref><figref num="3-3">FIG. 3 includes BLAST alignment analysis of the identified GAS40 coiled coil region with other streptococci.</figref><figref num="3-4">FIG. 3 includes BLAST alignment analysis of the identified GAS40 coiled coil region with other streptococci.</figref><figref num="4-1">FIG. 4 shows the predicted secondary structure of the amino acid sequence of GAS40.</figref><figref num="4-2">FIG. 4 shows the predicted secondary structure of the amino acid sequence of GAS40.</figref><figref num="5-1">FIG. 5 schematically shows the position of GAS40 in the GAS genome. Also included is a comparative schematic showing GAS suddenly displaced species lacking GAS40. These schematics are further detailed with the prospect that GAS40 was acquired by a horizontal transition due to a transposon factor.</figref><figref num="5-2">FIG. 5 schematically shows the position of GAS40 in the GAS genome. Also included is a comparative schematic showing GAS suddenly displaced species lacking GAS40. These schematics are further detailed with the prospect that GAS40 was acquired by a horizontal transition due to a transposon factor.</figref><figref num="6">Figure 6 shows a comparative analysis of FACS showing the surface exposure of GAS40 in wild-type strains (GAS40-deficient mutants have no surface exposure).</figref><figref num="7-1">FIG. 7 shows data on opson phagocytosis in GAS40 (in various expression constructs).</figref><figref num="7-2">FIG. 7 shows data on opson phagocytosis in GAS40 (in various expression constructs).</figref><figref num="8">FIG. 8 shows immunization and antigen infusion data for some GAS antigens of the invention.</figref>