USOO6270775B1 (12) United States Patent (10) Patent No.: US 6,270,775 B1 Cleary (45) Date of Patent: *Aug. 7, 2001

(54) STREPTOCOCCAL C5A PEPTIDASE Berg, A., et al., “Streptococcal CySteine Proteinase Releases WACCINE Biologically Active Fragments of Streptococcal Surface Proteins”, The Journal Of Biological Chemistry, 270(17), 9862–9867, (1995). (75) Inventor: Paul Patrick Cleary, Shoreview, MN Bessen, D., et al., “Influence of Intranasal Immunization (US) with Synthetic Peptides Corresponding to Conserved Epitodes of M Protein on Mucosal Colonization by Group A (73) Assignee: Regents of the University of Streptococci”, Infection and Immunity, 56(10), 2666-2672, Minnesota, Minneapolis, MN (US) (1988). (*) Notice: Subject to any disclaimer, the term of this Bessen, D., et al., “Synthetic Peptide Vaccine Against patent is extended or adjusted under 35 Mucusal Colonization by Group A Streptococci. I. Protec U.S.C. 154(b) by 0 days. tion Against a Heterologous M Serotype with Shared C Repeat Region Epitopes”, J Immunol, 145(4), 1251-1256, This patent is Subject to a terminal dis (1990). claimer. Booth, S., et al., “DapSone Suppresses Integrin-Mediated Neutrophil Adherence Function”, The Journal of Investiga (21) Appl. No.: 09/206,800 tive Dermatology, 98(2), 135-140, (1992). Boyle, M., et al., “Measurement of Leukocyte Chemotaxis (22) Filed: Dec. 7, 1998 in Vivo”, Methods in Enzymology, 162, 101-114, (1988). Related U.S. Application Data Bronze, M., et al., “Epitopes of group A Streptococcal M protein that evoke cross-protective local immune 62) Division of applicationpp No. 08/589,756, filed on Jan. 22, responses”, J. Immunol., 148(3), 888-893, (1992). 1996, now Pat. No. 5,846,547. Bronze, M., et al., “Protective Immunity Evoked by Locally (51) Int. Cl...... A61K 39/09; A61K 39/02; Administered Group A Strepticoccal Vaccines in Mice”, J. A61K 39/00; A61K 38/00; C12N 1/20 Immunol., 141 (8), 2767-2770, (1988). (52) U.S. Cl...... 424/244.1; 424/234.1; 424/1841; 424/93; 424/177; 435/252.3; Brummer, E., et al., “Immunological Activation of Polymor 514/2; 514/13 phonuclear Neutrophilis for Fungal Killing: Studies with (58) Field of Search ...... 424/244.1, 234.1, Murine Cells and Blastomyces Dermatitidis In Vitro”, Jour 424/1841, 93,177; 435/252.3; 514/2, 13 nal of Leukocyte Biology, 36,505–520, (1984). Chen, C.C., et al., “Complete Nucleotide Sequence of the (56) References Cited Streptococcal C5A Peptidase Gene of Streptococcus pyo genes', The Journal of Bilogical Chemistry, 265(6), U.S. PATENT DOCUMENTS 3161–3167, (1990). 4,454,121 6/1984 Beachey ...... 424/177 Clark, J., et al., “A New Method for Quantitation of Cell 4,695,562 9/1987 Beachey et al...... 514/13 Mediated Immunity in the Mouse”, Journal of the Reticu 4,772,584 9/1988 Cleary et al...... 514/2 loendothelial Society, 25(3), 255–267, (1979). 5,124,153 6/1992 Beachy et al...... 424/93 P 5,162,226 11/1992 Beachey et al...... 435/252.3 Cleary, P., et al., “A Streptococcal Inactivator of Chemot 5,846,547 * 12/1998 Cleary et al...... 424/244.1 axis: A new Virulence Factor Specific to Group A Strepto cocci”, Recent Advances in Streptococci and Streptococcal Diseases (Y. Kimura, S. Kotami and Y. Shiokawa (ed). FOREIGN PATENT DOCUMENTS Reedbooks ltd; Berkshire, England, 179-180, (1984). 89/09064 10/1989 (WO). 93/14198 7/1993 (WO). (List continued on next page.) 93/21220 10/1993 (WO). 94/06421 3/1994 (WO). 94/06465 3/1994 (WO). Primary Examiner Nita Minnifield 95/28960 11/1995 (WO). ASSistant Examiner Padma Baskar 97/26008 1/1996 (WO) ...... A61 K/39/09 (74) Attorney, Agent, or Firm-Schwegman, Lundberg, Woessner & Kluth, PA. OTHER PUBLICATIONS (57) ABSTRACT Brown protective immunity administered A Strept vaccines Novel vaccines for use against B-hemolytic StreptococcuS J. of Imm 141-2767-2770, 1988.* colonization or infection are disclosed. The vaccines contain Carter, P., et al., “Dissecting the catalytic triad of a Serine an immunogenic amount of Streptococcal C5a peptidase, or protease”, Nature, vol. 332, XP002135964, 564-568, (Apr. a fragment or mutant thereof. Also disclosed is a method of 7, 1988). protecting a Susceptible mammal against B-hemolytic Strep Stafslien, D.K., et al., “Site Directed Mutagenesis of the tococcuS colonization or infection by administering Such a Streptococcal C5a Peptidase”, Abstracts of the General Mtg Vaccine. of the Am. Soc for Microbiology, vol. 98, XP002135963, 59, (May, 1998). 42 Claims, 8 Drawing Sheets US 6,270,775 B1 Page 2

OTHER PUBLICATIONS Massell, B., et al., “Rheumatic Fever Following Streptococ Cleary, P., et al., “Similarity Between the Group B and A cal Vaccination”, JAMA, 207(6), 1115–1119, (1969). Streptococcal C5a Peptidase Genes”, Infection and Immu Mcghee, J., et al., “New Perspective in Mucosal Immunity nity, 60(10), 4239-4244, (1992). with Emphasis on Vaccine Development”, Seminars in Cleary, P., et al., “Streptococcal C5a Peptidase is a Highly Hematology, 30(4), 3–15, (1993). Specific Endopeptidase”, Infection and Immunity, 60(12), Medaglini, D., et al., “Mucosal and Systemic immune 5219–5223, (1992). responses to a recombinant protein expressed on the Surface Cleary, P., et al., “Virulent Human Strains of Group G of the oral commensal bacterium StreptococcuS gordonii Streptococci Express a C5a Peptidase Enzyme Similar to after oral colonization”, Proc. Natl. Acad. Sci. USA, 92(15), that Produced by Group A Streptococci”, Infection and 6868–6872, (1995). Immunity, 59(7), 2305-2310, (Jul 1991). Oconnor, S., et al., “In Vivo Streptococcus Pyogenes C5a Courtney, H.S., et al., “Analysis of the role of M24 Protein Peptidase Activity: Analysis Using Transposon- and in group A Streptococcal adhesion and colonization by use Nitrosoguanidine-Induced Mutants”, The Journal of Infec of omega'-interpoSon mutagenesis”, Infect. Immun., tious Diseases, 156(3), 495-504, (1987). 62(11), 4868–4873, (1994). Oconnor, S., et al., “The Human Antibody Response to FenderSon, P., et al., “Tropomyosin Shares immunology Streptococcal C5a Peptidase”, The Journal of Infection Epitopes with Group A Streptococcal M Proteins”, The Diseases, 163, 109-116, (1991). Journal of Immunology, 142(7), 2475–2481, (Apr. 1989). Fischetti, V., “Streptococcal M Protein: Molecular Design Podbielski, A., et al., “The Group A Streptococcal VirR49 and Biological Behavior”, Clinical Microbiology Reviews, Gene Controls Expression of Four Structural Vir Regulon 2(3), 285-314, (Jul. 1989). Genes”, Infection and Immunity, 63(1), 9–20, (1995). Fischetti, V.A., et al., “Protection against Streptoccal pha Raeder, R., et al., “Properties of IgG-binding proteins ryngeal colonization with vaccines composed of M protein expressed by StreptococcuS pyogenes isolates are predictive conserved regions”, Adv. Exp. Med. Bio., 303, 159-167, of invasive potential”, J. Infect. Dis., 173(4), 888-895, (1991). (1996). Fischetti, V.A., et al., “Protection against Streptococcal pha Springer, T., et al., “Mac-1: A Macrophage Differentiation ryngeal colinization with a vaccinia: M protein recombi Antigen Identified by Monoclonal Antibody”, European nant", Science, 244, 1487–1490, (1989). Journal of Immunology, 9, 301-306, (1979). Hill, H., et al., “Group B Streptococci Inhibit the Chemot Sriskandan, S., et al., “Streptococcal pyrogenic eXotoxin A actic Activity of the Fifth Component of Complement”, The (SPEA) release, distribution, and role in a murine model of Journal of Immunology, 141(10), 3551-3556, (1988). fasciitis and multi-organ failure due to StreptococcuS pyO Hope-Simpson, R., "StreptococcuS Pyogenes in the Throat: genes”, J. Infect. Dis., 173(6), 1399–1407, (1996). A study in a small population, 1962-1975, J. Hyg. Camb., Sriskandan, S., et al., “The role of nitric oxide in experi 87, 109–129, (1981). mental murine Sepsis due to pyrogenic eXotoxin A-produc Ji, Y., et al., “C5a peptidase alters clearance and trafficking ing Streptococcus pyogenes', Infect. Immun., 65(5), of group A Streptococci by infected mice’, Infection and 1767–1762, (1997). Immunity, 64(2), 503–510, (1996). Stevens, D., “Invasive Group A Streptococcus Infections”, Ji, Y., et al., “Intranasal immunization with C5a peptidase prevents nasopharyngeal colonization of mice by the group Clinical Infectious Diseases, 14, 2-13, (1992). A streptococcus”, Infect. Immun., 65(6), 2080-2087, (1997). Suvorov, A., et al., “C5a Peptidase Gene from Group A Kapur, V., et al., "Vaccination with Streptococcal extracel Streptococci', Genetics and Molecular Biology of Strepto lular cysteine protease (interleukin-1B convertase) protects COcci, Lactococci, and EnterOCOcci, Washington, D.C., mice against challenge with heterologous group A Strepto 230–232, (1991). cocci”, Microbial. Path., 16(6), 443–450, (1994). Vugia, D.J., et al., “Invasive group A Streptococcal infec Laemmli, U.K., “Cleavage of Structural Proteins during the tions in children with varicella in Southern California’, Assembly of the Head of Bacteriophage T4, Nature, 227, Pediatr. Infect. Dis. J., 15(2), 146-150, (1996). 680–685 (1970). Wessels, M., et al., “Critical role of the group A streptococ Lee, P., et al., “Quantification and Toxicity of Group A cal capsule in pharyngeal colonization and infection in Streptococcal Pyrogenic Exotoxins in an Animal Model of mice", Proc. Natl. Acad. Sci. USA, 91(25), 12238-12242, Toxic Shock Syndrome-Like Illness”, Journal of Clinical (1994). Microbiology, 27(8), 1890–1892, (1989). Wexler, D., et al., “Mechanism of Action of the Group A Martin, T., et al., “The Effect of Type-Specific Polysaccha Streptococcal C5a Inactivator', Proc. Natl. Acad. Sci. 82, ride Capsule on the Clearance of Group B Sreptococci from 8144-8148, (1985). the Lungs of Infant and Adult Rats', The Journal of Infec tious Diseases, 165, 306–314, (1992). * cited by examiner U.S. Patent Aug. 7, 2001 Sheet 1 of 8 US 6,270,775 B1

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3 8 O O US 6,270,775 B1 1 2 STREPTOCOCCAL CSA PEPTIDASE M protein: molecular design and biological behavior, Clin. WACCINE Microbiol. Rev., 2:285-314 (1989). Thus, inoculation with one serotype-specific M protein will not likely be effective This application is a divisional of U.S. patent application in protecting against other M Serotypes. The Second problem Ser. No. 08/589,756 filed on Jan. 22, 1996, now U.S. Pat. 5 relates to the Safety of an M protein vaccine. Several regions No. 5,846,547. of the M protein contain antigenic epitopes which are The invention was made with the Support of NIH Grant immunologically croSS-reactive with human tissue, particu No. AI 20016-11. The U.S. government has certain rights in larly heart tissue. The N-termini of M proteins are highly the invention. variable in Sequence and antigenic specificity. Inclusion of more than 80 different peptides, representing this variable BACKGROUND OF THE INVENTION Sequence, in a vaccine would be required to achieve broad There are Several different B-hemolytic Streptococcal Spe protection against group A Streptococcal infection. New cies that have been identified. StreptococcuS pyogenes, also variant M proteins would still continue to arise, requiring called group A Streptococci, is a common bacterial pathogen ongoing Surveillance of Streptococcal disease and changes in of humans. Primarily a disease of children, it causes a 15 the vaccine composition. In contrast, the carboxyl-termini of variety of infections including pharyngitis, impetigo and M proteins are conserved in Sequence. This region of the M Sepsis in humans. Subsequent to infection, autoimmune protein, however, contains an amino acid Sequence which is complications Such as rheumatic fever and acute glomeru immunologically cross-reactive with human heart tissue. lonephritis can occur in humans. This pathogen also causes This property of M protein is thought to account for heart Severe acute diseases Such as Scarlet fever, necrotizing Valve damage associated with rheumatic fever. P. FenderSon fasciitis and toxic shock. et al., “Tropomyosinsharies immunologic epitopes with Sore throat caused by group A Streptococci, commonly group A Streptococcal M proteins, J. Immunol. called “strep throat,” accounts for at least 16% of all office 142:2475-2481 (1989). In an early trial, children who were calls in a general medical practice, depending on the Season. vaccinated with M protein in 1979 had a ten fold higher Hope-Simpson, E., "StreptococcuS pyogenes in the throat: A incidence of rheumatic fever and associated heart Valve study in a small population, 1962-1975, ” J. Hyg. Camb., damage. Massell, B. F., et al., “Rheumatic fever following 87: 109-129 (1981). This species is also the cause of the streptococcal vaccination, JAMA, 207:1115-1119 (1969). recent resurgence in North America and four other conti Other proteins under consideration for vaccine develop nents of toxic shock associated with necrotizing fasciitis. ment are the erythrogenic toxins, Streptococcal pyrogenic Stevens, D. L., “Invasive group A StreptococcuS infections,” eXotoxin A and Streptococcal pyrogenic eXotoxin B. Lee, P. Clin. Infect. Dis., 14:2-13 (1992). Also implicated in caus K., et al., “Quantification and toxicity of group A Strepto ing Strep throat and occasionally in causing toxic shock are coccal pyrogenic eXotoxins in an animal model of toxic groups C and G Streptococci. Hope-Simpson, E., "Strepto shock syndrome-like illness,” J. Clin. Microb., coccus pyogenes in the throat: A Study in a Small population, 27:1890–1892 (1989). Immunity to these proteins could 35 prevent the deadly Symptoms of toxic shock, but will not 1962–1975, "J. Hyg. Camb., 87:109–129 (1981). prevent colonization by Streptococci, nor likely lower the Group B Streptococci, also known as StreptococcuS incidence of Strep throat. Estimates Suggest that the inci agalactiae, are responsible for neonatal Sepsis and menin dence of toxic shock infections is 10 to 20 cases per 100,000 gitis. T. R. Martin et al., “The effect of type-specific polysac population; therefore, use of these proteins to immunize the charide capsule on the clearance of group B Streptococci 40 general population against toxic Shock is neither practical from the lung of infant and adult rats”, J. Infect Dis., 165:306–314 (1992). Although frequently a member of nor economically feasible. vaginal mucosal flora of adult females, from 0.1 to 0.5/1000 Thus, there remains a continuing need for an effective newborns develop Serious disease following infection dur means to prevent or ameliorate Streptococcal infections. ing delivery. In spite of the high mortality from group B More specifically, a need exists to develop compositions 45 useful in vaccines to prevent or ameliorate colonization of Streptococcal infections, mechanisms of the pathogenicity host tissues by Streptococci, thereby reducing the incidence are poorly understood. Martin, T. R., et al., “The effect of of Strep throat and impetigo. Elimination of Sequelae Such as type-specific polysaccharide capsule on the clearance of rheumatic fever, acute glomerulonephritis, Sepsis, toxic Group B Streptococci from the lung of infant and adult rats,” Shock and necrotizing fasciitis would be a direct conse J. Infect. Dis., 165:306–314 (1992). 50 quence of reducing the incidence of acute infection and Streptococcal infections are currently treated by antibiotic carriage of the organism. A need also exists to develop therapy. However, 25-30% of those treated have recurrent compositions useful in vaccines to prevent or ameliorate disease and/or shed the organism in mucosal Secretions. At infections caused by all B-hemolytic Streptococcal Species, present no means is available to prevent Streptococcal infec namely groups A, B, C and G. tions. Historically, Streptococcal vaccine development has 55 focused on the bacterium's cell Surface M protein. Bessen, SUMMARY OF THE INVENTION D., et al., “Influence of intranasal immunization with Syn The present invention provides a vaccine, and methods of thetic peptides corresponding to conserved epitopes of M vaccination, effective to prevent or reduce the incidence of protein on mucosal colonization by group A Streptococci,” B-hemolytic StreptococcuS in Susceptible mammals, includ Infect. Immun., 56:2666-2672 (1988); Bronze, M. S., et al., 60 ing humans, and domestic animals Such as dogs, cows, pigs “Protective immunity evoked by locally administered group and horses. The vaccine contains an immunogenic amount A Streptococcal vaccines in mice,” Journal of Immunology, of streptococcal C5a peptidase (SCP), or one or more 141:2767-2770 (1988). immunogenic fragments or mutants thereof in combination Two major problems will limit the use, marketing, and with a physiologically-acceptable, non-toxic vehicle. The possibly FDA approval, of a M protein vaccine. First, more 65 vaccine may comprise a fragment or mutant SCP that lackS than 80 different M serotypes of S. pyogenes exist and new SCP enzymatic activity (dSCP). It may also contain an Serotypes continually arise. Fischetti, V. A., “Streptococcal immunological adjuvant. The vaccine can be used to prevent US 6,270,775 B1 3 4 colonization of group A Streptococcus, group B polymorphonuclear leukocytes (PMNs) and mononuclear Streptococcus, group C StreptococcuS or group G Strepto cells at the Site of infection. Attraction of these cells is coccuS. The vaccine may comprise an immunogenic recom mediated by chemotactic Stimuli, Such as host factors or binant Streptococcal C5a peptidase conjugated or linked to factorS Secreted by the invading organism. The C5a an immunogenic peptide or to an immunogenic polysaccha 5 chemoattractant is pivotal to the Stimulation of this inflam ride. matory response in mammals. C5a is a 74 residue glyco peptide cleaved from the fifth component (C5) of comple The Streptococcal C5a peptidase vaccine can be admin ment. Phagocytic cells respond in a directed manner to a istered by Subcutaneous or intramuscular injection. gradient of C5a and accumulate at the Site of infection. C5a Alternatively, the vaccine can be administered by oral inges may be the most immediate attractant of phagocytes during tion or intranasal inoculation. inflammation. As PMNs infiltrate an inflammatory lesion As described in the working examples below, an SCP they Secrete other chemokines, Such as IL8, which further gene (ScpA49) was cloned into an E. coli expression vector intensify the inflammatory response. (pGex-4T-1). The transferase-SCP fusion from the E. coli clone was expressed and purified. The purified recombinant Streptococcal C5a peptidase (SCP) is a proteolytic 15 enzyme located on the Surface of pathogenic Streptococci SCP (dSCP) was then used to immunize mice. The vacci where it destroys C5a, as C5a is locally produced. SCP nated mice and a control group of mice were then challenged specifically cleaves the C5a chemotaxin at the PMN binding with wild-type Streptococci. The mice receiving the recom site (between His'7-Lys' residues of C5a) and removes the binant SCP vaccine were free of streptococci soon after seven most C-terminal residues of C5a. This cleavage of the infection, whereas 30-50% of the control group were culture PMN binding site eliminates the chemotactic signal. Cleary, positive for many days. Therefore, the recombinant SCP was P., et al., “Streptococcal C5a peptidase is a highly specific effective as a vaccine against 3-hemolytic Streptococci. endopeptidase,” Infect. Immun., 60:5219–5223 (1992); Wexler, D. E., et al., “Mechanism of action of the group A BRIEF DESCRIPTION OF THE DRAWINGS streptococcal C5a inactivator,” Proc. Natl. Acad. Sci. USA, FIG. 1. Architecture of C5a peptidase from B-hemolytic 25 82:8144–8148 (1985). Streptococci. D indicates an aspartic acid residue, H indi SCP from group A Streptococci is a Subtilisin-like Serine cates histidine, S indicates Serine; L indicates leucine, P protease with an M of 124,814 da and with a cell wall indicates proline; T indicates threonine; and N indicates anchor motif which is common to many gram bacterial asparagine. R, R2, Ra and R indicate repeated Sequences. Surface proteins. The architecture of C5a peptidase is given The numbers indicate the amino acid residue position in the in FIG. 1. The complete nucleotide Sequence of the Strep peptidase. tococcal C5a peptidase gene of StreptococcuS pyogenes has FIG. 2. Alignment of the amino acid sequence of SCP been published. Chen, C., and Cleary, P., “Complete nucle from group A SEQ ID NO:6 corresponds to the region otide Sequence of the Streptococcal C5a peptidase gene of consisting of amino acid residues 130 through 512 of SEQ Streptococcus pyogenes,” J. Biol. Chem., 265:3161-3167 ID NO:1. SEQID NO:7 corresponds to the region consisting 35 (1990). In contrast to Subtilisins, SCP has a very narrow of amino acid residues 130 through 512 of SEQ ID NO:2. Substrate Specificity. This narrow Specificity is Surprising in SEQ ID NO:8 corresponds to the region consisting of amino light of the marked Similarities between their catalytic acid residues 130 through 512 of SEQ ID NO:3. SEQ ID domains. Cleary, P., et al., “Streptococcal C5a peptidase is a NO:9 corresponds to amino acid residues 130 through 1050 highly Specific endopeptidase, Infect. Immun., of SEQID NO:1. SEQID NO:10 corresponds to amino acid 40 60:5219–5223 (1992). Residues involved in charge transfer residues 130 through 1050 of SEQ ID NO:2. SEQ ID NO:11 are conserved, as are residues on both sides of the binding corresponds to amino acid residues 130 through 1050 of pocket, however, the remaining amino acid Sequence of SCP SEQ ID NO:3. Streptococci strain 49, group A streptococci is unrelated to that of Subtilisins. More than 40 serotypes of strain 12 and group B streptococci (SEQ ID NO:1, SEQ ID Group A streptococci were found to produce SCP protein or NO:2, and SEQ ID NO:3, respectively). The sequences are 45 to harbor the gene. Cleary, P., et al., “A Streptococcal identical except for the indicated amino acid positions. The inactivator of chemotaxis: a new virulence factor Specific to triangle (A) indicates the predicted cleavage point of the group A Streptococci,” in Recent Advances in Streptococci Signal peptidase. Amino acids predicted to be in the and Streptococcal Disease p. 179-180 (S. Kotami and Y. enzyme’s active site are marked by asteriskS. Deletions in Shiokawa ed., Reedbooks Ltd., Berkshire, England; 1984); the amino acid Sequence are indicated by dots and are boxed. 50 Podbielski, A., et al., “The group A streptococcal virR49 FIG. 3. Construction of SCP insertion and deletion gene controls expression of four structural Vir regulon mutants. Black box indicates deleted region. genes.” Infect. Immun., 63:9-20 (1995). FIG. 4. Single color FACS analysis. Fluorescence data A C5a peptidase enzyme associated with group B Strep were analyzed by gating on PMNS. A Second gate was Set to tococci has also been identified. Hill, H. R., et al., “Group B count high Staining cells defined by the first gate. Air Sacs 55 streptococci inhibit the chemotactic activity of the fifth were inoculated with 1x10 CFU. component of complement,” J. Immunol. 141:3551-3556 (1988). Restriction mapping and completion of the scpB FIG. 5. Persistence of Wild type and SCPA following nucleotide sequence showed that scpb is 97-98% similar to intranasal infection. ScpA. See FIG. 2 for comparison of the amino acid Sequence FIG. 6. Intranasal immunization of CD-1 mice with SPCA 60 of SCP from group A Streptococci Strain 49, group A protein interferes with oral colonization by M type 49 Streptococci strain 12 and group B Streptococci (SEQ ID Streptococci. NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively). DETAILED DESCRIPTION OF THE More than 30 Strains, representing all Serotypes of group B INVENTION streptococci carry the scpB gene. Cleary P. P., et al. “Simi 65 larity between the Group B and A Streptococcal C5a Pepti An important first line of defense against infection by dase genes.” Infect. Immun. 60:4239-4244 (1992); Suvorov many bacterial pathogens is the accumulation of phagocytic A. N., et al., “C5a peptidase gene from group B US 6,270,775 B1 S 6 streptococci,” in Genetics and Molecular Biology of healthy individuals. Sera containing high concentrations of Streptococci, Lactococci, and Enterococci p. 230-232 (G. anti-SCP immunoglobulin were capable of neutralizing SCP Dunny, P. Cleary and L. McKay (ed.); American Society for activity. Detection of this antibody in >90% of the saliva Microbiology, Washington, D.C.; 1991). Specimens obtained from children who had recently expe rienced Streptococcal pharyngitis demonstrated that children Human isolates of groups G and C Streptococci also can produce an antibody response. harbor ScpA-like genes. Some group G Strains were shown Even though the human Subjects produced IgG and IgA to express C5a Specific protease activity on their Surface. against SCP in response to a natural Streptococcal infection, Cleary, P. P., et al., “Virulent human strains of group G it was not known whether the anti-SCP immunoglobulin Streptococci express a C5a peptidase enzyme similar to that provides any protection against infection. The basis for produced by group A Streptococci.” Infect. Immun., immunity to Streptococcal infection following natural infec 59:2305-2310 (1991). Therefore, all serotypes (>80) of tion is poorly understood. Further, it was not known if the group A Streptococci, group B Streptococci, group C Strep SCP protein could act as a vaccine against 3-hemolytic tococci and group G Streptococci produce the SCP enzyme. Streptococcal colonization or infection. First, a study was SCP assists Streptococci to colonize a potential infection 15 performed to examine the role of SCP in colonization of the Site, Such as the nasopharyngeal mucosa, by inhibiting the nasopharynx. Following intranasal infection with live group influx of phagocytic white cells to the site of infection. This A Streptococci, throat cultures were taken daily for up to ten impedes the initial clearance of the Streptococci by the host. days. Wild type and isogenic SCP-deficient mutant strepto The impact of SCP on inflammation, C5a leukocyte cocci were compared for the ability to persist in the throat chemotaxis and Streptococcal virulence was examined using over this ten day period. As predicted, the SCP-deficient Streptococcal Strains with well-defined mutations in the mutant Streptococci were cleared from the nasopharynx protease Structural gene. SCP mutants were constructed by more rapidly. targeted plasmid insertion and by replacement of the Wild The Same intranasal mouse model was used to test the type gene with ScpA containing a specific internal deletion. capacity of SCP to induce immunity which will prevent Mutants lacked C5a protease activity and did not inhibit the 25 colonization. A mutant form of the recombinant ScpA49 chemotactic response of human or mouse PMNs to C5a in gene (lacking 848-1033 nucleotides from the 5' end and vitro. 3941-4346 nucleotides from the 3' end of the gene) was A mouse connective tissue air Sac model was used to cloned into and expressed from the high expression vector confirm that SCP retards the influx of phagocytic cells and pGEX-4T-1. Enzymatically defective SCP protein (dSCP) clearance of Streptococci from the Site of infection. A was purified from an E. coli recombinant by affinity chro connective tissue air Sac is generated by injecting a Small matography. Sera from rabbits vaccinated intradermally amount of air and PBS (with or without streptococci in it) with this protein preparation neutralized SCP activity in with a 25-gauge needle under the skin on the back of a vitro. Purified protein (40 ug) was administered intranasally mouse. Boyle, M. D. P. et al., “Measurement of leukocyte to mice over a period of five weeks. Immunized mice cleared chemotaxis in vivo.” Meth. Enzymol., 162:101:115 (1988). 35 Streptococci in 1–2 days, whereas, throat cultures of non At the end of the experiment, the mice were euthanized by immunized mice remained positive for up to 10 days. The cervical dislocation, the air SacS dissected from the animals, experiment was repeated on three Sets of mice, vaccinated and the air SacS homogenized in buffer. An advantage of the with three separate preparations of a SCP protein. air Sac model is that the air Sac remains inflated for Several The present invention thus provides a vaccine for use to days and free of inflammation, unless an irritant is injected. 40 protect mammals against B-hemolytic StreptococcuS colo Thus, injected bacteria and the resulting inflammatory nization or infection. In one embodiment of this invention, response remains localized over short periods of infection. as is customary for vaccines, the Streptococcal C5a The air Sac model was modified to compare clearance of peptidase, variant or fragment thereof, can be delivered to a wild type SCP" and SCP streptococci, (i.e., group A strep mammal in a pharmacologically acceptable vehicle. AS one tococci which carried a mutant non-functional form of SCP) 45 skilled in the art will appreciate, it is not necessary to use the and to analyze the cellular infiltrate at an early Stage of entire protein. A Selected portion of the polypeptide (for infection. Tissue Suspensions were assayed for viable Strep example, a Synthetic immunogenic polypeptide correspond tococci on blood agar plates and the cellular infiltrate was ing to a portion of the Streptococcal C5a peptidase) can be analyzed by fluorescent cell sorting (FACS). In FACS used. analysis, individual cells in Suspension are labelled with 50 AS one skilled in the art will also appreciate, it is not Specific fluorescent monoantibodies. Aliquots of labelled necessary to use a polypeptide that is identical to the native cells are injected into a FAC-Scan flowcytometer, or fluo SCP amino acid Sequence. The amino acid Sequence of the rescent cell Sorter, which counts cells based on their unique immunogenic polypeptide can correspond essentially to the fluorescence. The experiments using the air Sac model native SCP amino acid Sequence. AS used herein “corre indicated that streptococci that were SCP were more viru 55 spond essentially to refers to a polypeptide Sequence that lent than streptococci that were SCP. will elicit a protective immunological response at least A Study was performed to measure production of human Substantially equivalent to the response generated by native antibody, both IgG and IgA, against SCP in human Sera and SCP. An immunological response to a composition or vac saliva. O’Connor, SP, et al., “The Human Antibody cine is the development in the host of a cellular and/or Response to Streptococcal C5a Peptidase,” J. Infect. Dis. 60 antibody-mediated immune response to the polypeptide or 163:109-16 (1991). Generally, sera and saliva from young, vaccine of interest. Usually, Such a response consists of the uninfected children lacked antibody to SCP. In contrast, Subject producing antibodies, B cell, helper T cells, Suppres most Sera and Saliva Specimens from healthy adults had Sor T cells, and/or cytotoxic T cells directed Specifically to measurable levels of anti-SCP IgG and SCP-specific secre an antigen or antigens included in the composition or tory IgA (anti-SCPSigA). Paired acute and convalescent Sera 65 vaccine of interest. Vaccines of the present invention can from patients with Streptococcal pharyngitis possessed Sig also include effective amounts of immunological adjuvants, nificantly higher levels of anti-SCP IgG than did sera from known to enhance an immune response. US 6,270,775 B1 7 8 Alternatively, the SCP can be conjugated or linked to product may be incorporated into liposomes for use in a another peptide or to a polysaccharide. For example, immu vaccine formulation, or may be conjugated to proteins Such nogenic proteins well-known in the art, also known as as keyhole limpet hemocyanin (KLH) or human Serum “carriers, may be employed. Useful immunogenic proteins albumin (HSA) or other polymers. include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, human serum albumin, human The application of SCP, subunit or mutant thereof, for gamma globulin, chicken immunoglobulin G and bovine vaccination of a mammal against colonization offers advan gamma globulin. Useful immunogenic polysaccharides tages over other vaccine candidates. Prevention of coloni include group A Streptococci poly Saccharide, Zation or infection by inoculation with a single protein will C-polysaccharide from group B Streptococci, or the capsular not only reduce the incidence of the very common problems polysaccharide of Streptococci pnuemoniae. Alternatively, of Strep throat and impetigo, but will also eliminate Sequelae polysaccharides of other pathogens that are used as Vaccines Such as rheumatic fever, acute glomerulonephritis, Sepsis, can be conjugated or linked to SCP toxic Shock and necrotizing fasciitis. The following examples are intended to illustrate but not To immunize a subject, the SCP or an immunologically limit the invention. active fragment or mutant thereof, is administered 15 parenterally, usually by intramuscular or Subcutaneous EXAMPLE 1. injection in an appropriate vehicle. Other modes of administration, however, Such as oral delivery or intranasal Construction of Insertion and Deletion Mutants in delivery, are also acceptable. Vaccine formulations will ScpA49 and ScpA6 contain an effective amount of the active ingredient in a a) Bacterial Strains and culture conditions. S. pyogenes vehicle, the effective amount being readily determined by strain CS101 is a serotype M49, and OF" strain. CS159 is a one skilled in the art. The active ingredient may typically clinical isolate with a deletion which extends through the M range from about 1% to about 95% (w/w) of the gene cluster and ScpA. A Spontaneous, Streptomycin resis composition, or even higher or lower if appropriate. The tant derivative of strain CS101, named CS101Sm, was quantity to be administered depends upon factorS Such as the 25 Selected by plating Streptococci from a Stationary phase age, weight and physical condition of the animal or the culture on tryptose blood agar containing Streptomycin (200 human Subject considered for vaccination. The quantity also ug/ml). CS101::pGhost5 is strain CS101 with pGhost5 depends upon the capacity of the animal’s immune System integrated into the chromosome at an unknown location, but to Synthesize antibodies, and the degree of protection outside ScpA and the emm gene cluster. Escherichia coli desired. Effective dosages can be readily established by one strain ER1821 (from New England Biolabs, Inc. Beverly, of ordinary skill in the art through routine trials establishing Mass.) was used as the recipient for the Suicide vector, dose response curves. The Subject is immunized by admin plasmid pChostS. Plasmid pC"hostS was obtained from istration of the SCP or fragment thereof in one or more Appligene, Inc. Pleasanton, Calif. Streptococci were grown doses. Multiple doses may be administered as is required to in Todd-Hewitt broth supplemented with 2% neopeptone or maintain a State of immunity to Streptococci. 35 1% yeast extract, or on tryptose agar plates with 5% sheep Intranasal formulations may include vehicles that neither blood. E. coli strain ER1821 containing plasmid pChostS cause irritation to the nasal mucosa nor Significantly disturb was grown in LB broth with erythromycin (300 tug/ml). ciliary function. Diluents Such as water, aqueous Saline or Streptococci with plasmid pChostS were cultured in Todd other known Substances can be employed with the Subject Hewitt broth with 1% yeast extract (THY) containing 1 invention. The nasal formulations may also contain preser 40 Aug/ml of erythromycin (Erm). Vatives Such as, but not limited to, chlorobutanol and ben SCP refers to streptococcal C5a peptidase from Zalkonium chloride. A Surfactant may be present to enhance B-hemolytic Streptococcus generally. SCPA12, SCPA49, absorption of the Subject proteins by the nasal mucosa. SCPA6 are the Specific peptidases from group A Strepto Oral liquid preparations may be in the form of, for coccus M type 12, 49 and 6 strains, respectively. The term example, aqueous or oily Suspension, Solutions, emulsions, 45 ScpA refers to the gene encoding SCP from group A Strep Syrups or elixirs, or may be presented dry in tablet form or tococci. ScpA12, ScpA6 and ScpA49 are the genes encoding a product for reconstitution with water or other suitable the SCPA12, SCPA49 and SCPA6 peptidases. SCPB and vehicle before use. Such liquid preparations may contain ScpB refer to the peptidase and gene from group B Strepto conventional additives Such as Suspending agents, emulsi cocci. The amino acid sequences for SCPA49 (SEQ ID fying agents, non-aqueous vehicles (which may include 50 NO:1), SCPA12 (SEQ ID NO:2) and SCPB (SEQ ID NO:3) edible oils), or preservative. are given in FIG. 2. To prepare a vaccine, the purified SCP, subunit or mutant b) Construction of ScpA insertion mutant. Well-defined thereof, can be isolated, lyophilized and stabilized. The SCP insertion mutants of ScpA were constructed using plasmid peptide may then be adjusted to an appropriate insertion and gene replacement methods. An internal concentration, optionally combined with a Suitable vaccine 55 ScpA49 BglII-BamHI fragment, the insertion target, was adjuvant, and packaged for use. Suitable adjuvants include ligated into the thermosensitive shuttle vector pGhostS to but are not limited to Surfactants, e.g., hexadecylamine, form plasmid pG::ScpA1.2 and transformed into E. coli octadecylamine, lySolecithin, dimethyldioctadecylammo ER1821 (FIG. 3). The pGhost5 vector contains an E. coli nium bromide, N,N-dioctadecyl-N'-N-bis(2-hydroxyethyl origin of replication that is active at 39 C., a temperature propane di-amine), methoxyhexadecyl-glycerol, and plu 60 sensitive Gram" origin of replication (active at 30° C. and ronic polyols, polanions, e.g., pyran, dextran Sulfate, poly inactive at 39 C. in Streptococci), and an erythromycin IC, polyacrylic acid, carbopol; peptides, e.g., muramyl resistance gene for Selection. High temperature forces the dipeptide, aimethylglycine, tuftsin, oil emulsions, alum, and plasmid to integrate into the chromosomal DNA of group A mixtures thereof. Other potential adjuvants include the B Streptococci by homologous recombinant at frequencies peptide subunits of E. coli heat labile toxin or of the cholera 65 ranging from 10° to 10. toxin. McGhee, J. R., et al., “On vaccine development,” Recombinant plasmid DNA poi::ScpA1.2 was electropo Sem. Hematol., 30:3-15 (1993). Finally, the immunogenic rated into CS101 recipient cells. Transformants were US 6,270,775 B1 10 Selected on THY-agar plates containing 1 lug/ml erythromy C. overnight. The culture pellet was washed two times in 5 cin at 30° C. Chromosomal integrants which resulted from ml cold 0.2 M NaAcetate (pH 5.2), then suspended in 1 ml recombination between the plasmid insert and the chromo TE-sucrose buffer (20% sucrose 10 mM Tris, 1 mM EDTA, Somal ScpA were selected by erythromycin resistance at 39 pH 7.0) and 40 ul Mutanolysin. The mixture was rotated at C. Two insertion mutants, M14 and M16, were analyzed. 5 37° C. for 2 hr, then centrifuged 5 min at 4500 rpm. EmrS revertants of strain M14 and M16 were obtained by Supernatants contained protease inhibitor, 100 mM phenyl passage in THY without antibiotic at 30° C. and finally methylsulfonyl fluoride (PMSF). Electrophoresis and West plated at 37°C. without Erm selection. Colonies that had lost ern blotting methods were performed as described in the plasmid were isolated to confirm that the mutant phe Laemmli, U.K., "Cleavage of Structural proteins during the notype resulted from insertion of the plasmid into ScpA49, assembly of the head of bacteriophage T4, ' Nature rather than from a Simultaneous unrelated mutation. 227:680–685 (1970). For colony blots, colonies were grown c) Introduction of a defined deletion into ScpA. A mutant on THY-agar plates, printed onto nitrocellulose membrane Strain with a defined deletion internal to ScpA was con (BioBlot-Nc, Costor, Cambridge, Mass.), fixed under an Structed to eliminate the possibility that insertions in ScpA infrared lamp for 10 min. and exposed to antibody. could be polar and reduce expression of downstream genes, 15 O'Connor, S. P. and Cleary, P. P., “In vivo Streptococcus unknown genes which could also contribute to the organ pyogenes C5a peptidase activity,” J. Infect. Dis. ism's virulence. First, a defined deletion in Bg|II-HindIII 156:495-506 (1987). The primary antiserum used to detect fragment of ScpA was produced by inside-out PCR with SCP protein on Western and colony blots was prepared by primer 1. (5'-GGGGGGGAATTC immunization of a rabbit with purified recombinant SCP GTAGCGGGTATCATGGGAC-3), SEQ ID NO:4, and protein. Binding was detected by anti-rabbit antibody alka primer 2 (5'-GGGGGGGAATTC line phosphatase conjugate. GGGTGCTGCAATATCTGGC-3"), SEQ ID NO:5. Under C5a peptidase activity was measured using a PMN adher lined nucleotides correspond to ScpA sequences with coor ence assay. Booth, S.A. et al., “DapSone Suppresses integrin dinates 2398 and 2322, respectively, and the bold faced mediated neutrophil adherence finction,” J. Invest. Derma nucleotides correspond to a EcoRI recognition Site. The 25 tol. 98:135-140 (1992). After incubation of C5a (Sigma, St. primers were Selected to produce an in-frame deletion in the Louis, Mo.) with Streptococcal extracts or purified protease, ScpA gene. These primerS copy plasmid DNA in opposite residual C5a can activate PMNs to become adherent to BSA directions and define the boundaries of the deletion. Innis, coated wells. First, microtiter wells were coated with 0.5% M. A., et al., eds., PCR Protocols A Guide to Methods and BSA in PBS and incubated for 1 hr at 37° C. Human PMNS Applications (Academic Press, 1990). Plasmid pG:scpA1.2 were isolated by centrifugation in Ficoll Hypague (Sigma, DNA was used as template. St. Louis, Mo.). 40 ul of intact Streptococci or protein The amplified product was digested with EcoRI and extracts were incubated with 20 ul of 5 uM C5a in 340 ul of ligated to plasmid pChosts. The resulting plasmid PBS with 1% glucose and 0.1% CaCl at 37° C. for 45 min. pG::AScpA1.1 contained an 76 bp deletion internal to ScpA. BSA-coated wells were washed with PBS, and resuspended This in-frame deletion removed 25 amino acids, including 35 PMNs and residual C5a were added to wells. The mixture the Serine which forms part of the predicted catalytic center was incubated for 45 min at 37° C. in 7% CO. Finally, wells of Serine proteases. Chen, C., and Cleary, P., “Complete were washed to remove nonadherent PMNS. Adherent nucleotide Sequence of the Streptococcal C5a peptidase gene PMNs were stained with crystal violet and the ODzo was of Streptococcus pyogenes,” J. Biol. Chem., 265:3161-3167 read in an ELISA reader. The optical density is proportional (1990). An EcoRV site was created at the point of deletion. 40 DNA which overlaps the deletion was sequenced to confirm to the amount of residual C5a or inversely proportional to the boundaries of the deletion. the amount of SCP activity. Insertion mutants completely lacked SCPA49 antigen; The plasmid pG:ScpA1.1, which contains the deletion, whereas, deletion mutants MJ2-5 and MJ3-15, as expected was transformed into E. coli ER1821. Colonies were produced SCP antigen. Both whole cells and mutanolysin Selected for ErmR and then Screened for the appropriate 45 protein extracts from M14, M16, M2-5 and MJ3-15 lacked ScpA deletion using miniprep plasmid DNA restricted by the ability to destroy rC5a activated adherence of PMNs to EcoRI. The precise boundaries of the deletion were con microtiter plates. A Small amount of residual inhibitory firmed by DNA sequencing. Plasmid pC::AScpA1.1 was activity (10-15%) associated with mutant extracts may be electroporated into strain CS101Sm as described above, then due to toxic effects of the extract on the neutrophils. integrants were selected by grown on Erm at 39 C. Inte 50 gration of the plasmid into the chromosome of the M49 Strain CS101sm using high temperature Selection. The inser EXAMPLE 2 tion location was confirmed by PCR. Growth of CS101Sm SCP Delays Recruitment of Phagocytes and (p.G::ScpA1.1) at low temperature without erythromycin Clearance of Streptococci from Subdermal Sites of Selection resulted in high frequency Segregation of ErmS 55 Infection revertants which have lost the plasmid by random deletion event or by excision due to recombination between the In order to verify that SCP was responsible for the duplicated ScpA Sequences created by the insertion. Two inactivation of C5a, the insertion and deletion mutants of deletion mutants were identified, MJ2-5 and MJ3-15, and ScpA were constructed as described in Example 1 above, and were studied further. The chromosomal deletion left behind 60 tested for activity. When insertions or deletions were intro by recombinational excision of plasmid pG::ScpA1.1 was duced into ScpA, the mutant SCP was not able to destroy defined by PCR and Southern hybridization to EcoRV C5a-activated adherence of PMNs to microtiter plates. digested DNA. The impact of mutations in ScpA on virulence was tested d) In vitro effects on SCP. The impact of insertions and using an animal model where Streptococci remained deletions on the expression of SCP antigen and peptidase 65 localized, and where the influx of inflammatory cells could activity was assessed by Western blot and PMNs adherence be analyzed. To test the hypothesis that SCP functions very assays. Streptococci were incubated in 100 ml THY at 37 early to retard initial clearance of the organism, the fate of US 6,270,775 B1 11 12 SCP and SCP streptococci just 4 hours after inoculation of connective tissue air SacS was compared. Moreover, the TABLE 1. dissemination of Streptococci to lymph nodes and Spleens after this short period of infection was also assessed. CD1 Distribution of SCP' and SCP streptococci male outbred mice (25 g) obtained from Charles River 4 hours after air sac infection Breeding Laboratory, Wilmington, Mass. were used for all No. of No. of positive cultures Homogenized experiments. A connective tissue air Sac was generated by injecting 0.9 ml of air and 0.1 ml group A Streptococci Strains Mice spleen lymph node Air Sac diluted in PBS with a 25-gauge needle under the skin on the CS101pG 8 7 2 1.3 x 10' + 2.2 x 107 back of the mouse. In some experiments the SCP 1O (SCP') M16 (SCP) 8 O 5 6.0 x 107 + 1.3 x 107 CS101::pGhost5 was used as a positive control. In other CS101Sm 8 6 2 1.6 x 10' + 2.6 x 107 experiments strain CS101Sm was used as the positive con (SCP') trol. Mice were euthanized by cervical dislocation 4 hours M3-15 8 1. 5 3.7 x 107 - 1.5 x 107 after infection. Where indicated all four inguinal lymph (SCP) nodes, Spleen and air Sac were dissected from the animals 15 Each mouse was inoculated with 3 x 10 CFU of stationary phase strep and homogenized in PBS. Tissue Suspensions were assayed tococci. for viable colony forming unit (CFU) on blood agar plates Difference in the frequency of isolation of SCP' streptococci from containing 1 lug/ml erythromycin or 200 lug/ml Streptomycin. spleens relative to SCP streptococci was statistically significant (P < 0.05) for each experiment by the Fisher's exact test. In a preliminary experiment air SacS were fixed on Slides, Differences in CFU isolated from homogenized air sacs (means + SEMs) Stained with Wright's Stain and examined microscopically. were significant, strains CS101 pCi (SCP) and M16 (SCP) and MJ3-15 Although counts of granulocytes by this method were (SCP) (P<0.001) for each experiment by unpaired t test. unreliable, there appeared to be significantly fewer residual The more rapid clearance of Streptococci from air Sacs SCP than wild type streptococci in fixed tissue. Additional experiments were performed in an attempt to measure this resulted from more intense recruitment of PMNS. The total difference. Dispersed cell populations of air SacS were 25 cell population, the percentage of Mac-1 positive granulo prepared by grinding the air Sac in PBS and passing them cytes (Springer, G. et al., “Mac-1:macrophage differentia through Nylon monofilament mesh (TETKO Co. New tion antigen identified by monoclonal antibody, Eur: J. York). Immunol. 9:301-306 (1979)), and the percentage of Gr-1 The cells were pelleted by centrifugation 5 min at 300xg positive PMN (Brummer, E. et al., “Immunological activa and resuspended at 5x10"/ml in FACS buffer (Hank's bal tion of polymorphonuclear neutrophils for fungal killing: anced salt solution without phenol red, 0.1% NaNa, 1.0% Studies with murine cells and blastomyces dermatitidis in BSA fraction V). Cells (10x10) were stained directly with vitro,” J. Leuko. Bio. 36:505–520 (1984)) in air sacs were 1 tug FITC anti-mouse Mac-1 or indirectly with 1 lug Biotin compared by single color FACS analysis. Clark, J. M., “A conjugated anti-mouse Gr-1 followed by 1 ug Streptavidin 35 new method for quantitation of cell-mediated immunity in labelled with fluorescene or FITC. Monoclonal antibodies, the mouse,” J. Reticuloendothel. Soc. 25:255-267 (1979). Mac-1 and Gr-1, were obtained from Pharmingen, Inc. CA. Briefly, in a FACS analysis, individual cells in Suspension Labeled cells were fixed in 1.0% paraformaldehyde. Fluo are labelled with specific fluorescent monoantibodies. Ali rescence profiles were generated using a FAC-Scan flow quots of labelled cells are injected into a FAC-Scan flow cytometer and Consort 32 software (Becton Dickinson). 40 Mouse PMNs were purified from whole blood by Ficoll cytometer or fluorescent cell Sorter which counts cells based Hypadue density gradient centrifugation and used as a on their unique fluorescence. standard to defined PMNs in mixed populations. For mea Air Sacs infected with the SCP deletion mutant contained Surement of Specifically labeled cells, the mean fluorescence twice as many inflammatory cells as those inoculated with for each antibody marker was determined and gates were Set 45 SCP" streptococci (FIG. 4). A hundred-fold increase in the to reflect intensely labeled cells. Controls included unstained inoculum size did not alter this difference. Air SacS infected cells, and cells exposed to only streptavidin FITC. with 1x10 SCP cells, strain MJ3-15, contained three times Two experiments were performed. The first compared the more Gr-1 positive cells than those inoculated with the SCP ScpA49 insertion mutant M16 to its SCP parent culture, culture. In airs sacs inoculated with SCP streptococci strain CS101. The second compared the ScpA49 deletion 50 approximately 6% of the cells were PMNs and 21% were mutant MJ3-15, to its parent, strain CS101Sm. (Table 1) In both experiments homogenized air SacS from mice inocu other kinds of Mac-1" granulocytes, including PMNs. In lated with SCP streptococci contained fewer numbers of contrast, air SacS inoculated with SCP Streptococci con Streptococci after 4 hours than air SacS inoculated with wild tained predominately PMNS. Gr-1 positive cells were equal type Streptococci. The first experiment showed a two-fold 55 to or greater than the number of Mac-1 positive cells. Flow reduction and the Second showed a four-fold reduction. cytometer gates were set to measure only high Staining These differences were statistically significant at P-0.05 and granulocytes. The remaining 70-80% of cells not stained P<0.001, respectively, using an Unpaired t-test. It was also with either antibody were likely either low staining observed that wild type SCP streptococci were found in granulocytes, red blood cells or lymphocytes. Large num Spleen homogenates from 7 of 8 mice and 6 of 8 mice; 60 bers of lymphocytes were observed microscopically in whereas, the SCP mutants were rarely found in the spleen. Wrights Stained air Sac preparations. The opposite was true for lymph node homogenates. Nodes from 10 of 16 mice infected with SCP streptococci har SCP colonies of streptococci that emerged from spleen bored viable streptococci; whereas, only 4 of 16 nodes from homogenates were highly encapsulated, resembling water mice infected with wild type Streptococci contained viable 65 drops. In contrast the few SCP colonies arising from lymph bacteria. This difference was determined to be statistically nodes, were more like the inoculum. They were mixtures of significant at P-0.05 using the Fisher's exact test. non-mucoid and moderately mucoid colonies. These data US 6,270,775 B1 13 14 Suggest that MSCP encapsulated Streptococci can adapt, streptococci (dSCP). This fragment was amplified by PCR multiply and invade the bloodstream within 4 hours after using a forward primer beginning at nucleotide 1033 and a infection. The basis for differential trafficking of mutant and reverse primer beginning at nucleotide 3941 (numbering wild type Streptococci may be due to the more vigorous corresponding to that of Chen, C., and Cleary, P., “Complete influx of phagocytic cells in response to SCP bacteria. nucleotide Sequence of the Streptococcal C5a peptidase gene Macrophages and/or skin dendritic cells may more rapidly of Streptococcus pyogenes,” J. Biol. Chem., 265:3161-3167 engulfed SCP streptococci and delivered them to lymph (1990)). The fragment was ligated to the thrombin binding nodes. Reduction of mutant Streptococci relative to wild Site of glutathione transferase gene on the pGeX-4T-1 high type is an unexpected finding, because SCP Streptococci are expression vector from Pharmacia Inc. The plasmid con M" and resistant to phagocytosis by human neutrophils in taining ScpA designated pCO has been deposited in the vitro. American Type Culture Collection, Rockville, Md., under the provision of the Budapest Treaty, and assigned ATCC EXAMPLE 3 accession number 98225. SCP is Required for Colonization of the Mouse 15 The transferase-SCP fusion protein from one E. coli clone Nasopharynx was expressed and purified by affinity chromatography on a Mice were inoculated intranasally to evaluate the relative glutathione Sepharose 4b column. All methods are described capacity of wild type (SCP) and SCP streptococci to by the manufacturer (Pharmacia). The dSCP was cleaved colonize the nasopharynx. Streptomycin resistant M49 Strain from the hybrid protein by thrombin digestion. The thrombin CS101 and deletion mutant MJ3-15 were used in these was removed from eluted SCP by chromatography on a experiments. Cultures were not mouse passed in order to benzamidine Sepharose 6B column (Pharmacia). The affin avoid Selection of variants that might be uniquely mouse ity purified protein was confirmed to be pure SCPA49 by SDS-PAGE and by Western blot. Hyperimmune antiserum, virulent, but no longer depend on M protein and/or SCP for 25 persistence in the animal. directed against purified SCPA49 was prepared in rabbits. CD1 outbred mice were intranasally inoculated with The recombinant SCP was not functional as a peptidase. 2x10 stationary phase CFU. The nasopharynxes of anes Two groups of mice were immunized by administration of thetized mice were Swabbed daily for 8-10 days and 10 ul into each nostril, a total of 40 ug of protein, four times Streaked on blood agar containing Streptomycin. Differences over a period of five weeks. Control mice received only between SCP' and SCP were evident by day 1, however, PBS. Prior to infection sera pooled from groups of 5 mice Statistically significant differences were not observed until were determined by ELISA to have high titers of anti days 3 and 4 (FIG. 5). By day four 9/18 mice infected with SCPA49 antibody. See Table 2. MSCP streptococci produced positive throat cultures, 35 whereas only 2/18 mice infected with MSCP strain TABLE 2 retained streptococci in their throats. Four of 18 mice died Titers of antibodies (IgG) against SCP from infection with SCP streptococci. None of the mice Before After following infection with SCP bacteria succumbed to the Exp Sample Immunization Immunization infection. The numbers of colonies on the blood agar plates 40 I pool I &1:10 1:320-1:640 were also consistent with more rapid clearance of SCP ScP pool II &1:10 1:32O Streptococci. For example, on the third day cultures from pool III &1:10 1:640-1:1,280 seven mice contained >100 SCP CFU, whereas, only one I pool I &1:10 &1:10 PBS pool II &1:10 &1:10 mouse inoculated SCP streptococci contained >100 CFU. 45 pool III &1:10 <1.10 Because M49 streptococci are more often associated with II pool I &1:10 1:5,120 PBS pool II &1:10 1:2,560 skin infections the above experiments were repeated with an pool III &1:10 1:5,120 M6 Strain, a Serotype more often associated with throat II pool I &1:10 &1:10 infections. An insertion mutant, Strain AK1.4, was con PBS pool II &1:10 &1:10 50 pool III &1:10 &1:10 structed using the M6 strain UAB200 and the strategy III pool I &1:10 1:800 previously described in Example 1. Strain AK1.4 was also SCP pool II &1:10 1:800 cleared more rapidly than the wild type M6 culture from the III PBS pool I &1:10 &1:10 nasopharynx. The above experiments confirm that group A Streptococci are dependent upon SCP for persistence in the 55 Mice were challenged with 3x10 CFU of the wild type, mouse nasopharynx. All SCP mutants used in the above CS101sm strain, 7 days after the last vaccine booster. In two experiments were M, i.e. they resisted phagocytosis by Separate experiments immunized mice were free of Strepto fresh human blood. Yet, they were cleared from the nasopha cocci 48 hrs after infection (FIG. 6; Tables 3 and 4). In ryngeal mucosa. contrast 30-50% of non-vaccinated controls remained cul 60 EXAMPLE 4 ture positive for six days, and Some were Still positive ten days after infection. Differences were determined to be Intranasal Immunization of Mice with Purified Statistically Significant by the Fisher exact test. Infection of Recombinant SCPA49 Blocks Colonization a third group of immunized and control mice produced Following Intranasal Challenge 65 Similar results. A PCR fragment which corresponds to a deleted form of High titer rabbit Serum directed against this mutant the ScpA49 gene was cloned from CS101 M49 group A SCPA49 protein was able to neutralize peptidase activity US 6,270,775 B1 15 16 associated with intact M1, M12, and M6 streptococci in Vitro, confirming that peptidase lackS Serotype specificity. TABLE 3-continued Therefore, even SCP which is not functional as a peptidase Throat cultures for Streptococci after intranasal challenge of mice is effective as a vaccine. It should be noted that pre- vaccinated intranasally with PBS or SCP expressed in E. coli DH5C. incubation of M49 streptococci with rabbit anti-SCP prior to 5 (CFU after vaccine) in. inoculation of mice did not reduce colonization. Days after challenge TABLE 3 Mice 1 2 3 4 5 6 7 8 9 10 Throat cultures for Streptococci after intranasal challenge of mice 1O No. of 8 6 5 5 4 4 2 3 2 2 vaccinated intranasally with PBS or SCP expressed in E. coli DH5C. positive CFU after vaccine SCP AD Days after challenge II

Mice 1 2 3 4 5 6 7 8 9 10 15 O O O O O O O O O O PBSCT 2 O O O O O O O O O O II 3 O O O O O O O O O O 4 O O O O O O O O O O 1. O O O O O O O O O O 5 35 O O O O O O O O O 2 3 O O O O O O O O O 20 6 O O O O O O O O O O 3 77 >200 150 4 11 3 O 51 97 53 7 O O O O O O O O O O 4 9 >2OO >2OO 3 11 3 O O O O 8 O O O O O O O O O O 5 O O O O O O O O O O 9 O O O O O O O O O O 6 4 6 45 47 3 >2OO 29 200 83 70 1O O O O O O O O O O 7 15 194 s2OO 9 172 10 5 3 O O 11 O O O 21 O O O O O O 8 O O O O O O O O O O 9 O 32 4 4 O O O O O O 25 12 O O O O O O O O O O 1O 2 O O O O O O O O O 13 O O O O O O O O O O 11 3 O O O O O O O O O No. of 1 O O 1. O O O O O O 12 O O O O O O O O O O positive 13 127 4 O O O O O O O O

TABLE 4 Throat cultures for Streptococci after intranasal challenge of mice vaccinated intranasally with PBS or SCP expressed in E. coli DH5C. CFU after vaccine Days after challenge

Mice: 1. 2 3 4 5 6 7 8 9 1O

PBSCTI

1. 112 143 85 16 O O O O O O 2 127 27 18 89 3 7 7 7 70 3 3 >2OO >2OO >2OO >2OO >2OO >2OO >2OO 108 >2OO 66 4 31 2OO 4 2 O O O O O O 5 4 O O 3 3 O O O O O 6 O O O O O O O O O O 7 >2OO >2OO 120 125 91 145 >2OO >2OO >200 166 8 2 O O O O O O O O O 9 O O O O O O O O O O 1O O O O O O O O O O O 11 37 >2OO 194 16 >2OO 47 >2OO 101 >2OO >2OO No. of 8 6 6 7 5 4 4 4 4 4 positive SCPAD

1. 6 O O O O O O O O O 2 1OS 41 O O O O O O O O 3 O O O O O O O O O O 4 2 O O O O O O O O O 5 2 O O O O O O O O O 6 9 O 11 O O O O O O O 7 O O O O O O O O O O 8 26 O O O O O O O O O 9 O 19 O O 5 57 O O 21 91 1O O O O O O O O O O O 11 7 O O O O O O O O O No. of 7 2 1. O 1. 1. O O 1. 1. positive *Mice were inoculated twice, because the dose of bacteria was too low at first time inocula tion. US 6,270,775 B1 17 18 EXAMPLE 5 genes. Alignment of the Sequences showed that ScpA12 is actually phylogenetically closer to ScpB than it is to ScpA49. C5a Peptidase from Group B Streptococci is Nearly Thirty Strains, representing Serotypes III, III/R, II, Ia/c, Identical in Sequence to those from M12 and M49 NT/c, NT/c/R1 carry a copy of scpB. Group A Streptococci Recombinant SCP was expressed in E. coli using expres The group B streptococci C5a peptidase (SCPB) gene was sion vector plasmid pGEX-4T-1 (ATCC accession number cloned, Sequenced and compared to that from Serotype 98225) and was shown to be identical to the enzyme group A streptococci M12 and M49. The entire ScpB gene extracted from the parental group B Streptococcal Strain was amplified by PCR using primers which correspond to portions of the ScpA12 Sequence using the method described 78-471 (Type II a+b). Western blot analysis suggested the in Example 4 above. The SCPB gene encodes an open recombinant SCP is identical to the C5ase enzyme previ reading frame (ORF) of 3450 bp which specifies a protein of ously purified from group B Streptococci. 1150 amino acids with Mr of 126,237da. The amino acid All publications, patents and patent documents are incor sequence of SCPB is shown in FIG. 2. Comparison of the porated by reference herein, as though individually incor ScpB nucleotide and deduced amino acid Sequence to those 15 porated by reference. The invention has been described with from M12 and M49 group A streptococci showed high reference to various Specific and preferred embodiments and similarities, 98% and 97%, respectively. ScpB contained a 50 techniques. However, it should be understood that many bp deletion which Overlapped two of the C-terminal repeats, variations and modifications may be made while remaining and had Several other minor differences relative to ScpA within the scope of the invention.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 11 <210> SEQ ID NO 1 <211& LENGTH: 1164 &212> TYPE PRT <213> ORGANISM: Streptococcus pyogenes, strain 49 <400 SEQUENCE: 1

Leu Arg Lys Lys Glin Lys Leu Pro Phe Asp Leu Ala Ile Ala Leu 1 5 10 15

Met Ser Thr Ser Ile Teu Lleu. Asn Ala Glin Ser Asp Ile Lys Ala Asn 25 30

Thr Wall Thr Glu Asp Thr Pro Ala Thr Glu Glin Ala Wall Glu Thr Pro 35 40 45

Glin Pro Thir Thr Wall Ser Glu Glu Wall Pro Ser Ser Lys Glu Thr 5 O 55 60

Thr Pro Glin Thr Pro Asp Asp Ala Glu Glu Thr Wall Ala Asp Asp Ala 65 70 75 8O

Asn Asp Leu Ala Pro Glin Ala Pro Ala Lys Thr Pro Thr Ser Ala 85 90 95

Thr Ser Thir Ile Arg Asp Teu Asn Asp Pro Ser Glin Wall 105 110

Thr Teu Glin Glu Ala Gly Lys Gly Ala Gly Thr Wall Wall Ala Wall 115 120 125

Ile Asp Ala Gly Phe Asp Lys Asn His Glu Ala Trp Arg Teu Thr Asp 130 135 14 O

Lys Ala Arg Tyr Glin Ser Lys Glu Asp Telu Glu Ala Lys 145 15 O 155 160

Glu His Gly Ile Thr Tyr Gly Glu Trp Val Asn Wall Ala 1.65 17 O 175

His Asp Ser Lys Asp Gly Thr Ala Wall Asp Glin Glu 18O 185 190

His Gly Thir His Ser Gly Ile Teu Ser Gly Asn Ala Pro Ser Glu 195 200 2O5

Thr Lys Glu Pro Arg Teu Glu Gly Ala Met Pro Glu Ala Glin Leu 210 215 220 US 6,270,775 B1 19 20

-continued

Teu Telu Met Arg Wall Glu Ile Wall Asn Gly Teu Ala Asp Tyr Ala Arg 225 230 235 240

Asn Ala Glin Ala Ile Arg Asp Ala Wall Asn Teu Gly Ala Lys Wall 245 250 255

Ile Asn Met Ser Phe Gly Asn Ala Ala Telu Ala Ala Asn Telu Pro 260 265 27 O

Asp Glu Thr Pro Phe Wall Ala Ser Lys Wall Arg 275 280 285

Ile Wall Thr Thr Ala Gly Asn Asp Ser Ser Phe Gly Gly Tys Thr Arg 29 O 295

Teu Pro Telu Ala Asp His Pro Asp Gly Wall Wall Gly Thr Pro Ala 305 310 315 320

Ala Ala Asp Ser Thr Teu Thr Wall Ala Ser Ser Pro Asp Asn Glin 325 330 335

Teu Thr Glu Thr Ala Met Wall Thr Asp Asp Glin Glin Asp Lys Glu 340 345 350

Met Pro Wall Telu Ser Thr Asn Arg Phe Glu Pro Asn Lys Ala Tyr Asp 355 360 365

Ala Ala Asn Arg Gly Met Glu Asp Asp Phe Wall 370 375

Lys Gly Ile Ala Teu Ile Glu Arg Ser Asp Ile Asp Phe Thr Asp 385 390 395 400

Ile Ala Asn Ala Ala Gly Ala Wall Gly Wall Teu Ile 405 410 415

Asp Asn Glin Asp Gly Phe Pro Ile Glu Teu Pro Asn Wall Asp Glin 420 4.25 430

Met Pro Ala Ala Phe Ile Ser Arg Asp Gly Teu Teu Teu Lys Asp 435 4 40 4 45

Asn Ser Glin Thr Ile Thr Phe Asn Ala Thr Pro Wall Telu Pro 450 455 460

Thr Ala Ser Gly Thr Lys Teu Ser Arg Phe Ser Ser Trp Telu Thr 465 470 475 480

Ala Asp Gly Asn Ile Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile 485 490 495

Teu Ser Ser Ala Ala Asn Asn Tyr Ala Teu Ser Gly Thr Ser 5 OO 505 510

Met Ser Ala Pro Teu Wall Ala Wall Ile Met Gly Teu Teu Glin Lys Glin 515 525

Glu Thr Glin Pro Asp Met Thr Glin Ser Glu Arg Teu Asp Telu 530 535 540

Ala Wall Teu Met Ser Ser Ala Thr Ala Teu Asp Glu Asp 545 550 555 560

Glu Ala Phe Ser Pro Arg Glin Glin Gly Ala Gly Ala Wall Asp 565 570 575

Ala Ala Ser Glu Ala Thr Met Wall Thr Asp Tys Asp Asn 58O 585 59 O

Thr Ser Ser Wall His Teu Asn Asn Wall Ser Asp Lys Phe Glu Wall 595 600 605

Thr Wall Thr Wall His Asn Lys Ser Asp Pro His Glu Teu Tyr 610 615 62O

Glin Ala Thr Wall Glin Thr Asp Wall Asp Gly His Phe Ala Telu 625 630 635 640 US 6,270,775 B1 21 22

-continued Ala Pro Ala Teu Ile Glu Thr Ser Trp Glin Lys Ile Thr Ile Pro 645 650 655

Ala Asn Ser Ser Glin Wall Thr Ile Pro Ile Asp Ile Ser Glin Phe 660 665 670

Ser Asp Telu Teu Ala Glin Met Asn Gly Tyr Phe Teu Glu Gly 675 680 685

Phe Wall Arg Ile Glin Asp Pro Thr Glu Glu Teu Met Ser Ile 69 O. 695 7 OO

Pro Ile Gly Phe Arg Gly Asp Phe Gly Asn Teu Ser Ala Telu Glu 705 710 715 720

Pro Telu Asp Ser Asp Gly Ser Ser His Glu Glu 725 730 735

Ile Ser Asp Ala Asp Glin Telu Asp Gly Asp Gly Teu Glin Phe Tyr 740 745 750

Ala Telu Lys Asn Asp Phe Thr Ala Telu Thr Thr Glu Ser Asn Pro Trp 755 760 765

Thr Ile Ile Asn Wall Wall Lys Glu Gly Wall Glu Asn Ile Glu Asp Ile 770 775

Glu Ser Ser Glu Ile Thr Glu Thr Ile Phe Ala Gly Thr Phe Ala Lys 785 790 795

Glin Asp Asp Asp Arg His Ile His Arg His Ala Asn Gly Lys 805 810 815

Pro Ala Ala Ile Ser Pro Asn Gly Asp Gly Asn Arg Asp Wall 820 825 830

Glin Phe His Gly Thr Phe Teu Arg Asn Ala Asn Teu Wall Ala Glu 835 840 845

Wall Telu Asp Glu Gly Asn Wall Wall Trp Thr Ser Glu Wall Thr Glu 85 O 855 860

Glin Wall Wall Asn Tyr Asn Asn Asp Telu Ala Ser Thr Teu Gly Ser 865 870 875 88O

Thr Arg Phe Glu Ile Ser Arg Trp Asp Gly Lys Asp Asp Ala Lys 885 890 895

Wall Wall Ala Asn Gly Thr Thr Tyr Arg Wall Arg Thr Pro Ile 9 OO 905 910

Ser Ser Gly Ala Glu Glin His Thr Asp Phe Asp Wall Ile Wall Asp 915 920 925

Asn Thr Thr Pro Glu Wall Ala Thr Ser Ala Thr Phe Ser Thr Glu Asp 930 935 940

Arg Arg Telu Thr Teu Ala Ser Pro Glin Thr Ser Glin Pro Wall Tyr 945 950 955 96.O

Arg Glu Arg Ile Ala Thr Met Asp Glu Asp Teu Pro Thr Thr 965 970 975

Glu Ile Ser Pro Asn Glu Asp Gly Thr Phe Thr Teu Pro Glu Glu 985 99 O

Ala Glu Thr Met Glu Gly Ala Thr Wall Pro Teu Lys Met Ser Asp Phe 995 10 OO 1005

Thr Tyr Wall Wall Glu Asp Met Ala Gly Asn Ile Thr Tyr Thr Pro Wall 1010 1015 1020

Thr Lys Lieu Telu Glu Gly His Ser Asn Lys Pro Glu Glin Asp Gly Ser 1025 1030 1035 1040

Asp Glin Ala Pro Asp Lys Lys Pro Glu Thr Lys Pro Glu Gln Asp Gly 1045 105 O 1055 US 6,270,775 B1 23 24

-continued Ser Asp Glin Ala Pro Asp Lys Lys Pro Glu Thir Lys Pro Gly Glin Asp 1060 1065 1070 Gly Ser Gly Glin Thr Pro Asp Llys Llys Pro Glu Thr Lys Pro Glu Lys 1075 1080 1085 Asp Ser Ser Gly Glin Thr Pro Gly Lys Thr Pro Gln Lys Gly Glin Pro 1090 1095 1100

Ser Arg Thr Lieu Glu Lys Arg Ser Ser Lys Arg Ala Leu Ala Thr Lys 1105 1110 1115 1120 Ala Ser Thr Arg Asp Gln Leu Pro Thr Thr Asn Asp Lys Asp Thr Asn 1125 1130 1135

Arg Lieu. His Lieu Lleu Lys Lieu Val Met Thir Thr Phe Phe Leu Gly Lieu 1140 1145 1150

Val Ala His Ile Phe Lys Thr Lys Arg Thr Glu Asp 1155 1160

<210> SEQ ID NO 2 &2 11s LENGTH 1167 &212> TYPE PRT <213> ORGANISM: Streptococcus pyogenes, strain 12 <400 SEQUENCE: 2

Leu Arg Lys Lys Glin Teu Pro Phe Asp Teu Ala Ile Ala Leu 1 5 10 15

Met Ser Thr Ser Ile Teu Teu Asn Ala Glin Ser Asp Ile Lys Ala Asn 2O 25 30

Thr Val Thr Glu Asp Thr Pro Wall Thr Glu Glin Ala Wall Glu Thr Pro 35 40 45

Gln Pro Thr Ala Wall Ser Glu Glu Wall Pro Ser Ser Lys Glu Thir Lys 50 55 60

Thr Pro Glin Thr Pro Asp Asp Ala Glu Glu Thr Ile Ala Asp Asp Ala 65 70 75 8O

Asn Asp Leu Ala Pro Glin Ala Pro Ala Lys Thr Ala Asp Thr Pro Ala 85 90 95

Thr Ser Lys Ala Thr Ile Arg Asp Telu Asn Asp Pro Ser Glin Val Lys 105 110

Thr Lieu Glin Glu Lys Ala Gly Lys Gly Ala Gly Thr Wal Wall Ala Wall 115 120 125

Ile Asp Ala Gly Phe Asp Lys Asn His Glu Ala Trp Arg Lieu. Thir Asp 130 135 1 4 0

Lys. Thir Lys Ala Arg Tyr Glin Ser Glu Asp Teu Glu Lys Ala Lys 145 15 O 155 160

Lys Glu His Gly Ile Thr Gly Glu Trp Wall Asn Asp Llys Val Ala 1.65 170 175

Tyr Tyr His Asp Tyr Ser Asp Gly Lys Thr Ala Val Asp Glin Glu 18O 185 190

His Gly. Thr His Val Ser Gly Ile Telu Ser Gly Asn Ala Pro Ser Glu 195 200 2O5

Thr Lys Glu Pro Tyr Arg Teu Glu Gly Ala Met Pro Glu Ala Glin Leu 210 215 220

Leu Lleu Met Arg Val Glu Ile Wall Asn Gly Teu Ala Asp Tyr Ala Arg 225 230 235 240

Asn Tyr Ala Glin Ala Ile Arg Asp Ala Wall Asn Teu Gly Ala Lys Val 245 250 255

Ile Asn Met Ser Phe Gly Asn Ala Ala Telu Ala Ala Asn Lieu Pro 260 265 27 O US 6,270,775 B1 25 26

-continued

Asp Glu Thr Ala Phe Asp Ala Lys Ser Lys Gly Wall Ser 275 280 285

Ile Wall Thr Ser Ala Gly Asn Asp Ser Ser Phe Gly Gly Tys Thr Arg 29 O 295

Teu Pro Telu Ala Asp His Pro Asp Gly Wall Wall Gly Thr Pro Ala 305 310 315 320

Ala Ala Asp Ser Thr Teu Thr Wall Ala Ser Ser Pro Asp Lys Glin 325 330 335

Teu Thr Glu Thr Ala Met Wall Thr Asp Asp Glin Glin Asp Lys Glu 340 345 350

Met Pro Wall Telu Ser Thr Asn Arg Phe Glu Pro Asn Lys Ala Tyr Asp 355 360 365

Ala Ala Asn Arg Gly Met Glu Asp Asp Phe Wall 370 375

Lys Gly Ile Ala Teu Ile Glu Arg Gly Asp Ile Asp Phe Lys Asp 385 390 395 400

Wall Ala Asn Ala Ala Gly Ala Wall Gly Wall Teu Ile 405 410 415

Asp Asn Glin Asp Gly Phe Pro Ile Glu Teu Pro Asn Wall Asp Glin 420 425 430

Met Pro Ala Ala Phe Ile Ser Arg Asp Gly Teu Teu Teu Lys Asp 435 4 40 4 45

Asn Pro Glin Thr Ile Thr Phe Asn Ala Thr Pro Wall Telu Pro 450 455 460

Thr Ala Ser Gly Thr Lys Teu Ser Arg Phe Ser Ser Trp Telu Thr 465 470 475 480

Ala Asp Gly Asn Ile Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile 485 490 495

Teu Ser Ser Wall Ala Asn Asn Tyr Ala Teu Ser Gly Thr Ser 5 OO 505 510

Met Ser Ala Pro Teu Wall Ala Gly Ile Met Gly Teu Teu Glin Lys Glin 515 525

Glu Thr Glin Pro Asp Met Thr Pro Ser Glu Arg Teu Asp Telu 530 535 540

Ala Wall Teu Met Ser Ser Ala Thr Ala Teu Asp Glu Asp 545 550 555 560

Glu Ala Phe Ser Pro Arg Glin Glin Gly Ala Gly Ala Wall Asp 565 570 575

Ala Ala Ser Ala Ala Thr Met Wall Thr Asp Tys Asp Asn 585 59 O

Thr Ser Ser Wall His Teu Asn Asn Wall Ser Asp Lys Phe Glu Wall 595 600 605

Thr Wall Thr Wall His Asn Lys Ser Asp Pro Glin Glu Teu Tyr 610 615 62O

Glin Ala Thr Wall Glin Thr Asp Wall Asp Gly His Phe Ala Telu 625 630 635 640

Ala Pro Wall Teu Glu Ala Ser Trp Glin Ile Thr Ile Pro 645 650 655

Ala Asn Ser Ser Glin Wall Thr Wall Pro Ile Ala Ser Phe 660 665 670

Ser Asp Telu Teu Ala Glin Met Asn Gly Phe Teu Glu Gly 675 680 685 US 6,270,775 B1 27 28

-continued

Phe Wall Arg Phe Glin Asp Pro Thr Glu Glu Leu Met Ser Ile 69 O. 695 7 OO

Pro Ile Gly Phe Arg Gly Asp Phe Gly Asn Teu Ser Ala Wall Glu 705 710 715 720

Pro Ile Asp Ser Asp Gly Ser Ser His Glu Ala 725 730 735

Asn Ser Asp Ala Asp Glin Telu Asp Gly Asp Gly Teu Glin Phe Tyr 740 745 750

Ala Telu Lys Asn Asn Phe Thr Ala Telu Thr Thr Glu Ser Asn Pro Trp 755 760 765

Thr Ile Ile Ala Wall Lys Glu Gly Wall Glu Asn Ile Glu Asp Ile 770 775

Glu Ser Ser Glu Ile Thr Glu Thr Ile Phe Ala Gly Thr Phe Ala Lys 785 790 795 8OO

Glin Asp Asp Asp Ser His Ile His Arg His Ala Asn Gly Glu 805 810 815

Pro Ala Ala Ile Ser Pro Asn Gly Asp Gly Asn Arg Asp Tyr Val 820 825 830

Glin Phe Glin Gly Thr Phe Teu Arg Asn Ala Asn Teu Wall Ala Glu 835 840 845

Wall Telu Asp Glu Gly Asn Wall Wall Trp Thr Ser Glu Wall Thr Glu 85 O 855 860

Glin Wall Wall Asn Tyr Asn Asn Asp Telu Ala Ser Thr Teu Gly Ser 865 870 875 88O

Thr Arg Phe Glu Lys Thr Arg Trp Asp Gly Lys Asp Asp Gly Lys 885 890 895

Wall Wall Ala Asn Gly Thr Thr Tyr Arg Wall Arg Thr Pro Ile 9 OO 905 910

Ser Ser Gly Ala Glu Glin His Thr Asp Phe Asp Wall Ile Val Asp 915 920 925

Asn Thr Thr Pro Glu Wall Ala Thr Ser Ala Thr Phe Ser Thr Glu Asp 930 935 940

Arg Arg Telu Thr Teu Ala Ser Pro Thr Ser Glin Pro Val Tyr 945 950 955 96.O

Arg Glu Arg Ile Ala Thr Met Asp Glu Asp Teu Pro Th Thr 965 970 975

Glu Ile Ser Pro Asn Glu Asp Gly Thr Phe Thr Teu Pro Glu Glu 985 99 O

Ala Glu Thr Met Glu Gly Ala Thr Wall Pro Teu Lys Met Ser Asp Phe 995 10 OO 1005

Thr Tyr Wall Wall Glu Asp Met Ala Gly Asn Ile Thr Tyr Thr Pro Wall 1010 1015 1020

Thr Lys Telu Telu Glu Gly His Ser Asn Lys Pro Glu Glin Asp Gly Ser 1025 1030 1035 1040

Gly Glin Thr Pro Asp Lys Lys Pro Glu Ala Lys Pro Glu Glin Asp Gly 1045 105 O 1055

Ser Asp Glin Ala Pro Asp Lys Lys Pro Glu Ala Lys Pro Glu Glin Asp 1060 1065 1070

Gly Ser Gly Glin Thr Pro Asp Lys Llys Pro Glu Thir Lys Pro Glu Lys 1075 1080 1085 Asp Ser Ser Gly Glin Thr Pro Gly Lys Thr Pro Gln Lys Gly Glin Pro 1090 1095 1100 US 6,270,775 B1 29 30

-continued Ser Arg Thir Lieu Glu Lys Arg Ser Ser Lys Arg Ala Leu Ala Thr Lys 1105 1110 1115 1120

Ala Ser Thr Arg Asp Gln Leu Pro Thir Thr Asn Asp Lys Asp Thr Asn 1125 1130 1135 Arg Lieu His Lieu Lleu Lys Lieu Val Met Thr Thr Phe Phe Phe Gly Leu 1140 1145 1150 Wall Ala His Ile Phe Lys Thr Lys Arg Gln Lys Glu Thir Lys Lys 1155 1160 1165

<210> SEQ ID NO 3 <211& LENGTH: 11 5 O &212> TYPE PRT <213> ORGANISM Streptococcus agalactiae

<400 SEQUENCE: 3

Leu Arg Lys Lys Glin Teu Pro Phe Asp Lys Teu Ala Ile Ala Telu 1 5 10 15

Met Ser Thr Ser Ile Teu Teu Asn Ala Glin Ser Asp Ile Tys Ala Asn 25 30

Thir Wall Thr Glu Asp Thr Pro Ala Thr Glu Glin Thr Wall Glu Thr Pro 35 40 45

Gln Pro Thr Ala Wall Ser Glu Glu Ala Pro Ser Ser Glu Thr 50 55 60

Thr Pro Glin Thr Pro Ser Asp Ala Gly Glu Thr Wall Ala Asp Asp Ala 65 70 75 8O

Asn Asp Telu Pro Glin Ala Pro Ala Lys Thr Ala Asp Thr Pro Ala 85 90 95

Thir Ser Thr Ile Arg Asp Telu Asn Asp Pro Ser Glin Wall 105 110

Thir Leu Glin Ala Gly Lys Gly Ala Gly Thr Wall Wall Ala Wall 115 120 125

Ile Asp Ala Phe Asp Lys Asn His Glu Ala Trp Arg Teu Thr Asp 130 135 1 4 0

Lys Thr Arg Tyr Glin Ser Glu Asp Teu Glu Ala Lys 145 15 O 155 160

Lys Glu His Ile Thr Gly Glu Trp Wall Asn Asp Wall Ala 1.65 170 175

His Asp Ser Asp Gly Thr Ala Wall Asp Glin Glu 18O 185 190

His Gly Thr His Ser Gly Ile Telu Ser Gly Asn Ala Pro Ser Glu 195 200

Thr Lys Glu Pro Arg Teu Glu Gly Ala Met Pro Glu Ala Glin Telu 210 215 220

Teu Telu Met Arg Wall Ile Wall Asn Gly Teu Ala Asp Ala Arg 225 235 240

Asn Tyr Ala Glin Ala Arg Asp Ala Ile Asn Teu Gly Ala Lys Wall 245 250 255

Ile Asn Met Ser Phe Asn Ala Ala Telu Ala Ala Asn Telu Pro 260 265 27 O

Asp Glu Thr Phe Asp Ala Ser Lys Wall Ser 275 280 285

Ile Wall Thr Ser Ala Asn Asp Ser Ser Phe Gly Gly Tys Thr 29 O 295 3OO

Leu Pro Telu Ala Asp Pro Asp Gly Wall Wall Gly Thr Pro Ala 305 315 320 US 6,270,775 B1 31 32

-continued

Ala Ala Asp Ser Thr Teu Thr Wall Ala Ser Ser Pro Asp Lys Glin 325 330 335

Teu Thr Glu Thr Wall Arg Wall Thr Asp Glin Glin Asp Lys Glu 340 345 350

Met Pro Wall Telu Ser Thr Asn Arg Phe Pro Asn Lys Ala Tyr Asp 355 360 365

Ala Ala Asn Arg Gly Thr Asp Asp Phe Tys Wall 370 375

Lys Gly Ile Ala Teu Ile Glu Arg Asp Ile Asp Phe Lys Asp 385 390 395 400

Ile Ala Ala Ala Gly Wall Gly Wall Teu Ile 405 415

Asp Asn Glin Asp Gly Phe Pro Ile Teu Pro Asn Wall Asp Glin 420 425 430

Met Pro Ala Ala Phe Ile Ser Arg Asp Gly Teu Teu Teu Lys Asp 435 4 40 4 45

Asn Pro Glin Thr Ile Thr Phe Asn Ala Thr Pro Wall Telu Pro 450 455 460

Thr Ala Ser Gly Thr Lys Teu Ser Arg Phe Ser Ser Trp Telu Thr 465 470 475 480

Ala Asp Gly Asn Ile Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile 485 490 495

Teu Ser Ser Wall Ala Asn Asn Tyr Ala Teu Ser Gly Thr Ser 5 OO 505 510

Met Ser Ala Pro Teu Wall Ala Gly Ile Met Gly Teu Teu Glin Lys Glin 515 52O 525

Glu Thr Glin Pro Asp Met Thr Pro Ser Glu Arg Teu Asp Telu 530 535 540

Ala Wall Teu Met Ser Ser Ala Thr Ala Teu Asp Glu Asp 545 550 555 560

Glu Ala Phe Ser Pro Arg Glin Glin Gly Ala Gly Ala Wall Asp 565 570 575

Ala Ala Ser Ala Ala Thr Met Wall Thr Asp Tys Asp Asn 585 59 O

Thr Ser Ser Wall His Teu Asn Asn Wall Ser Asp Lys Phe Glu Wall 595 600 605

Thr Wall Asn Wall His Asn Lys Ser Asp Pro Glin Glu Teu Tyr 610 615 62O

Glin Ala Thr Wall Glin Thr Asp Wall Asp His Phe Ala Telu 625 630 640

Ala Pro Wall Teu Glu Ala Ser Trp Ile Thr Ile Pro 645 650 655

Ala Asn Ser Ser Glin Wall Thr Wall Pro Ala Ser Phe 660 665 670

Ser Asp Telu Teu Ala Glin Met Asn Phe Teu Glu Gly 675 680 685

Phe Wall Arg Phe Glin Asp Pro Glu Teu Met Ser Ile 69 O. 695 7 OO

Pro Ile Gly Phe Arg Gly Asp Phe Gly Teu Ser Ala Telu Glu 705 710 720

Pro Ile Asp Ser Asp Gly Ser His Glu Ala 725 730 735 US 6,270,775 B1 33 34

-continued Asn Ser Asp Ala Asp Glin Telu Asp Gly Asp Gly Leu Glin Phe Tyr 740 745 750

Ala Lieu Lys Asn Asn Phe Thr Ala Telu Thr Thr Glu Ser Asn Pro Trp 755 760 765

Thir Ile Ile Ala Wall Lys Glu Gly Wall Glu Asn Ile Glu Asp Ile 770 775

Glu Ser Ser Glu Ile Thr Glu Thr Ile Telu Ala Gly Thr Phe Ala Lys 785 790 795 8OO

Glin Asp Asp Asp Ser His Ile His Arg His Ala Asn Gly Lys 805 810 815

Pro Tyr Ala Ala Ile Ser Pro Asn Gly Asp Gly Asn Arg Asp Tyr Val 820 825 830

Glin Phe Glin Gly Thr Phe Teu Arg Asn Ala Asn Teu Wall Ala Glu 835 840 845

Val Lieu. Asp Glu Gly Asn Wall Wall Trp Thr Ser Glu Wall Thr Glu 85 O 855 860

Glin Wal Wall Asn Tyr Asn Asn Asp Telu Ala Ser Thr Teu Gly Ser 865 870 875 88O

Thr Arg Phe Glu Lys Thr Arg Trp Asp Gly Asp Asp Gly Lys 885 890 895

Wal Wall Ala Asn Gly Thr Thr Tyr Arg Wall Arg Thr Pro Ile 9 OO 905 910

Ser Ser Gly Ala Glu Glin His Thr Asp Phe Asp Wall Ile Val Asp 915 920 925

Asn. Thir Thr Pro Glu Wall Ala Thr Ser Ala Thr Phe Ser Thr Glu Asp 930 935 940

Arg Arg Lieu Thr Teu Ala Ser Pro Thr Ser Glin Pro Val Tyr 945 950 955 96.O

Arg Glu Arg Ile Ala Thr Met Asp Glu Asp Teu Pro Th Thr 965 970 975

Glu Tyr Ile Ser Pro Asn Glu Asp Gly Thr Phe Thr Teu Pro Glu Glu 985 99 O

Ala Glu Thr Thr Glu Gly Ala Thr Wall Pro Teu Lys Met Ser Asp Phe 995 10 OO 1005

Thr Tyr Val Wall Glu Asp Met Ala Gly Asn Ile Thr Tyr Thr Pro Wall 1010 1015 1020

Thr Lys Lieu Telu Glu Gly His Ser Asn Lys Pro Glu Glin Asp Gly Ser 1025 1030 1035 1040

Asp Glin Ala Pro Asp Lys Lys Pro Glu Ala Lys Pro Glu Glin Asp Gly 1045 105 O 1055

Ser Gly Glin Thr Pro Asp Lys Lys Thr Glu Thr Lys Pro Glu Lys Asp 1060 1065 1070

Ser Ser Gly Glin Thr Pro Gly Lys Thr Pro Gln Lys Gly Glin Pro Ser 1075 1080 1085

Arg Thr Lieu Glu Lys Arg Ser Ser Lys Arg Ala Leu Ala Thr Lys Ala 1090 1095 1100

Ser Thr Arg Asp Glin Leu Pro Thr Thr Asn Asp Lys Asp Thr Asn Arg 1105 1110 1115 1120 Lieu. His Lieu Leu Lys Leu Val Met Thr Thr Phe Phe Leu Gly Leu Val 1125 1130 1135 Ala His Ile Phe Lys Thr Lys Arg Gln Lys Glu Thir Lys Lys 1140 1145 1150 US 6,270,775 B1 35 36

-continued SEQ ID NO 4 LENGTH: 31 TYPE ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: A primer <400 SEQUENCE: 4 ggggggga at tcgtag cqgg tat catggga C

SEQ ID NO 5 LENGTH: 31 TYPE ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: A primer <400 SEQUENCE: 5 ggggggga at tcgggtgctg. Caatatotgg C

SEQ ID NO 6 LENGTH 38 3 TYPE PRT ORGANISM Streptococcus pyogenes, strain 49

<400 SEQUENCE: 6

Asp Ala Gly Phe Asn His Glu Ala Trp Arg Teu Thr Asp Lys 1 10 15

Ala Lys Ala Arg Ser Glu Asp Teu Glu Ala Lys 2O 25 30

Glu His Gly Ile Thr Gly Glu Trp Wall ASn Asp Lys Wall Ala Tyr 35 40 45

His Asp Ser Asp Gly Lys Thr Ala Wall Asp Glin Glu His 50 55 60

Thr His Ser Gly Ile Telu Ser Gly Asn Ala Pro Ser Glu Thr 70 75

Glu Pro Arg Teu Glu Gly Ala Met Pro Glu Ala Glin Telu Telu 85 90 95

Teu Met Arg Wall Glu Ile Wall Asn Gly Telu Ala Asp Ala Arg Asn 100 105 110

Ala Glin Ala Ile Arg Asp Ala Wall Asn Teu Gly Ala Wall Ile 115 120 125

Asn Met Ser Phe Gly Asn Ala Ala Telu Ala Ala Asn Teu Pro Asp 130 135 1 4 0

Glu Thr Pro Phe Wall Ala Ser Gly Wall Arg Ile 145 15 O 155 160

Wall Thr Thr Ala Gly Asn Asp Ser Ser Phe Gly Gly Lys Thr Arg Telu 1.65 170 175

Pro Telu Ala Asp His Pro Asp Gly Wall Wall Gly Thr Pro Ala Ala 18O 185 190

Ala Asp Ser Thr Teu Thr Wall Ala Ser Ser Pro Asp Asn Glin Telu 195 200

Thr Glu Thr Ala Met Wall Lys Thr Asp Asp Glin Glin Asp Glu Met 210 215 220

Pro Wall Telu Ser Thr Asn Arg Phe Glu Pro Asn Ala Tyr Tyr 225 230 235 240

Ala Ala Asn Arg Gly Met Glu Asp Asp Phe Asp Wall 245 250 255 US 6,270,775 B1 37 38

-continued Gly Lys Ile Ala Lieu. Ile Glu Arg Ser Asp Ile Asp Phe Thr Asp Lys 260 265 27 O Ile Ala Asn Ala Lys Lys Ala Gly Ala Val Gly Val Lieu. Ile Tyr Asp 275 280 285 Asn Glin Asp Lys Gly Phe Pro Ile Glu Lieu Pro Asn. Wall Asp Gln Met 29 O 295 3OO Pro Ala Ala Phe Ile Ser Arg Lys Asp Gly Lieu Lleu Lleu Lys Asp Asn 305 310 315 320 Ser Gln Lys Thr Ile Thr Phe Asn Ala Thr Pro Lys Val Leu Pro Thr 325 330 335 Ala Ser Gly. Thr Lys Leu Ser Arg Phe Ser Ser Trp Gly Leu Thr Ala 340 345 350 Asp Gly Asn. Ile Lys Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile Leu 355 360 365 Ser Ser Ala Ala Asn. Asn Lys Tyr Ala Lys Lieu Ser Gly. Thir Ser 370 375 38O

<210 SEQ ID NO 7 &2 11s LENGTH 383 &212> TYPE PRT <213> ORGANISM: Streptococcus pyogenes, strain 12 <400 SEQUENCE: 7 Asp Ala Gly Phe Asp Lys Asn His Glu Ala Trp Arg Lieu. Thir Asp Lys 1 5 10 15 Thr Lys Ala Arg Tyr Glin Ser Lys Glu Asp Leu Glu Lys Ala Lys Lys 2O 25 30 Glu His Gly Ile Thr Tyr Gly Glu Trp Val Asn Asp Lys Val Ala Tyr 35 40 45 Tyr His Asp Tyr Ser Lys Asp Gly Lys Thr Ala Val Asp Glin Glu His 50 55 60 Gly Thr His Val Ser Gly Ile Leu Ser Gly Asn Ala Pro Ser Glu Thr 65 70 75 8O Lys Glu Pro Tyr Arg Lieu Glu Gly Ala Met Pro Glu Ala Glin Leu Lieu 85 90 95 Leu Met Arg Val Glu Ile Val Asn Gly Lieu Ala Asp Tyr Ala Arg Asn 100 105 110 Tyr Ala Glin Ala Ile Arg Asp Ala Val Asn Lieu Gly Ala Lys Val Ile 115 120 125 Asn Met Ser Phe Gly Asn Ala Ala Lieu Ala Tyr Ala Asn Lieu Pro Asp 130 135 1 4 0 Glu Thir Lys Lys Ala Phe Asp Tyr Ala Lys Ser Lys Gly Wal Ser Ile 145 15 O 155 160 Val Thir Ser Ala Gly Asn Asp Ser Ser Phe Gly Gly Lys Thr Arg Lieu 1.65 170 175 Pro Leu Ala Asp His Pro Asp Tyr Gly Val Val Gly Thr Pro Ala Ala 18O 185 190 Ala Asp Ser Thr Lieu. Thr Val Ala Ser Tyr Ser Pro Asp Lys Glin Lieu 195 200 2O5 Thr Glu Thir Ala Met Val Lys Thr Asp Asp Glin Glin Asp Lys Glu Met 210 215 220 Pro Val Leu Ser Thr Asn Arg Phe Glu Pro Asn Lys Ala Tyr Asp Tyr 225 230 235 240 Ala Tyr Ala Asn Arg Gly Met Lys Glu Asp Asp Phe Lys Asp Wall Lys 245 250 255 US 6,270,775 B1 39 40

-continued Gly Lys Ile Ala Lieu. Ile Glu Arg Gly Asp Ile Asp Phe Lys Asp Lys 260 265 27 O Val Ala Asn Ala Lys Lys Ala Gly Ala Val Gly Val Lieu. Ile Tyr Asp 275 280 285 Asn Glin Asp Lys Gly Phe Pro Ile Glu Lieu Pro Asn. Wall Asp Gln Met 29 O 295 3OO Pro Ala Ala Phe Ile Ser Arg Lys Asp Gly Lieu Lleu Lleu Lys Asp Asn 305 310 315 320 Pro Glin Lys Thr Ile Thr Phe Asn Ala Thr Pro Lys Val Leu Pro Thr 325 330 335 Ala Ser Gly. Thr Lys Leu Ser Arg Phe Ser Ser Trp Gly Leu Thr Ala 340 345 350 Asp Gly Asn. Ile Lys Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile Leu 355 360 365 Ser Ser Val Ala Asn. Asn Lys Tyr Ala Lys Lieu Ser Gly. Thir Ser 370 375 38O

<210 SEQ ID NO 8 &2 11s LENGTH 383 &212> TYPE PRT <213> ORGANISM: Streptococcus agalactiae <400 SEQUENCE: 8 Asp Ala Gly Phe Asp Lys Asn His Glu Ala Trp Arg Lieu. Thir Asp Lys 1 5 10 15 Thr Lys Ala Arg Tyr Glin Ser Lys Glu Asp Leu Glu Lys Ala Lys Lys 2O 25 30 Glu His Gly Ile Thr Tyr Gly Glu Trp Val Asn Asp Lys Val Ala Tyr 35 40 45 Tyr His Asp Tyr Ser Lys Asp Gly Lys Thr Ala Val Asp Glin Glu His 50 55 60 Gly Thr His Val Ser Gly Ile Leu Ser Gly Asn Ala Pro Ser Glu Thr 65 70 75 8O Lys Glu Pro Tyr Arg Lieu Glu Gly Ala Met Pro Glu Ala Glin Leu Lieu 85 90 95 Leu Met Arg Val Glu Ile Val Asn Gly Lieu Ala Asp Tyr Ala Arg Asn 100 105 110 Tyr Ala Glin Ala Ile Arg Asp Ala Ile Asn Lieu Gly Ala Lys Val Ile 115 120 125 Asn Met Ser Phe Gly Asn Ala Ala Lieu Ala Tyr Ala Asn Lieu Pro Asp 130 135 1 4 0 Glu Thir Lys Lys Ala Phe Asp Tyr Ala Lys Ser Lys Gly Wal Ser Ile 145 15 O 155 160 Val Thir Ser Ala Gly Asn Asp Ser Ser Phe Gly Gly Lys Thr Arg Lieu 1.65 170 175 Pro Leu Ala Asp His Pro Asp Tyr Gly Val Val Gly Thr Pro Ala Ala 18O 185 190 Ala Asp Ser Thr Lieu. Thr Val Ala Ser Tyr Ser Pro Asp Lys Glin Lieu 195 200 2O5 Thr Glu Thr Val Arg Val Lys Thr Ala Asp Glin Glin Asp Lys Glu Met 210 215 220 Pro Val Leu Ser Thr Asn Arg Phe Glu Pro Asn Lys Ala Tyr Asp Tyr 225 230 235 240 Ala Tyr Ala Asn Arg Gly Thr Lys Glu Asp Asp Phe Lys Asp Wall Lys 245 250 255 US 6,270,775 B1 41 42

-continued Gly Lys Ile Ala Lieu. Ile Glu Arg Gly Asp Ile Asp Phe Lys Asp Lys 260 265 27 O Ile Ala Lys Ala Lys Lys Ala Gly Ala Val Gly Val Lieu. Ile Tyr Asp 275 280 285 Asn Glin Asp Lys Gly Phe Pro Ile Glu Lieu Pro Asn. Wall Asp Gln Met 29 O 295 3OO Pro Ala Ala Phe Ile Ser Arg Lys Asp Gly Lieu Lleu Lleu Lys Asp Asn 305 310 315 320 Pro Glin Lys Thr Ile Thr Phe Asn Ala Thr Pro Lys Val Leu Pro Thr 325 330 335 Ala Ser Gly. Thr Lys Leu Ser Arg Phe Ser Ser Trp Gly Leu Thr Ala 340 345 350 Asp Gly Asn. Ile Lys Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile Leu 355 360 365 Ser Ser Val Ala Asn. Asn Lys Tyr Ala Lys Lieu Ser Gly. Thir Ser 370 375 38O

<210 SEQ ID NO 9 &2 11s LENGTH 921 &212> TYPE PRT <213> ORGANISM: Streptococcus pyogenes, strain 49 <400 SEQUENCE: 9 Asp Ala Gly Phe Asp Lys Asn His Glu Ala Trp Arg Lieu. Thir Asp Lys 1 5 10 15 Ala Lys Ala Arg Tyr Glin Ser Lys Glu Asp Leu Glu Lys Ala Lys Lys 2O 25 30 Glu His Gly Ile Thr Tyr Gly Glu Trp Val Asn Asp Lys Val Ala Tyr 35 40 45 Tyr His Asp Tyr Ser Lys Asp Gly Lys Thr Ala Val Asp Glin Glu His 50 55 60 Gly Thr His Val Ser Gly Ile Leu Ser Gly Asn Ala Pro Ser Glu Thr 65 70 75 8O Lys Glu Pro Tyr Arg Lieu Glu Gly Ala Met Pro Glu Ala Glin Leu Lieu 85 90 95 Leu Met Arg Val Glu Ile Val Asn Gly Lieu Ala Asp Tyr Ala Arg Asn 100 105 110 Tyr Ala Glin Ala Ile Arg Asp Ala Val Asn Lieu Gly Ala Lys Val Ile 115 120 125 Asn Met Ser Phe Gly Asn Ala Ala Lieu Ala Tyr Ala Asn Lieu Pro Asp 130 135 1 4 0 Glu Thir Lys Llys Pro Phe Val Tyr Ala Lys Ser Lys Gly Val Arg Ile 145 15 O 155 160 Val Thr Thr Ala Gly Asn Asp Ser Ser Phe Gly Gly Lys Thr Arg Leu 1.65 170 175 Pro Leu Ala Asp His Pro Asp Tyr Gly Val Val Gly Thr Pro Ala Ala 18O 185 190 Ala Asp Ser Thr Lieu. Thr Val Ala Ser Tyr Ser Pro Asp Asn Glin Lieu 195 200 2O5 Thr Glu Thir Ala Met Val Lys Thr Asp Asp Glin Glin Asp Lys Glu Met 210 215 220 Pro Val Leu Ser Thr Asn Arg Phe Glu Pro Asn Lys Ala Tyr Asp Tyr 225 230 235 240 Ala Tyr Ala Asn Arg Gly Met Lys Glu Asp Asp Phe Lys Asp Wall Lys 245 250 255 US 6,270,775 B1 43 44

-continued

Gly Ile Ala Teu Ile Glu Arg Ser Asp Ile Asp Phe Thr Asp 260 265 27 O

Ile Ala Asn Ala Ala Gly Ala Wall Gly Wall Teu Ile Tyr Asp 275 280 285

Asn Glin Asp Gly Phe Pro Ile Glu Telu Pro Asn Wall Asp Glin Met 29 O 295 3OO

Pro Ala Ala Phe Ile Ser Arg Asp Gly Teu Teu Teu Asp Asn 305 310 315 320

Ser Glin Thr Ile Thr Phe Asn Ala Thr Pro Wall Teu Pro Thr 325 330 335

Ala Ser Gly Thr Teu Ser Arg Phe Ser Ser Trp Gly Teu Thr Ala 340 345 350

Asp Gly Asn Ile Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile Telu 355 360 365

Ser Ser Ala Ala Asn Asn Lys Ala Teu Ser Gly Thr Ser Met 370 375 38O

Ser Ala Pro Telu Wall Ala Wall Ile Met Gly Teu Teu Glin Glin Tyr 385 390 395 400

Glu Thr Glin Pro Asp Met Thr Glin Ser Glu Arg Teu Asp Telu Ala 405 410 415

Wall Telu Met Ser Ser Ala Thr Ala Teu Asp Glu Asp Glu 420 425 430

Ala Tyr Phe Ser Pro Arg Glin Glin Gly Ala Gly Ala Wall Asp Ala 435 4 40 4 45

Lys Ala Ser Glu Ala Thr Met Wall Thr Asp Asp Asn Thr 450 455 460

Ser Ser Wall His Teu Asn Asn Ser Asp Phe Glu Wall Thr 465 470 475 480

Wall Thr Wall His Asn Ser Asp Pro His Glu Teu Tyr Glin 485 490 495

Ala Thr Wall Glin Thr Asp Wall Asp Gly His Phe Ala Telu Ala 5 OO 505 510

Pro Ala Telu Ile Glu Thr Ser Trp Glin Ile Thr Ile Pro Ala 515 52O 525

Asn Ser Ser Glin Wall Thr Ile Pro Ile Asp Ile Ser Glin Phe Ser 530 535 540

Lys Asp Telu Telu Ala Glin Met Asn Gly Tyr Phe Teu Glu Gly Phe 545 550 555 560

Wall Arg Ile Glin Asp Pro Thr Glu Glu Teu Met Ser Ile Pro 565 570 575

Ile Gly Phe Arg Gly Asp Phe Gly Asn Teu Ser Ala Teu Glu Lys 58O 585 59 O

Pro Telu Tyr Asp Ser Asp Gly Ser Ser His Glu Glu Ile 595 600 605

Ser Asp Ala Asp Glin Teu Asp Gly Asp Gly Teu Glin Phe Ala 610 615 62O

Teu Asn Asp Phe Thr Ala Telu Thr Thr Glu Ser Asn Pro Trp Thr 625 630 635 640

Ile Ile Asn Wall Wall Glu Gly Wall Glu Asn Ile Glu Asp Ile Glu 645 650 655

Ser Ser Glu Ile Thr Glu Thr Ile Phe Ala Gly Thr Phe Ala Glin 660 665 670 US 6,270,775 B1 45 46

-continued Asp Asp Asp Arg His Tyr Tyr Ile His Arg His Ala Asn Gly Lys Pro 675 680 685 Tyr Ala Ala Ile Ser Pro Asn Gly Asp Gly Asn Arg Asp Tyr Val Glin 69 O. 695 7 OO Phe His Gly Thr Phe Leu Arg Asn Ala Lys Asn Lieu Val Ala Glu Val 705 710 715 720 Leu Asp Lys Glu Gly Asn Val Val Trp Thr Ser Glu Val Thr Glu Gln 725 730 735 Val Val Lys Asn Tyr Asn. Asn Asp Leu Ala Ser Thr Lieu Gly Ser Thr 740 745 750 Arg Phe Glu Ile Ser Arg Trp Asp Gly Lys Asp Lys Asp Ala Lys Val 755 760 765 Val Ala Asn Gly Thr Tyr Thr Tyr Arg Val Arg Tyr Thr Pro Ile Ser 770 775 78O Ser Gly Ala Lys Glu Gln His Thr Asp Phe Asp Val Ile Val Asp Asn 785 790 795 8OO Thir Thr Pro Glu Val Ala Thr Ser Ala Thr Phe Ser Thr Glu Asp Arg 805 810 815 Arg Leu Thir Leu Ala Ser Lys Pro Gln Thr Ser Gln Pro Val Tyr Arg 820 825 830 Glu Arg Ile Ala Tyr Thr Tyr Met Asp Glu Asp Leu Pro Thr Thr Glu 835 840 845 Tyr Ile Ser Pro Asn Glu Asp Gly Thr Phe Thr Leu Pro Glu Glu Ala 85 O 855 860 Glu Thr Met Glu Gly Ala Thr Val Pro Leu Lys Met Ser Asp Phe Thr 865 870 875 88O Tyr Val Val Glu Asp Met Ala Gly Asn Ile Thr Tyr Thr Pro Val Thr 885 890 895 Lys Lieu Lieu Glu Gly His Ser Asn Lys Pro Glu Glin Asp Gly Ser Asp 9 OO 905 910 Glin Ala Pro Asp Lys Lys Pro Glu Thr 915 920

<210> SEQ ID NO 10 &2 11s LENGTH 921 &212> TYPE PRT <213> ORGANISM: Streptococcus pyogenes, strain 12 <400 SEQUENCE: 10 Asp Ala Gly Phe Asp Lys Asn His Glu Ala Trp Arg Lieu. Thir Asp Lys 1 5 10 15 Thr Lys Ala Arg Tyr Glin Ser Lys Glu Asp Leu Glu Lys Ala Lys Lys 2O 25 30 Glu His Gly Ile Thr Tyr Gly Glu Trp Val Asn Asp Lys Val Ala Tyr 35 40 45 Tyr His Asp Tyr Ser Lys Asp Gly Lys Thr Ala Val Asp Glin Glu His 50 55 60 Gly Thr His Val Ser Gly Ile Leu Ser Gly Asn Ala Pro Ser Glu Thr 65 70 75 8O Lys Glu Pro Tyr Arg Lieu Glu Gly Ala Met Pro Glu Ala Glin Leu Lieu 85 90 95 Leu Met Arg Val Glu Ile Val Asn Gly Lieu Ala Asp Tyr Ala Arg Asn 100 105 110 Tyr Ala Glin Ala Ile Arg Asp Ala Val Asn Lieu Gly Ala Lys Val Ile 115 120 125 US 6,270,775 B1 47 48

-continued

Asn Met Ser Phe Gly Asn Ala Ala Telu Ala Ala Asn Lieu Pro Asp 130 135 1 4 0

Glu Thr Ala Phe Asp Ala Ser Lys Gly Wall Ser Ile 145 15 O 155 160

Wall Thr Ser Ala Gly Asn Asp Ser Ser Phe Gly Gly Lys Thr Arg Telu 1.65 170 175

Pro Telu Ala Asp His Pro Asp Gly Wall Wall Gly Thr Pro Ala Ala 18O 185 190

Ala Asp Ser Thr Teu Thr Wall Ala Ser Ser Pro Asp Glin Telu 195 200

Thr Glu Thr Ala Met Wall Lys Thr Asp Asp Glin Glin Asp Glu Met 210 215 220

Pro Wall Telu Ser Thr Asn Arg Phe Glu Pro Asn Ala Tyr 225 230 235 240

Ala Ala Asn Arg Gly Met Glu Asp Asp Phe Asp Wall 245 250 255

Gly Ile Ala Teu Ile Glu Arg Gly Asp Ile Asp Phe Tys Asp 260 265 27 O

Wall Ala Asn Ala Ala Gly Ala Wall Gly Wall Teu Ile Asp 275 280 285

Asn Glin Asp Gly Phe Pro Ile Glu Telu Pro Asn Wall Asp Glin Met 29 O 295 3OO

Pro Ala Ala Phe Ile Ser Arg Asp Gly Teu Teu Teu Asp Asn 305 310 315 320

Pro Glin Thr Ile Thr Phe Asn Ala Thr Pro Wall Teu Pro Thr 325 330 335

Ala Ser Gly Thr Teu Ser Arg Phe Ser Ser Trp Gly Teu Thr Ala 340 345 350

Asp Gly Asn Ile Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile Telu 355 360 365

Ser Ser Wall Ala Asn Asn Lys Ala Teu Ser Gly Thr Ser Met 370 375 38O

Ser Ala Pro Telu Wall Ala Gly Ile Met Gly Teu Teu Glin Glin Tyr 385 390 395 400

Glu Thr Glin Pro Asp Met Thr Pro Ser Glu Arg Teu Asp Telu Ala 405 410 415

Wall Telu Met Ser Ser Ala Thr Ala Teu Asp Glu Asp Glu 420 425 430

Ala Tyr Phe Ser Pro Arg Glin Glin Gly Ala Gly Ala Wall Asp Ala 435 4 40 4 45

Lys Ala Ser Ala Ala Thr Met Wall Thr Asp Asp Asn Thr 450 455 460

Ser Ser Wall His Teu Asn Asn Wall Ser Asp Phe Glu Wall Thr 465 470 475 480

Wall Thr Wall His Asn Ser Asp Pro Glin Glu Teu Tyr Glin 485 490 495

Ala Thr Wall Glin Thr Asp Wall Asp Gly His Phe Ala Telu Ala 5 OO 505 510

Pro Wall Telu Glu Ala Ser Trp Glin Ile Thr Ile Pro Ala 515 525

Asn Ser Ser Glin Wall Thr Wall Pro Ile Asp Ala Ser Arg Phe Ser 530 535 540 US 6,270,775 B1 49 SO

-continued Lys Asp Lieu Lieu Ala Gln Met Lys Asn Gly Tyr Phe Lieu Glu Gly Phe 545 550 555 560 Val Arg Phe Lys Glin Asp Pro Thr Lys Glu Glu Leu Met Ser Ile Pro 565 570 575 Tyr Ile Gly Phe Arg Gly Asp Phe Gly Asn Lieu Ser Ala Val Glu Lys 58O 585 59 O Pro Ile Tyr Asp Ser Lys Asp Gly Ser Ser Tyr Tyr His Glu Ala Asn 595 600 605 Ser Asp Ala Lys Asp Glin Lieu. Asp Gly Asp Gly Lieu Glin Phe Tyr Ala 610 615 62O Leu Lys Asn Asn Phe Thr Ala Leu Thir Thr Glu Ser Asn Pro Trp Thr 625 630 635 640 Ile Ile Lys Ala Wall Lys Glu Gly Val Glu Asn. Ile Glu Asp Ile Glu 645 650 655 Ser Ser Glu Ile Thr Glu Thir Ile Phe Ala Gly Thr Phe Ala Lys Glin 660 665 670 Asp Asp Asp Ser His Tyr Tyr Ile His Arg His Ala Asn Gly Glu Pro 675 680 685 Tyr Ala Ala Ile Ser Pro Asn Gly Asp Gly Asn Arg Asp Tyr Val Glin 69 O. 695 7 OO Phe Glin Gly Thr Phe Leu Arg Asn Ala Lys Asn Lieu Val Ala Glu Val 705 710 715 720 Leu Asp Lys Glu Gly Asn Val Val Trp Thr Ser Glu Val Thr Glu Gln 725 730 735 Val Val Lys Asn Tyr Asn. Asn Asp Leu Ala Ser Thr Lieu Gly Ser Thr 740 745 750 Arg Phe Glu Lys Thr Arg Trp Asp Gly Lys Asp Lys Asp Gly Lys Val 755 760 765 Val Ala Asn Gly Thr Tyr Thr Tyr Arg Val Arg Tyr Thr Pro Ile Ser 770 775 78O Ser Gly Ala Lys Glu Gln His Thr Asp Phe Asp Val Ile Val Asp Asn 785 790 795 8OO

Thir Thr Pro Glu Val Ala Thr Ser Ala Thr Phe Ser Thr Glu Asp Arg 805 810 815 Arg Leu Thir Leu Ala Ser Lys Pro Llys Thr Ser Gln Pro Val Tyr Arg 820 825 830 Glu Arg Ile Ala Tyr Thr Tyr Met Asp Glu Asp Leu Pro Thr Thr Glu 835 840 845

Tyr Ile Ser Pro Asn Glu Asp Gly Thr Phe Thr Leu Pro Glu Glu Ala 85 O 855 860

Glu Thr Met Glu Gly Ala Thr Val Pro Leu Lys Met Ser Asp Phe Thr 865 870 875 88O Tyr Val Val Glu Asp Met Ala Gly Asn Ile Thr Tyr Thr Pro Val Thr 885 890 895 Lys Lieu Lieu Glu Gly His Ser Asn Lys Pro Glu Glin Asp Gly Ser Gly 9 OO 905 910 Glin Thr Pro Asp Lys Lys Pro Glu Ala 915 920

<210> SEQ ID NO 11 &2 11s LENGTH 921 &212> TYPE PRT <213> ORGANISM: Streptococcus agalactiae US 6,270,775 B1 S1 52

-continued <400 SEQUENCE: 11

Asp Ala Gly Phe Asn His Glu Ala Trp Arg Teu Thr Lys 1 10 15

Thr Lys Ala Arg Ser Glu Asp Teu Glu Ala Lys 2O 25 30

Glu His Gly Ile Thr Gly Glu Trp Wall Asn Asp Lys Wall Ala Tyr 35 40 45

His Asp Ser Asp Gly Lys Thr Ala Wall Asp Glin Glu His 50 55 60

Thr His Ser Gly Ile Telu Ser Gly Asn Ala Pro Ser Glu Thr 70 75

Glu Pro Arg Teu Glu Gly Ala Met Pro Glu Ala Glin Telu Telu 85 90 95

Teu Met Arg Wall Glu Ile Wall Asn Gly Telu Ala Asp Ala Arg Asn 100 105 110

Ala Glin Ala Ile Arg Asp Ala Ile Asn Teu Gly Ala Wall Ile 115 120 125

Asn Met Ser Phe Gly Asn Ala Ala Telu Ala Ala Asn Teu Pro Asp 130 135 1 4 0

Glu Thr Ala Phe Asp Ala Ser Gly Wall Ser Ile 145 15 O 155 160

Wall Thr Ser Ala Gly Asn Asp Ser Ser Phe Gly Gly Lys Thr Arg Telu 1.65 170 175

Pro Telu Ala Asp His Pro Asp Gly Wall Wall Gly Thr Pro Ala Ala 18O 185 190

Ala Asp Ser Thr Teu Thr Wall Ala Ser Ser Pro Asp Glin Telu 195 200 2O5

Thr Glu Thr Wall Arg Wall Lys Thr Asp Glin Glin Asp Glu Met 210 215 220

Pro Wall Telu Ser Thr Asn Arg Phe Pro Asn Ala Tyr 225 230 235 240

Ala Ala Asn Arg Gly Thr Asp Asp Phe Asp Wall 245 250 255

Gly Lys Ile Ala Teu Ile Glu Arg Asp Ile Asp Phe Tys Asp Lys 260 27 O

Ile Ala Lys Ala Ala Gly Wall Gly Wall Teu Ile Tyr Asp 275 280 285

Asn Glin Asp Gly Phe Pro Ile Telu Pro Asn Wall Asp Glin Met 29 O 295

Pro Ala Ala Phe Ile Ser Arg Asp Gly Teu Teu Teu Asn 305 310 315 320

Pro Glin Thr Ile Thr Phe Asn Ala Thr Pro Wall Teu Pro Thr 325 330 335

Ala Ser Gly Thr Teu Ser Arg Phe Ser Ser Trp Gly Teu Thr Ala 340 345 350

Asp Gly Asn Ile Pro Asp Ile Ala Ala Pro Gly Glin Asp Ile Telu 355 360 365

Ser Ser Wall Ala Asn Asn Lys Ala Teu Ser Gly Thr Ser Met 370 375 38O

Ser Ala Pro Telu Wall Ala Gly Ile Met Gly Teu Teu Glin Glin Tyr 385 390 395 400

Glu Thr Glin Pro Asp Met Thr Pro Ser Glu Arg Teu Asp Telu Ala 405 410 415 US 6,270,775 B1 S3

-continued

Wall Telu Met Ser Ser Ala Thr Ala Teu Tyr Asp Glu Asp Glu 420 425 430

Ala Tyr Phe Ser Pro Arg Glin Glin Gly Ala Gly Ala Wall Asp Ala 435 4 40 4 45

Lys Ala Ser Ala Ala Thr Met Wall Thr Asp Lys Asp Asn Thr 450 455 460

Ser Ser Wall His Teu Asn Asn Ser Asp Lys Phe Glu Wall Thr 465 470 475 480

Wall Asn Wall His Asn Ser Asp Pro Glin Glu Teu Tyr Glin 485 490 495

Ala Thr Wall Glin Thr Asp Wall Asp Gly His Phe Ala Telu Ala 5 OO 505 510

Pro Wall Telu Glu Ala Ser Trp Glin Ile Thr Ile Pro Ala 515 525

Asn Ser Ser Glin Wall Thr Wall Pro Ile Asp Ala Ser Arg Phe Ser 530 535 540

Lys Asp Telu Telu Ala Glin Met Asn Gly Tyr Phe Teu Glu Gly Phe 545 550 555 560

Wall Arg Phe Glin Asp Pro Glu Glu Teu Met Ser Ile Pro 565 570 575

Ile Gly Phe Arg Gly Asp Phe Gly Asn Teu Ser Ala Teu Glu 58O 585 59 O

Pro Ile Tyr Asp Ser Asp Gly Ser Ser His Glu Ala Asn 595 600 605

Ser Asp Ala Asp Glin Teu Asp Gly Asp Gly Teu Glin Phe Tyr Ala 610 615 620

Teu Asn Asn Phe Thr Ala Telu Thr Thr Glu Ser Asn Pro Trp Thr 625 630 635 640

Ile Ile Ala Wall Glu Gly Wall Glu Asn Ile Glu Asp Ile Glu 645 650 655

Ser Ser Glu Ile Thr Glu Thr Ile Telu Ala Gly Thr Phe Ala Lys Glin 660 665 670

Asp Asp Asp Ser His Ile His Arg His Ala Asn Lys Pro 675 680 685

Ala Ala Ile Ser Pro Asn Gly Asp Gly Asn Arg Asp Wall Glin 69 O. 695 7 OO

Phe Glin Gly Thr Phe Teu Arg Asn Ala Asn Teu Wall Ala Glu Wall 705 710 715 720

Teu Asp Glu Gly Asn Wall Wall Trp Thr Ser Glu Wall Thr Glu Glin 725 730 735

Wall Wall Asn Asn Asn Asp Telu Ala Ser Thr Teu Gly Ser Thr 740 745 750

Arg Phe Glu Thr Arg Trp Asp Gly Asp Asp Lys Wall 755 760 765

Wall Ala Asn Thr Thr Arg Wall Arg Tyr Thr Pro Ile Ser 770 775 78O

Ser Gly Ala Glu Glin His Thr Asp Phe Asp Wall Ile Wall Asp Asn 785 790 795

Thr Thr Pro Glu Wall Ala Thr Ser Ala Thr Phe Ser Thr Glu Asp Arg 805 810 815

Arg Telu Thr Telu Ala Ser Pro Lys Thr Ser Glin Pro Wall Tyr 820 825 830 US 6,270,775 B1 SS S6

-continued Glu Arg Ile Ala Tyr Thr Tyr Met Asp Glu Asp Leu Pro Thir Thr Glu 835 840 845

Ile Ser Pro Asn Glu Asp Gly Thr Phe Thr Leu Pro Glu Glu Ala 85 O 855 860

Thir Thr Glu Gly Ala Thr Val Pro Leu Lys Met Ser Asp Phe Thr 870 875 88O

Wal Wall Glu Asp Met Ala Gly Asn Ile Thr Tyr Thr Pro Wall Thr 885 890 895

Telu Telu Glu Gly His Ser Asn Lys Pro Glu Glin Asp Gly Ser Asp 9 OO 905 910

Glin Ala Pro Asp Lys Pro Glu Ala 915 920

What is claimed is: 16. The method according to claim 9 wherein the mammal 1. A vaccine comprising an immunogenic amount of an is a human, dog, bovine, porcine, or horse. enzymatically inactive Streptococcal C5a peptidase (SCP) 17. The method according to claim 16 wherein the mam peptide, wherein the peptide comprises the catalytic domain mal is human. of SCP, which amount is effective to prevent or ameliorate 18. The method of claim 9, wherein the vaccine comprises B-hemolytic StreptococcuS colonization or infection in a an enzymatically inactive Streptococcal C5a peptidase, con Susceptible mammal in combination with a physiologically 25 jugated or linked to a peptide. acceptable, non-toxic Vehicle. 19. The method of claim 9, wherein the vaccine comprises 2. The vaccine of claim 1 which further comprises an an enzymatically inactive C5a peptidase, conjugated or effective amount of an immunological adjuvant. linked to a polysaccharide. 3. The vaccine of claim 1 wherein the mammal is a 20. The vaccine of claim 1, wherein the catalytic domain human, dog, bovine, porcine or horse. is the Sequence corresponding essentially to amino acid 4. The vaccine of claim 3 wherein the mammal is human. residue 130 to amino acid residue 512 of SEQ ID NO:1, 5. The vaccine of claim 1 wherein the B-hemolytic SEO ID NO:2 or SEO ID NO:3. StreptococcuS is group A Streptococcus, group B 21. The vaccine of claim 1, wherein the catalytic domain Streptococcus, group C StreptococcuS or group G Strepto corresponds to SEQ ID NO:6, SEQ ID NO:7 or SEQ ID COCCS. 35 NO:8. 6. A method according to claim 5, wherein the 22. The vaccine of claim 1, wherein the enzymatically B-hemolytic StreptococcuS is Group A StreptococcuS. inactive peptide further comprises the region of SCP 7. The vaccine of claim 1, which comprises an enzymati between the catalytic domain and the cell wall insert. cally inactive Streptococcal C5a peptidase, conjugated or 23. The vaccine of claim 22, wherein the enzymatically linked to a peptide. 40 inactive peptide is the Sequence corresponding essentially to 8. The vaccine of claim 1, which comprises an enzymati amino acid residue 130 to amino acid residue 1050 of SEQ cally inactive Streptococcal C5a peptidase, conjugated or ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. linked to a polysaccharide. 24. The vaccine of claim 22, wherein the enzymatically 9. A method of protecting a Susceptible mammal against inactive peptide corresponds to SEQ ID NO:9, SEQ ID B-hemolytic StreptococcuS colonization or infection com NO:10 or SEO ID NO:11. prising administering to the mammal an effective amount of 45 25. A vaccine comprising an immunogenic amount of an a vaccine comprising an immunogenic amount of an enzy enzymatically inactive peptide comprising the catalytic matically inactive streptococcal C5a peptidase (SCP) domain of streptococcal C5a peptidase (SCP), wherein the peptide, wherein the peptide includes the catalytic domain of peptide is capable of generating antibody Specifically rec SCP, which amount is effective to prevent or ameliorate ognizes a streptococcal C5a peptidase (SCP), and wherein StreptococcuS colonization or infection in the Susceptible 50 the amount is effective to prevent or ameliorate B-hemolytic mammal in combination with a physiologically-acceptable, StreptococcuS colonization or infection in a Susceptible non-toxic vehicle. mammal in combination with a physiologically-acceptable, 10. The method of claim 9 wherein the vaccine further non-toxic vehicle. comprises an effective amount of an immunological adju 26. The vaccine of claim 25, wherein the catalytic domain Vant. 55 is the Sequence corresponding essentially to amino acid 11. The method of claim 9 wherein the vaccine is admin residue 130 to amino acid residue 512 of SEQ ID NO:1, istered by Subcutaneous or intramuscular injection. SEO ID NO:2 or SEO ID NO:3. 12. The method of claim 9 wherein the vaccine is admin 27. The vaccine of claim 25, wherein the catalytic domain istered by oral ingestion. corresponds to SEQ ID NO:6, SEQ ID NO:7 or SEQ ID 13. The method of claim 9 wherein the vaccine is admin 60 NO:8. istered intranasally. 28. The vaccine of claim 25, wherein the enzymatically 14. A method according to claim 9, wherein the inactive peptide further comprises the region of SCP B-hemolytic StreptococcuS is group A Streptococcus, group between the catalytic domain and the cell wall insert. B Streptococcus, group C StreptococcuS or group G Strep 29. The vaccine of claim 28, wherein the enzymatically toCOCCuS. 65 inactive peptide is the Sequence corresponding essentially to 15. A method according to claim 14, wherein the amino acid residue 130 to amino acid residue 1050 of SEQ B-hemolytic StreptococcuS is group A StreptococcuS. ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. US 6,270,775 B1 57 58 30. The vaccine of claim 28, wherein the enzymatically tion or infection in a Susceptible mammal in combination inactive peptide corresponds to SEQ ID NO:9, SEQ ID with a physiologically-acceptable, non-toxic Vehicle. NO:10 or SEO ID NO:11. 37. The method of claim 36, wherein the catalytic domain 31. The method of claim 9, wherein the catalytic domain is the Sequence corresponding essentially to amino acid is the Sequence corresponding essentially to amino acid residue 130 to amino acid residue 512 of SEQ ID NO:1, residue 130 to amino acid residue 512 of SEQ ID NO:1, SEO ID NO:2 or SEO ID NO:3. SEO ID NO:2 or SEO ID NO:3. 38. The method of claim 36, wherein the catalytic domain corresponds to SEQ ID NO:6, SEQ ID NO:7 or SEQ ID 32. The method of claim 9, wherein the catalytic domain NO:8. corresponds to SEQ ID NO:6, SEQ ID NO:7 or SEQ ID 39. The method of claim 36, wherein the enzymatically NO:8. inactive peptide further comprises the region of SCP 33. The method of claim 9, wherein the enzymatically between the catalytic domain and the cell wall insert. inactive peptide further comprises the region of SCP 40. The method of claim 39 wherein the enzymatically between the catalytic domain and the cell wall insert. inactive peptide is the Sequence corresponding essentially to 34. The method of claim 33, wherein the enzymatically amino acid residue 130 to amino acid residue 1050 of SEQ inactive peptide is the Sequence corresponding essentially to 15 amino acid residue 130 to amino acid residue 1050 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 41. The method of claim 39, wherein the enzymatically 35. The method of claim 33, wherein the enzymatically inactive peptide corresponds to SEQ ID NO:9, SEQ ID inactive peptide corresponds to SEQ ID NO:9, SEQ ID NO:10 or SEO ID NO:11. NO:10 or SEO ID NO:11. 42. A vaccine comprising an immunogenic amount of an 36. A method of protecting a Susceptible mammal against enzymatically inactive peptide comprising an epitope of the B-hemolytic StreptococcuS colonization or infection com catalytic domain of Streptococcal C5a peptidase (SCP), prising administering to the mammal an effective amount of wherein the peptide is capable of generating antibody that vaccine comprising an immunogenic amount of an enzy Specifically recognizes a Streptococcal C5a peptidase (SCP), matically inactive peptide comprising the catalytic domain 25 and wherein the amount is effective to prevent or ameliorate of streptococcal C5a peptidase (SCP), wherein the peptide is B-hemolytic StreptococcuS colonization or infection in a capable of generating antibody Specific to Streptococcal C5a Susceptible mammal in combination with a physiologically peptidase (SCP), and wherein the amount is effective to acceptable, non-toxic Vehicle. prevent or ameliorate B-hemolytic StreptococcuS coloniza k k k k k