AU2002318786B2 - Vaccine based on a cysteine protease or fragment thereof - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: BAYLOR COLLEGE OF MEDICINE Actual Inventors: JAMES A. MUSSER, VIVEK KAPUR Address for Service: CALLINAN LAWRTE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: VACCINE BASED ON A CYSTEINE PROTEASE OR FRAGMENT THEREOF The following statement is a full description of this invention, including the best method of performing it known to us:- 16/12/02,mc13102.cs,1 VACCINE BASED ON A CYSTEINE PROTEASE OR FRAGMENT THEREOF

INTRODUCTION

TECHNICAL FIELD The present invention relates generally to the fields of molecular bacteriology and infectious disease. More specifically, the present invention relates to a vaccine for group A Streptococcus, particularly S. pyogenes.

BACKGROUND

Streptococcuspyogenes is a Gram-positive bacterium that is the etiological agent of several diseases in humans, including pharyngitis and/or tonsillitis, skin infections (impetigo, erysipelas, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever poststreptococcal glomerulonephritis (PSGN), and a toxic-shock like syndrome (TSLS). On a global basis, ARF is the most common cause of pediatric heart disease. For example, it is estimated that in India more than six million school-aged children suffer from rheumatic heart disease. In the United States, "sore throat" is the third most common reason for physician office visits and S. pyogenes is recovered from about 30% of children with this complaint. There are about 25-35 million cases of streptococcal pharyngitis per year in the United States, responsible for about 1-2 billion dollars per year in health care costs.

In recent years, an intercontinental increase in streptococcal disease frequency and severity has occurred for unknown reasons, although two variant pyrogenic exotoxin A (SPEA) molecules have been implicated. The amino acid residues characterizing the mutant SPEA molecules are located in an area of the toxin that, based on recently published three-dimensional crystal structure of the related enterotoxin B from Staphylococcus aureus, form the T-cell receptor binding groove.

S. pyogenes synthesizes an extracellular zymogen of 371 amino acids (40,314 kDa) that can be transformed into an enzymatically active protease of 253 amino acids (27,588 kDa) by autocatalytic conversion. The zymogen contains one or more epitopes not associated with the truncated enzyme. Both the zymogen and active protease contain a single half-cysteine per molecule that is susceptible to sulfhydryl antagonists. In broth cultures, inactive precursor accumulates extracellularly during bacterial multiplication and reaches a maximum concentration at the end of logarithmic growth. Some strains yield up 16/12/02,mel 3102.speci,2 -3to 150 mg/liter of zymogen, and the molecule is a major extracellular protein. Thus, the streptococcal cysteine protease resembles many secreted bacterial extracellular protease virulence factors in having a specific signal peptide and a pro-sequence that is removed in an autocatalytic fashion to generate a fully active enzyme.

The continued great morbidity and mortality caused by S. pyogenes in developing nations, the significant health care financial burden attributable to Group A streptococci in the United States, and increasing levels of antibiotic resistance in this pathogen highlight the need for a fuller understanding of the molecular pathogenesis of streptococcal infection. Moreover, the recent disease increase underscores the lack of an efficacious vaccine, despite the repeated inclusion of S. pyogenes in lists of important human pathogens for which vaccines are needed.

Protection against systemic streptococcal infection is thought to be due predominantly to type-specific opsonic anti-M protein IgG. As a consequence, immunoprophylaxis research has been conducted almost exclusively in the context of formulating an M protein vaccine. However, two major theoretical and practical problems have hindered this approach. First, more than 100 distinctive M protein types have been described based on serological and gene sequencing studies. The occurrence of this extensive array of serotypic variants means either that an effective M protein vaccine must be heterogenous in composition or that conserved protective M protein elements must be used. Formulation of a highly polyvalent vaccine has generated little enthusiasm, and a conserved pan-protective M protein fragment has yet to be identified. A second problem that has plagued M protein vaccine research is the observation that M proteins contain epitopes that cross-react with heart and other human tissue. This fact, in concert with the presumed autoimmune aspects of several streptococcal diseases, has also slowed M protein vaccine development. Detection of Group A streptococcal infections has been hampered by the fact that currently available assays have relied upon detection of the Group A streptococcal M protein antigen. It therefore would be of interest to develop an effective vaccine for the immune prophylactic prevention of Group A streptococcal infection based upon other than the M protein and an efficient method of immunizing a human against Group A streptococcal infections. The extracellular protease, and antibodies generated against it, can provide the basis of screening assays. Likewise, PCR-based assays can be used for detection of nucleic acid sequences which encode the extracellular protease. It therefore is of interest to identify conserved epitopes, particularly immunodominant 16/12/02,mcl 3102.speei,3 -4conserved epitopes in the cysteine protease molecule, for the development of compositions and methods which can be used for screening for Group A streptococcus organisms.

RELEVANT LITERATURE Antibodies to molecules other than M proteins have been reported to provide protection against S. pyogenes infection as disclosed in the following references. Chappell and Stuart (Vaccine (1993) 11:643-8) found that intraperitoneal inoculation of washed bacterial cultures of an M-negative isolate protected mice against lethal challenge with S.

pyogenes strains expressing M3, M18, or type 28 surface molecules. The non-typespecific immunity was attributed to the presence of antibodies against three proteins of M, 32, 43 and 46 kDa. Stjernquist-Desatnik et al. (Vaccine (1990) 8:150-2) showed that intranasal vaccination with heat-killed isolates of M negative strains of S. pyogenes protected against intranasal challenge with an isolate expressing serotype M50. Rotta et al.

Exp. Med. (1965) 122:877-90) demonstrated heterologous protective immunity in mice immunized via the intraperitoneal route with streptococcal cell wall preparations or material enriched in peptidoglycan, a group A streptococcal cell wall component.

O'Connor and coworkers (J Infect. Dis. (1991) 163:109-16) have suggested that neutralizing antibodies directed against the streptococcal cell surface enzyme peptidase may provide protection against streptococcal colonization, but have not directly tested this hypothesis. A recent study by Dale et al. Infect. Dis. (1994) 169:319-23) suggested that organisms pretreated with antibodies to the group A streptococcal surface molecule lipoteichoic acid (LTA) are significantly attenuated in a mouse model of infection. However, due to the lack of inclusion of a non-specific antibody control in that study, it is not possible to determine if the protective effect of the antibodies was due to interaction with LTA, or was due merely to the non-specific binding ofimmunoglobulin to the bacterial surface.

S. pyogenes culture supernatants contain a protease that has fibrinolytic activity.

(Elliot (1945) J. Exp. Med. 81:573-92). The enzyme was purified, shown to be a cysteine protease (Lui et al. (1963) 238:251-6) and found to be identical to or an allelic variant of streptococcal pyrogenic exotoxin B (SPE (Gerlach et al. (1983) Zbl. Bakt. Hyg.

255:221-3; Hauser and Schlievert (1990) J Bacteriol. 172:4536-42).

16/12/02,mcl3102.speci,4 SUMMARY OF THE INVENTION The present application is a divisional application of Australian application No.

59321/91 (the "parent"), the parent specification of which is herein incorporated by reference, which is a divisional application of 35538/95 (the "grandparent"), the grandparent specification which is herein incorporated by reference.

Vaccines and methods for protecting against Group A streptococcal infection are provided. The vaccines inhibit replication of the Streptococcus and thus prevent or lessen the adverse clinical effects caused by streptococcal infections. The vaccines include a streptococcal extracellular protease or a peptide that includes either a region that is conserved among the different streptococcal extracellular protease alleles, or a region that varies among alleles. The extracellular protease in the vaccine composition is either alone or in conjunction with a streptococcal M-protein. The invention also provides a method of immunizing a mammal with the vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the processing sites, locations of amino acid variations found in the proteins made by the speB2 and speB4 alleles, and amino acids that are targets for mutation.

Figure 2 shows the generation of random mutations in speB.

Figure 3 shows protection with rabbit anti-cysteine protease IgG in mice. Mice were injected i.p. with 0.1 ml ofl mg rabbit antibody (IgG) in PBS (pH 7.4) raised against cysteine protease purified from strain MGAS 1719, which has speB7 allele and produces the SPE B 4 mature SPE B variant. The antibody was raised in rabbits against purified protease excised from SDS polyacrylamide gel. Control animals received either an equal amount of pre-immune antibody (0.1 ml containing 1 mg of IgG in PBS, pH 7.4) from the same rabbit used as the source for the anticysteine protease antibody, or 0.1 ml of PBS.

After 30 minutes, mice were injected i.p. with approximately 100 cfu of strain MGAS 315, which has the speB3 allele and produces the SPE BI mature SPEB variant and is an ET2/m3 organism from a case of TSLS in (Musser et al. 1993). The times required for 50% mortality for the mice groups are as follows: PBS, 26 hours; pre-immune, 25 hours; anti-cysteine protease, 55 hours.

Figure 4 shows alleles of speB. The polymorphic sites within the 160 bp upstream non-coding region and 1197 bp coding region of the speB gene are shown. The sequence described by Hauser and Schlievert Bacteriol (1990) 1972:4536-42)) was arbitrarily 16/12/02,mcl 3102.speci,5 -6designated as SpeB1, and the numbering of nucleotides and codons is cognate with that sequence. Only those nucleotides in the other alleles that differ from the speB 1 sequence are shown. The position of each polymorphic nucleotide site is shown above the 39 alleles and is numbered in vertical format. Non-synonymous nucleotide changes are underlined and the positions of the coding changes are designated by an asterisk above of the coding region polymorphic sites. The seven polymorphic nucleotide sites in the upstream noncoding region are shown in the left of the figure, and the asterisk in speB6 denotes a deletion of an adenine residue. The codon (numbered in vertical format) containing the polymorphic nucleotide sites is shown below the 39 alleles. The DNA sequence data for speB2 speB39 are available from EML/GenBank/DDBJ under accession numbers L26125-L26162. The DNA sequence data for speB1 was published in Hauser and Schlievert, J. Bacteriol (1990), 172:4536-42.

Figure 5 shows the results of passive administration of anti-protease antibody which protects mice against lethal challenge with heterologous Spyogenes. Intraperitoneal administration of rabbit antibody directed against streptococcal cysteine protease confers significant protection against lethal challenge with the highly virulent Spyogenes isolate MGAS 315 when compared with control animals that were given PBS or rabbit preimmune serum.

Figure 6 is a schematic representation of amino acid substitutions in the streptococcal cysteine protease and protease precursor in the challenge strain and the protease to which the antiserum was raised A short peptide fragment of the protease surrounding amino acid 308 contains immunodominant epitopes recognized by mouse polyclonal and monoclonal antibodies. The single letter amino acid abbreviations are A, Ala; V, Val; G, Gly; S, Ser.

Figure 7 shows the results of active immunization of mice with the streptococcal cysteine protease which protects against lethal challenge with heterologous S. pyogenes.

The data show that intraperitoneal immunization with purified streptococcal cysteine protease conferred significant protection (log rank test X 2 P 0.01) against lethal challenge with the highly virulent S. pyogenes isolate MGAS 315.

Figure 8 shows that rabbit serum to streptococcal M3 protein (lane T8 protein (lane or purified IgG from preimmune rabbits (lane does not react with the streptococcal protease. In contrast, purified IgG from rabbits immunized with the protease reacts specifically with the Mr 30 kDa streptococcal extracellular cysteine protease used in active immunization experiments (lane 4).

16/12/02,mcl 3102.speci,6 -7- BRIEF DESCRIPTION OF THE INVENTION The peptide used in the vaccine is derived from a cysteine protease or any portion of the protease that provokes an immune response, with consequent immunity, to Group A streptococcal infections. Generally, the cysteine protease is obtainable as a translated portion of the speB gene, or fragments or derivatives thereof, which provoke an immune response. The cysteine protease can be naturally occurring, or partially or wholly synthetic. A mammal can be immunized against Group A streptococcal infection by administration of the vaccine to the mammal in an amount sufficient to confer immunity to Group A streptococcal infection. The immunization methods will inhibit replication of Streptococcus species in the mammal and decrease symptoms that are associated with the disease, including phyaryngitis, tonsillitis, skin infections, scarlet fever, sepsis, erysipelis, fasciitis, pneumonia, acute rheumatic fever, poststreptococcal glomerulonephritis, cellulitis, bacteremia, and meningitis.

A vaccine based upon the speB gene product offers several advantages over those which are M-protein based. For example, antibodies to M-proteins are known to cross react with various host tissues, and there is considerable concern that an M-protein vaccine may evoke human autoimmune-mediated disease due to this sharing of epitopes. The greater than 100 identified M protein types elicit a predominantly type-specific immunity.

Thus, an effective M-protein based vaccine would need to be highly polyvalent or directed against as yet unidentified conserved pan-protective M-protein epitopes. The vaccine of the present invention is not type-specific and therefore is useful in the prevention of any Group A streptococcal infections. Antibodies to the cysteine protease or its zymogen are not known to cross-react with host tissues.

Streptococcus pyogenes is a gram positive coccus which is p-hemolytic, expresses group A antigen and is susceptible to bacitracin. For the purpose of this disclosure, a microorganism is considered to be the same as or equivalent to Streptococcuspyogenes if in its genome is a coding sequence for an extracellular protease which is involved in the pathogenesis of the microorganism. Accordingly, peptides are provided which immunologically mimic extracellular proteases encoded by the S. pyogenes bacterium, particularly proteins derived from the expression product of the speB region of the bacterial genome. To accommodate strain-to-strain variations among different isolates, adjustments for conservative substitutions and selection among the alternatives where nonconservative substitutions are involved, may be made. These peptides can be used individually or together for detection of the bacterium or of antibodies to the bacteria in a 16/12/02,mcl3102.speci,7 -8physiological sample. Depending upon the nature of the test protocol, the macromolecules may be labeled or unlabeled, bound to a solid surface, conjugated to a carrier as other compounds, or the like.

Of particular interest are peptides which are derived from the protein encoded by the speB gene, which encodes a zymogen and extracellular cysteine protease that cleaves human interleukin 11 precursor to form biologically active 1L-103, a major cytokine mediating inflammation and shock. The purified protease cleaves fibronectin and rapidly degrades vitronectin. The protein has no substantial activity against laminin. The cysteine protease also cleaves fibrotectin from human umbilical vein endothelial cells grown in vitro. Other organisms which produce extracellular proteases which are involved in pathogensis of the host organism may also be used as a source of the enzyme or fragments thereof, and of genetic material for use in the subject invention.

Several human pathogenic bacteria express extracellular proteases capable of degrading ECM proteins. Among these organisms are Pseudomonas aeruginosa, (Morihara Kamp Homma, In: Holder IA, ed. Bacterial enzymes and virulence, FL CRC Press, 1985; 41-75) and Porphyromonas (Bacteriodes) gingivalis, (Lantz et al, J. Bacteriol 1991; 173:495-504; and Otogoto and Kuramitsu, Infect Immun 1993; 61:117-23) two bacterial species that produce host tissue destruction by degradation of collagen and fibronectin, respectively. It is also noteworthy that Trypanosoma cruzi, the parasitic flagellate which causes American trypanosomiasis (Chagas' disease), expresses a cellsurface cysteine protease that is a major antigen in humans, (Gazzinelli et al, Infect Immun 1990; 58:1437-44; and Murta ACM et al, Molec Biochem Parasitol 1990; 43:27-38) and is thought to be an important virulence factor. (Eakin AE et al, J Biol Chem 1992; 267:7411- The enzyme (cruzipain) cleaves immunoglobulin G molecules and hydrolyzes the Fc fragment, (Murta ACM et al, Molec Biochem Parasitol 1990; 43:27-38) thereby assisting the organism to evade the immunological consequences of antibody binding. Entamoeba histolytica, the cause ofamebiasis, also produces an extracellular cysteine protease that is widely believed to be a major virulence factor. (Keene WE et al, J Exp Med 1986; 163:536-49.) Like the streptococcal cysteine protease, the E. histolytica enzyme degrades several ECM proteins, including type I collagen, fibronectin, and laminin. (Keene WE et al, J Exp Med 1986; 163:536-49.) The protease also causes cytopathic effect on cell culture monolayers, (Keene WE et al, Exp Parasitol 1990; 71: 199-206) and is involved with production of tissue necrosis in rat models of acute amebiasis. (Becker I et al, Exp Parasitol 1988; 67:268-80.) 16/12/02,mcl 3102.spcci,8 -9- The peptide includes at least five, sometimes six, sometimes eight, sometimes about 22 amino acids, but usually fewer than 50 amino acids, preferably fewer than about amino acids that comprise a subsequence of the protein encoded by the coding region of the cysteine protease gene. The peptide includes at least one linear epitope within the amino acid sequence which corresponds to the preproenzyme, the entire sequence of which shown below as SEQ ID The peptide will be as small as possible while still maintaining substantially all of the sensitivity of the larger peptide, the sequence of which is shown below as SEQ ID NO: In some cases there may be two or more smaller not overlapping peptides, each contained within the parent peptide, which separately or together provide equivalent sensitivity to the parent peptide. The peptide sequences may be modified by terminal-NH 2 acylation, terminal carboxy amidation, for example, ammonia methylamine, etc.

Furthermore, it will be appreciated that the amino acid sequence need not correspond exactly to any portion of the sequence given above, provided that the relevant epitope(s) are substantially retained. It also will be appreciated that the base pairs to which the above-cited peptide sequence corresponds may exhibit strain-to-strain variation.

16/12/02,mcl 3102.speci,9 uj tQ C SEQ ID 20~ 30 40 50 HNKKKLGIRL LSLLALGGFV LANPVFADQN FAflNEKEAKD SAITFIQKSA AIKAGARSAE DIKLDKVNLG GELSGSNMYV YNISTGGFVI VSGDKRSPEI LGYSTSGSFD ANGKENIASF 120 MESYVEQIKE NKILDTTYAG TAEIKQPVVK SLLDSKGIHY .NQGNPYNLLT PVIEKVKPGE 180 QSFVGQHAAT GCVATATAQI MKYHNYPNKG LKDYTYTLSS NNPYFNHPKN; LFAAISTRQY 240

NWMNII

1 PTYS GRESNVQK4A ISELMADVGI SVDMDYGPSS GSAGSSRVQR ALKENFGYNQ 300 310 320 330 340 350 360

SVHQINRSDF

GVSDGFFRLD

SKQDWEAQID

ALNPSALGTG

KELSQNQPVY

GGAGGFNGYQ

YQCGVGKVGGH

SAVVGIKP.

AFVIDGADGR

399 NFYHVNWGWG 360 -11- It should be understood that the polypeptides employed in the subject invention need not be identical to a cysteine protease obtainable from S. pyogenes, so long as the subject compounds are able to provide for immunological interaction with a cysteine protease obtainable from at least one of the strains of S. pyogenes. Therefore, the subject polypeptides may be subject to various changes such as insertions, deletions, and substitutions, other conservative or non-conservative, where such changes might provide for certain advantages in their use. By conservative substitution is intended combinations such as gly, ala; val, ile, leu; asp, glu; asn, gin; ses, thr; lys, arg; and phe tyr. Usually, the sequence will not differ by more than 20% from the sequence of at least one strain of S.

pyogenes except where additional amino acids may added at either terminus for the purpose of providing an "arm" by which the peptides of this invention may be immobilized conveniently. The arms usually will be at least 5 amino acids, and may be 50 or more amino acids.

Of particular interest as immunogens are peptides of at least 10 amino acids which include one or more of the following (numbers correspond to SEQ ID NO:5): amino acids 308-317, amino acids immediately adjacent to and to the left of residue 308, and amino acids immediately adjacent to and to the fight of residue 317 of S. pyogenes cysteine protease.

The peptides can be prepared in a wide variety of ways. The cysteine protease itself can be purified from natural sources, for example by binding to Red A, or by other means known to those skilled in the art of protein purification, such as affinity chromatography using antibody to the protein. The peptides, because of their relatively short size, also may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. The peptides are selected based upon the location of allelic variation and conservation and the cysteine protease antigenic index generated with a Jameson-Wolf plot. For example, peptides corresponding to the variable region (amino acids 308-317) of mature streptococcal cysteine protease are prepared.

Subsequently, peptides which correspond to the invariant calculated antigenic peaks in the cysteine protease are used for immunization. Examples of such sequences include P(171)- V-I-E-K-V-K-P-G-E-Q-S-F-V-G-Q (SEQ ID NO: Y(203)-H-N-Y-P-N-K-G-L-K-D-Y- T-Y-T-L (SEQ ID NO:2), P(247)-T-Y-S-G-R-E-S-N-V-Q-K-M-A-I (SEQ ID NO:3), I(344)-D-G-A-D-G-R-N-F-Y-H (SEQ ID NO:4). Naturally occurring variant zymogens of cysteine protease and cysteine protease itself display unique liner B-epitopes.

16/12/02,mc13102.speci,11 -12- To prepare synthetic peptides, overlapping 10-mer peptides are used which overlap 2 amino acid residues with the previous 10-mer in a consecutive primary sequence corresponding to the 371 amino acids of the mature cysteine protease zymogen (translated product minus leader sequence). Synthetic 10-mers corresponding to each variant amino acid residue are also used. The variant amino acids are positioned in the middle of the mer.

Once the 10-mer peptides are synthesized, an ELISA is used to examine the reactivity of each peptide using a panel of monoclonal antibodies raised against purified cysteine protease as well as sera obtained from patients with symptoms of S. pyogenes infection. The linear B cell epitopes are then determined; the same linear B cell epitopes will most likely be recognized by all of the sera from patients with S. pyogenes infection.

Both site-directed and random mutagenesis schemes can be employed to identify residues that disrupt cysteine protease function and zymogen processing, and to map regions that constitute antigenic domains of the protein. Figure 1 shows the processing sites, locations of amino acid variations found in the proteins made by the speB2 and speB4 alleles, and amino acids that are targets for mutation. Targets for functional amino acid replacement are based on biochemical analysis of cysteine protease (Tai, et al., J. Biol Chem.(1976) 251:1955-91 and by analogy with similar residues in the eukaryotic cysteine protease.

To create a stable zymogen to facilitate crystallographic studies and generate enzymatically deficient or inactive protease for structure-function studies, mutant forms of the cysteine protease protein are made and characterized. A targeted mutagenesis scheme creates changes that: disrupt protease activity; (ii) prevent zymogen processing; (iii) prevent substrate binding; and (iv) alter immunoreactivity. Amino acids are changed to structurally neutral alanine. As an example, a mutant protein that lacks protease activity, but which retains antigenicity, can be generated by mutagenesis of the single cysteine residue (Cys-192- Ala-192) at the catalytic site of the molecule. Also, His-340 and Gln- 185 and Asn-356 are mutagenized. These three changes are epistatic to the Cys-192 mutation, but may alone exhibit altered activity. Trp-357, thought to be involved in substrate binding and similarly positioned within papain, is also to be targeted.

A stable zymogen precursor is also created by mutating residues surrounding the protease cleavage site at Lys-145. In addition, mutagenesis of Cys-192 may prevent autoproteolysis, as occurred for a Cys- Ser mutant of papain, the prototype cysteine protease. Other mutagenesis targets include a putative nucleotide binding domain 16/1 202,mcl 3102.speci,12 13 (GVGKVG) (SEQ ID NO:6) and a potential collagen docking region [(GXX) 3 within the carboxy terminal portion of the protein. Site-directed mutagenesis is used, by the chargedto-alanine-scanning method, to substitute positively and negatively charged amino acids (often involved in recognition and activity) with alanine. Many of the charged residues (14 lysine, 7 arginine, 12 aspartate, and 7 glutamate residues in the mature peptide) are expected to lie on the surface of the cysteine protease structure, and some are expected to define epitopes on the molecule. In particular, a region of charged.amino acids, from 307 to 321 (8/15 charged), is examined; this region includes the site ofspeB2 and speB4 amino acid substitutions. Residues in antigenic regions identified in the epitope mapping studies are also mutated.

In order to create mutant speB proteins, as an example, first, the speB gene is amplified from a S. pyogenes strain, with PCR, and the product is cloned into a multicopy filamid vector such as pBluescript (Stratagene, La Jolla, CA). This vector is chosen because it carries the regulated lac promoter and can be replicated as a single-stranded molecule for site-directed mutagenesis. Cloning is designed to place the promoter, ribosome binding site and speB reading fame 3' to an inducible promoter, such a lac promoter, on the vector so that the protein can be conditionally over-expressed in a bacterial host such as an inducer of the inducible promoter is added to the culture broth.

For example, with the lac promoter, the E. coli when the lac inducer, IPTG, can be added.

The speB promoter is included requiring expression in S. pyogenes. Whole cell extracts and periplasmic shockates of E. coli cells carrying this primary speB clone are examined for the presence of the cysteine protease protein by SDS-PAGE and by Western blotting with anti-cysteine protease antibody. The resulting plasmid is the target for mutagenesis.

Oligonucleotide-directed mutations, such as substitutions, deletions and small insertions, are created on uracil-containing single-stranded templates by the method of Kunkel (Kunkel, 1985). When possible, mutagenic primers are designed to incorporate a unique restriction site into the speB gene for mapping and mutant selection. Both single and multiple alanine substitutions are created at the residues indicated above. Once residues critical to function are identified, small regions surrounding them are deleted or substituted, by using the same methods, to further characterize the region and to preclude reversion. When crystallographic data is available, additional amino acids are mutated.

A random mutagenesis scheme also can be employed. Variant proteins created by this method are most useful for epitope screening, although molecules with altered kinetics and substrate recognition also may be recovered. Regions of the speB sequence are 16/12/02,mcl3102.speci,13 -14randomized with mixed oligonucleotides in the primed-mutagenesis protocol, or short, inframe deletions within the gene are created with a modification of the DNAse I-linker insertion/deletion protocol of Palzkill and Bostein (Palzkill and Bostein, 1991).

To identify protease minus mutations, bacterial host cells producing potential mutant proteins are first screened for protease activity on casein agar plates. Since secretion of cysteine protease to the periplasm is expected, protease activity can be observed on plates. If this screening strategy is successful, thousands of colonies are rapidly examined for functional mutations in cysteine protease. Ifcysteine protease must be completely secreted from E. coli to exhibit activity, then osmotic shockates of each presumptive mutant strain are assayed for protease activity.

Alternatively, hybrid-DNA technology may be employed where a synthetic gene may be prepared by employing single strands which code for the polypeptide or substantially complementary strands thereof, where the single strands overlap and can be brought together in an annealing medium so as to hybridize. The hybridized strands may then be ligated to form the complete gene and by choice of appropriate termini, the gene may be inserted into an expression vectors. Alternatively, the region of the bacterial genome coding for the peptide may be cloned by conventional recombinant DNA techniques and expressed.

An example of a DNA coding sequence which may be used for expressing the cysteine protease is SEQ ID NO:7.

Fragments from this sequence can be employed for expression of peptide fragments, synonymous base changes can be made, where the modified codon code for the same amino acid(s), or non-synonymous changes in the coding sequence may be made, where the resulting amino acid may be a conservative or non-conservative change. The coding sequence is extended at either the or a 3'-terminus or both termini to extend the peptide while retaining its epitopic site. The extension may provide for an arm for linking, as a label, for example an enzyme, for joining two or all of the peptides together in the same chain, for providing antigenic activity, and the like. For expression, the coding sequence is provided with start and stop codons, promoter and terminator regions, and usually a replication system to provide an expression vector for expression in a cellular host, for example prokaryotic or eukaryotic, bacterial, yeast, and mammalian, etc. The sequences by themselves, fragments thereof, or larger sequences, usually at least 15 bases, preferably at least 18 bases, may be used as probes for detection of bacterial DNA.

16/12/02,mc13102.speci,14 Numerous techniques are described, such as the Southern technique, Northern technique, and improvements thereon, as well as other methodology.

Conveniently, the polypeptides may be prepared as fused proteins, where the polypeptide may be the N- or C-terminus of the fused polypeptide. The resulting fused protein can be used directly by itself as the reagent or the subject peptide may be cleaved from all or a portion of the remaining sequence of the fused protein. With a polypeptide where there are no internal methionines, by introducing a methionine at the fusion site, the polypeptide may be cleaved employing cyanogen bromide. Where there is an internal methionine, it will be necessary to provide for a proteolytic cleavage site, for example, poly-lysine, or -arginine, or combinations thereof A wide variety of proteases, including dipeptidases, are well known and the appropriate processing signal can be introduced at the proper site. The processing signal may have tandem repeats so as to insure cleavage, since the presence of one or more extraneous amino acids will not interfere with the utility of the subject polypeptides.

The subject peptides may be employed linked to a soluble macromolecular (220kd) carrier. Conveniently, the carrier may be a poly (amino acid) either naturally occurring or synthetic, to which antibodies are unlikely to be encountered in human serum. Illustrative polypeptides include poly-L-lysine, bovine serum albumin, keyhole limpet hemocyanin, bovine gammaglobulin, etc. The choice is primarily one of convenience and availability.

With such conjugates, there will be at least one molecule of at least one subject peptide per macromolecule, and not more than about one per 0.5kd, usually not more than one per 2kd of the macromolecule. One or more different peptides may be linked to the same macromolecule.

The manner of linking is conventional, employing such reagents aspmaleimidobenzioc acid, p-methyldithiobenzioc acid, maleic acid anhydride, succinic acid, anhydride, glutaraldehyde, etc. The linkage may occur at the N-terminus, C-terminus, or at a site intermediate the ends of the molecule. The subject peptide may be derivatized for linking, may be linked while bound to a support, or the like.

The compounds may be employed as labeled or unlabeled compounds as described in the parent application. Various assay protocols can be employed for detecting the presence of either antibodies to the cysteine protease or the cysteine protease itself, to the zymogen or antibodies to the zymogen as described in the parent application.

Antibodies to the peptides of the subject invention can be prepared in conventional ways as described in the parent application.

16/ 2/02,mcl 3102.speci,15 -16- The subject peptides find use by themselves or in combination to generate a protective immune response, and in vaccines, particularly for humans, but also in commercially valuable animals such as mink which are susceptible to group A streptococci infection, or other animals such as mice. The peptides are be formulated in a convenient manner, generally at concentrations in the range of 1 !tg to 20 mg-kg. Physiologically acceptable media may be used as carriers, such as sterile water, saline, phosphate buffered saline, and the like. Adjuvants may be employed, such as aluminum hydroxide gel, and the like. Administration may be by injection, for example intramuscularly, intraperitoneally, subcutaneously, intravenously, etc. Administration may be one or a plurality of times, usually at one to four week intervals. In addition, the efficacy of the vaccine may be enhanced by the addition of a streptococcal M protein antigen. In this vaccine, the conserved domain of the streptococcal M protein is combined with the cysteine protease. Preferably, the M protein that is used does not cross-react immunologically with host tissues. Production of antibodies may be used as a protection against group A Streptococcus, where protection may delay onset of death or prevent mortality due to infection.

Purified cysteine protease has several uses including use as a replacement for trypsin or other proteases in obtaining isolated cells, particularly those which are within a protein matrix, a collagen matrix, such as biopsy specimen, or adhered to a tissue culture plate or petri dish, or bound to an affinity column via a protein arm. The protease also finds use in the removal of excess scar tissue and for cleaning tissue from a biological substrate such as bone.

The DNA encoding the peptides of the subject invention find use as probes for identifying other sources of cysteine protease, and in PCR-based assays for identifying the presence of a group A Streptococcus, particularly S. pyogenes in a physiological sample.

The following examples are given for the purpose of illustrating various embodiments of the methods of the present invention and are not meant to limit the present invention in any fashion.

Examples 1 to 18 of the "parent" specification are specifically incorporated herein by reference.

EXAMPLE 1 Preparation of Synthetic Peptides ofCysteine Protease 17/12/02.mcl 3102.speci.16 17- Synthetic peptides based on cysteine protease may also be used as immunogens in the preparation of a vaccine against Group A streptococcal infections. Several synthetic peptides are selected based on the location of allelic variation and conservation and the cysteine protease antigenic index generated with a Jameson-Wolf plot. First, each of the following three peptides are used. These peptides correspond to the variable region (amino acids 308 to 317) in mature streptococcal cysteine protease containing two of the six major calculated antigenic peaks.

Peptide 1 (SEQ ID NO: 15): H-Q-I-N-R-S(308)-D-F-S-K-Q-D-W-E-A(317)-Q-I-

D-K-E

Peptide 2 (SEQ ID NO: 16): H-Q-I-N-G(308)-D-F-S-K-Q-D-W-E-A(317)-Q-I-D-

K-E

Peptide 3 (SEQ ID NO:17): H-Q-I-N-S(308)-D-F-S-K-Q-D-W-E-A(317)-Q-I-D-K-

E

Subsequently, each of the following four peptides, which correspond to four invariant calculated antigenic peaks are used for immunization.

Peptide 4 (SEQ ID NO: P(171)-V-I-E-K-V-K-P-G-E-Q-S-F-V-G-Q Peptide 5 (SEQ ID NO:2): Y(203)-H-N-Y-P-N-K-G-L-K-D-Y-T-Y-T-L Peptide 6 (SEQ ID NO:3): P(247)-T-Y-S-G-R-E-S-N-V-Q-K-M-A-I Peptide 7 (SEQ ID NO:4): I(344)-D-G-A-D-G-R-N-F-Y-H Naturally occurring variant zymogens and cysteine protease display unique linear B-cell epitopes.

Overlapping 10-mer peptides are used which overlap 2 amino acid residues with the previous one in the consecutive primary sequence corresponding to 371 amino acids of the mature cysteine protease zymogen (translated product minus leader sequence). Synthetic 10-mers corresponding to the 10 variant amino acid residues will also be used. The variant amino acids are positioned in the middle of the 10-mer. For example, if the sequence of a corresponding to one region of the SPEB1 variant is position 304-QINRSDFSKQ- 313 (SEQ ID NO:18), then 304-QINRGDFSKQ-313 (SEQ ID NO:19) is also examined, a that incorporates a variant amino acid found in the SPEB2 variant. Once the mer peptides are synthesized, an ELISA is used to examine the reactivity of all peptides with the following materials: rabbit polyclonal hyperimmune antiserum made against purified cysteine protease (positive control), (ii) rabbit pre-immune serum (negative control), (iii) our panel of 28 murine monoclonal antibodies raised against purified cysteine protease, (iv) acute and convalescent sera obtained from 20 patients with necrotizing 16/12/02,mcl 3102.speci,17 18fascitis and/or TSLS in Canada (obtained from D. Low, Mount Sinai Hospital, Ontario, Canada), 5 USA patients with TSLS characterized by extensive soft tissue destruction (obtained from D. Stevens, V.A. Hospital, Boise, Idaho), and 5 patients with ARF (obtained from A. Bisno, University of Miami Medical School). The great majority of the synthetic peptides usually are not reactive with each sera and there are a large number of internal redundant negative control peptides. Sera dilutions are used in these assays (1:1000 for hyperimmune rabbit antiserum, 1:500 for human serum, and 1:5 1:10 for MAb culture supernatants).

To determine the linear B-cell epitopes, for each sera and MAb tested, OD 405 is plotted versus 10-mer peptide number. The linear B-cell epitopes are displayed as a peak in the OD 405 values. In general, a peak is composed of several contiguous overlapping peptides, and the 10-mer peptide with the highest OD 405 value is defined as the parent peptide.

The pro region contains at least one unique linear B cell epitope. The same linear B cell epitopes will most likely be recognized by all 15 human convalescent sera specimens.

EXAMPLE 2 Creation of mutant speB proteins Figure 1 shows the processing sites, locations of amino acid variations found in the proteins made by the speB2 and speB4 alleles, and amino acids that are targets for mutation. Both site-directed and random mutagenesis schemes are employed to identify residues that disrupt cysteine protease function and zymogen processing, and to map regions that constitute antigenic domains of the protein. Targets for functional amino acid replacement are based on biochemical analysis of cysteine protease (Tai, et al., 1976) and by analogy with similar residues in eukaryotic cysteine protease.

To create a stable zymogen to facilitate crystallographic studies and generate enzymatically deficient or inactive protease for structure-function studies, mutant forms of the cysteine protease protein are made and characterized. A target mutagenesis scheme creates changes that: disrupt protease activity; (ii) prevent zymogen processing; (iii) prevent substrate binding; and (iv) alter immunoreactivity. Amino acids are changed to structurally neural alanine. A mutant protein that lacks protease activity, but which retains antigenicity, is generated by mutagenesis of the single cysteine residue (Cys-192- Ala- 192) at the catalytic site of the molecule. Also, His-340 and Gin-185 and Asn-356 are mutagenized. These three changes are epistatic to the Cys-192 mutation, but may alone 16/12/02,mcl3102.speci,18 -19exhibit altered activity. Trp-357, thought to be involved in substrate binding and similarly positions within papain, is also to be targeted. A stable zymogen precursor is also created by mutating residues surrounding the protease cleavage site at Lys-145. In addition, mutagenesis of Cys-192 may prevent autoproteolysis, as occurred for a Cys- Ser mutant of papain, the prototype cysteine protease. Other mutagenesis targets include a putative nucleotide binding domain (GVGKVG) and a potential collagen docking region [(GXX) 3 within carboxy terminal portion of the protein. Site-directed mutagenesis is used, by the charged-to-alanine-scanning method, to substitute positively and negatively charged amino acids (often involved in recognition and activity) with alanine. Many of the charged residues (14 lysine, 7 arginine, 12 aspartate, and 76 glutamate residues in the mature peptide) are expected to lie on the surface of the cysteine protease structure, and some are expected to define epitopes on the molecule. In particular, a region of charged amino acids, from 307 to 321 (8/15 charged), is examined; this region includes the site ofspeB and speB4 amino acid substitutions. Residues in antigenic regions identified in the epitope mapping studies are also mutated.

First, the speB gene is amplified from an ET1/M1 S. pyogenes strain, with PCR, and the product is cloned into a multicopy filamid vector such as pBluescript (strategene, La Jolla, Ca). This vector is chosen because it carries the regulated lac promoter and can be replicated as a single-stranded molecule for site-directed mutagenesis. Cloning is designed to place the promoter, ribosome binding site and speB reading frame 3' to the inducible lac promoter on the vector so that the protein can be conditionally overexpressed in E. coli when the lac inducer, IPTG, is added. The speB promoter is included requiring expressing in S. pyogenes. Whole cell extracts and periplasmic shockates of E.

coli cells carrying this primary speB clone are examined for the presence of the cysteine protease protein by SDS-PAGE and by Western blotting with anti-cysteine protease antibody. The resulting plasmid is the target for mutagenesis.

Oligonucleotide-directed mutations, such as substitutions, deletions and small insertions, are created on uracil-containing single-stranded templates by the method of Kunkel (Kunkel, 1985). When possible, mutagenic primers are designed to incorporate a unique restriction site into the speB gene for mapping and mutant selection. Both single and multiple alanine substitutions are created at the residues indicated above. Once residues critical to function are identified, small regions surrounding them are deleted or substituted, by using the same methods, to further characterize the region and to preclude reversion. When crystallographic data is available, additional amino acids are mutated.

16/12/02,mcl 3102.speci,19 A random mutagenesis scheme is also employed. Variant proteins created by this method are most useful for epitope screening, although molecules with altered kinetics and substrate recognitions may also be recovered. Regions of the speB sequence are randomized with mixed oligonucleotides in the primed-mutagenesis protocol, or short, inframe deletions within the gene are created with a modification of the DNAse 1-linker insertion/deletion protocol of Palzkill and Bostein (Palzkill and Bostein, 1991). Here, synthetic "excision linkers" are first ligated to randomly linearized target DNA, then excised with flanking nyucleotides to create small substitutions or deletions. For example, a linker with two copies of the recognition sequence for the enzyme Sapl (GCTCTTC) is used to create six base deletions (three bases on each end of the linker), or random two amino acid deletions, across the speB gene, as illustrated in Figure 2. Flanking bases are also randomized by filling the ends of the target sequence after linker excision, then inserting a second blunt end linker that includes a random sequence in place of the Ns.

The second linker is then removed by digestion with Sapl and the target sequence is ligated to generate substitutions.

To identify protease minus mutations, E. coli cells producing potential mutant proteins are first screened for protease activity on casein agar plates. Since secretion of cysteine protease to the periplasm is expected, it is possible that protease activity can be observed on plates. If this screening strategy is successful, then thousands of colonies are rapidly examined for functional mutations in cysteine protease. If cysteine protease must be completed secreted from E. coli to exhibit activity, then osmotic shockates of each presumptive mutant strain is assayed for proteolytic activity.

EXAMPLE 3 Passive immunization of mice Passive immunization with rabbit antibody directed against purified denatured cysteine protease partially protects mice against challenge with live S. pyogenes (Figures 3 and The ability of rabbit antibody raised against purified cysteine protease to protect mice challenged intraperitoneally with a lethal dose of live S. pyogenes was examined.

The method used is as follows.

S. pyogenes isolate MGAS 1719 is identical to strain B220. This isolate expresses T8 serotype, has the speB7 allele, and was used as the source strain for cysteine protease purification. Isolate MGAS 315 was recovered in the 1980s from a patient with toxicshock-like syndrome. The organism is electrophoretic type (ET) 2, expresses serotype M3 16/12/02,mcl3102.speci,20 -21protein, and was used as the challenge strain. This isolate also synthesizes SPEA and SSA.

Streptococcal cysteine protease was purified from strain MGAS 1719 culture supernatants with a combination of ultrafiltration and dye-ligand affinity chromatography, as described in Example 2 of the parent. SDS-polyacrylamide gel electrophoresis and coomassie blue staining of the resulting proteolytically active material showed a single major band of Mr kDa. The purified cysteine protease preparation does not react with rabbit antiserum raised against acid extracts ofM serotype 1 or 3, or type T8 S. pyogenes isolates by either western immunoblot or ELISA, but reacted strongly with rabbit antibodies raised against the streptococcal cysteine protease. (Figure 8).

The minimum lethal dose (MLD) of bacteria was calculated for isolate MGAS 315.

Bacteria were grown in brain heart infusion broth (Difco, MI) for 12 h at 37 C, the A 600 was adjusted to 0.7 (representing 10 3 cfu/ml) with sterile BHI broth, and 0.1 ml of 100 through 10 7 dilutions of bacteria made in sterile broth were injected intraperitoneally to each of five male 22-24 g CD-1 outbred mice (Charles River, MA). The bacterial suspensions were tested for purity before and after the inoculations, and the number of cfu injected per mouse was verified by colony counts. The mice were monitored for a period of 5 d post-inoculation and surviving mice were euthanized with methoxyflurane. Heart blood was collected for bacterial isolation from the mice that died or were sacrificed. The MLD was determined to be the highest dilution at or below which none of the mice in the group survived.

Minimum lethal dose The minimum lethal does (MLD), the highest dilution of S. pyogenes strain MGAS 315 at and below which there were no surviving mice, was approximately 10 organisms per inoculum, indicating that the strain MGAS 315 is unusually virulent to mice when administered via the intraperitoneal route. Pure cultures of group A streptococci were recovered from the heart blood of all mice that died after bacterial challenge.

The preparation of rabbit antiserum against streptococcal cysteine protease has been described in Kapur etal., Microb. Pathlg., (1993) 15:327-46. Male 22-24 g CD-1 outbred mice were intraperitoneally inoculated with 0.1 ml PBS (n 6)1 mg immune rabbit IgG in PBS (n or I mg pre-immune rabbit IgG in PBS (n 9) and challenged with the MLD of strain MGAS 315, 30 min after the administration of the treatment. The mice were monitored at 3 h intervals and mortality was recorded. Heart blood was collected from dead mice and plated onto BHI agar, and incubated for 48 h at 37 C in 5% CO 2 Kaplan- 16/12/02,mcl 3102.speci.21 22- Meier survival curves were plotted and the logrank test was employed to test for statistical differences in survival.

The results (Fig. 5) show that passive immunization with rabbit antibody directed against gel-purified denatured cysteine protease confers significant protection against challenge with highly virulent S. pyogenes strain MGAS 315 when compared with control animals given phosphate buffered saline (PBS) or pre-immune serum controls (log rank test; X2; P 00.001). The protection afforded by passively administered antiserum was considerably higher during earlier 65 h) rather than later time points (Fig. These results are especially significant because the experiment was specifically designed to minimize the likelihood of demonstrating protection since the rabbit antibody was raised against gel-purified denatured cysteine protease and not native zymogen or active protease forms (ii) in addition to the protease, the challenge strain is known to express pyrogenic exotoxin A (SPEA) and the recently described streptococcal superantigen, SSA, and (iii) the cysteine protease precursor made by the challenge strain (SPEBI) differs from the protease precursor variant against which the antiserum was raised (SPEB4) at two amino acid positions (Ala Val at position 111 and Ser Gly at position 308; Fig.

6).

EXAMPLE 4 Mouse immunization intranasal Intranasal immunization experiments are conducted essentially as described by Bessen and Fischetti (1988). Briefly, outbred Swiss CDI mice of the same gender, 4 to weeks old at the onset of immunization are used. Groups of 24 mice are immunized intranasally with 0 or 20 to 100 !tg of zymogen, mature, active protease or synthetic peptide. The mice are immunized once each on days 1, 3, and 5 are rested for 3 weeks and boosted i.n. with a single dose of antigen (20 or 40 The initial group of 20 immunized are randomized to two subgroups of 10 animals. Each subgroup is challenged with either the cysteine protease source strain (homologous challenge) or a strain expressing a distinct cysteine protease variant (heterologous challenge). Mice are given 10 ul of the bacterial suspension per nostril at 10 days after the cysteine protease boost. The vaccine is delivered i.n to unanesthetized mice (10 ul per nostril) through a model 750 Hamilton syringe equipped with a repeating dispenser and blunt-end needle. Throats are swabbed beginning 24 hours after challenge and at 24- and 48-hour intervals thereafter until day 11.

Additional throat cultures are taken on day 15. Throat swabs are cultured on blood agar 17/12/02,mcl3102.speci,22 -23plates overnight at 37 0 C in a CO 2 incubator and betahemolytic colonies are counted the following day.

EXAMPLE Bacterial challenge (intranasal) The initial group of 24 immunized mice is randomized to two subgroups of 12 animals. Each subgroup is then challenged with either the SPEB source strain (homologous challenge) or a strain expressing a distinct SPEB variant (heterologous challenge). Strains used for challenge are selected for resistance to 200 Pg/ml of streptomycin to facilitate recovery after challenge and if necessary are serially passaged by repeated i.p. injections in mice to increase ability to colonize and infect mice. A single stock of each challenge organism expressing SPEB is prepared from an overnight culture, concentrated 10-fold and stored at -80 0 C. Stocks are diluted 1:500 and grown overnight at 37 0 C in BHI broth, diluted 1:20 in fresh growth medium and cultured to an O.D.650 of The cells are harvested by centrifugation and suspended in saline to about 2.5 x 10 CFU/ml. Inasmuch as streptococci strains differ in ability to colonize mice intranasally, a challenge dose is used that reproducibly colonizes greater than 25% of a nonimmune mouse population. Animals are housed six per cage by cohort. Mice are given 10 .tl of the bacterial suspension per nostril at 10 days after the cysteine protease boost. Throats are swabbed beginning 24 hours after challenge and at 24- to 48-hour intervals thereafter until day 11. Additional throat cultures are taken on day 15. Throat swabs are cultured on blood agar plates with 200 [tg/ml of streptomycin, cultured overnight at 37 0 C in a CO 2 incubator and beta-hemolytic colonies are counted the following day.

EXAMPLE 6 Immunization of mice subcutaneous Immunization experiments are conducted in 4 to 5 week old outbred, immunocompetent, hairless mice (strain Crl:SKHI (hrhr)Br; Charles River) of the same gender. These mice are used because lesion size and character is easily cored and animals do not need to be shaved. Groups of 24 mice are immunized subcutaneously with 0, or 40 jtg of zymogen or mature, active protease. The mice are immunized once each on days 1, 7, 14, and 21, rested for 3 weeks and boosted with a single dose of protease 16/12/02,mcl 3102,speci,23 -24or 50 The mice are checked for seroconversion by a cysteine protease-specific

ELISA.

EXAMPLE 7 Bacterial challenge subcutaneous Immunized mice are randomized to two groups of 12 animals. Each group is then challenged with either the cysteine protease source strain (homologous challenge) or a strain expressing a distinct cysteine protease variant (heterologous challenge). A single stock of each challenge organism expressing cysteine protease is prepared from an overnight culture and adjusted to 10 6 CFU/ml. Mice (housed six per cage by cohort) are given 100 tL of the bacterial suspension mixed with an equal volume of sterile detran beads. The animals are inoculated s.c. on the right flank with a tuberculin syringe.

Bacterial dilutions are prepared at the time of challenge to determine the exact number of CFU used. Negative control animals consist of a group of 12 mice sham immunizations.

These mice are "challenged" with only sterile medium plus dextran beads.

EXAMPLE 8 Immunological assays Saliva and serum are collected from all immunized and control mice. Whole saliva is collected by pilocarpine stimulation 920 tg/mouse, subcutaneous) and centrifuged at 15,000 x g for 20 minutes. The material is divided and protease inhibitors are added to one of the aliquots. Storage is at -80°C. Serum is collected by bleeding from the tail vein.

Individual and pooled saliva and serum from mice assigned to each cohort, and control mice, are assayed for specific antibody to cysteine protease by ELISA.

EXAMPLE 9 Data analysis Animals are weighed immediately prior to challenge and every 24 hours postchallenge. Abscess volumes and area of dermo-necrosis will be calculated, and a lesionsize curve determined. Mean lesion sizes are compared statistically between groups by analysis of variance (ANOVA).

16/12/02,mcl 3102.speci,24 EXAMPLE Active immunization of mice Male Swiss CDI outbred mice were inoculated with either PBS (n 10) or 20 !tg of purified streptococcal cysteine protease in PBS (n 9) subcutaneously on day 1, followed with intraperitoneal inoculations of the same treatments at days 7, 14, 21, 42, 57, 63, and 79 for a total of nine immunizations. Serum antibody levels to the cysteine protease were checked at days 29, 71, and 84 by ELISA, and the mice were challenged with strain MGAS 315 on day 93, two weeks after the last immunization. The mice were monitored at 3 h intervals, mortality recorded, and Kaplan-Meier survival curves plotted and analyzed as described above. The results (Fig. 7) show that interperitoneal immunization with purified streptococcal cysteine protease also conferred significant protection (log rank test; X 2 P 0.01) against lethal challenge with the highly virulent S.

pyogenes isolate MGAS 315. It is noteworthy that immunization with the cysteine protease also conferred significant protection against S. pyogenes-induced early mortality in mice. For example, all 10 mice in the control group were dead by 28 h post challenge, but only 4 of 9 mice died in the protease immunized group (difference in proportions; z; p 0.003). Moreover, at the termination the experiment at 120 h, 2 of 9 mice in the protease-treated but none of 10 mice in the control group survived (difference in proportions; z; P 0.059). Thus, similar to the result observed with mice given immune rabbit serum, active immunization with the streptococcal cysteine protease conferred significant protection against lethal group A streptococcal infection.

EXAMPLE 11 PCR Assay for S. pyogenes Genomic DNA was prepared from isolates grown on brain heart infusion agar plates. In general, cells were scraped from one plate, suspended in 800 utl of 10 mM TrismM EDTA (ph 8.0) heated at 65 0 C for 15 min, washed, resuspended in 500 pl of TE containing 5 ltg of mutanolysin, and incubated at 37°C for 2 h. The cells were lysed by adding 100 il of 10% sodium dodecyl sulfate and heating at 65 0 C for 20 min. After centrifugation for 10 min, the supernatant was transferred to a clean tube and incubated overnight at 37 0 C with 100 ltg of RNase and 50 utg ofproteinase K. The DNA was then extracted with phenol-chloroform, precipitated with ethanol, and suspended in 100 .tl of

TE.

16/12/02,mcl 3102.speci,25 a -26- The cysteine protease structural gene was amplified by the polymerase chain reaction (PCR), with synthetic oligonucleotides. The oligonucleotide primers used to amplify speB and flanking regions were as follows: SPEB-X (SEQ ID NO:9), 5'-GTTGTCAGTGTCAACTAACCGT-3' and SPEB-2 (SEQ ID NO: 10), 5'-ATCTGTGTCTGATGGATAGCTT-3'.

PCR amplification of 1 jiL of chromosomal DNA was performed in 100 .1 of a mixture containing 50 mM KC1, 10 mM Tris-HCI, pH 8.3 1.5 mM MgCI 2 0.001% gelatin, 200 pM each of dATP, dCTP, dGTP, and dTTP, 200 nM each of SPEB-X and SPEB-2, and 2.5 units ofAmpliTaq DNA polymerase. The thermocycling parameters were denaturation at 94C for 1 min. annealing at 55 0 C for 2 min, and extension at 72 0 C for min for a total of 30 cyles. A final extension at 72 0 C for 15 min was used.

As shown above, cysteine protease and nucleic acid encoding it can be used for generating a protective immune response in a host mammal.

All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

16/12/02,mcl3102.speci,26 C- -58- Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

16/12/02,mcl 3102.speci,58

Claims (8)

1. A method of immunizing mammals comprising administering to a mammal a vaccine comprising a cysteine protease peptide, wherein said peptide is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4.

2. An isolated peptide comprising an amino sequence of SEQ ID NO:

3. An isolated peptide comprising an amino sequence of SEQ ID NO: 16.

4. An isolated peptide comprising an amino sequence of SEQ ID NO: 17. An isolated peptide comprising an amino sequence of SEQ ID NO: 1.

6. An isolated peptide comprising an amino sequence of SEQ ID NO: 2.

7. An isolated peptide comprising an amino sequence of SEQ ID NO: 3.

8. An isolated peptide comprising an amino sequence of SEQ ID NO: 4.

9. A vaccine comprising a cysteine protease peptide, wherein said peptide is selected from the group consisting of SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, substantially as herein described with reference to at least one of the accompanying Examples and/or Figures. A method of immunizing mammals comprising administering to a mammal a vaccine comprising a cysteine protease peptide, wherein said peptide is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 substantially as herein described with reference to at least one of the accompanying Examples and/or Figures. DATED this 1 7 th day of December, 2002 BAYLOR COLLEGE OF MEDICINE By their Patent Attorneys: CALLINAN LAWRIE 16/12/02,mcl 3102.speci,59