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Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

The HtrA serine protease has been shown to be essential for bacterial virulence and for survival after exposure to many types of environmental and cellular stresses. A Listeria monocytogenes 10403S htrA mutant was found to be sensitive to oxidative and puromycin-induced stress at high temperatures, showed a reduced ability to form biofilms, and was attenuated for virulence in mice.

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junin virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junin, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 μM), selective (concentration of compound that inhibited cell viability by 50% = >40 μM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10× 50% lethal dose (LD 50 ) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10× LD 50 ) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000× LD 50 of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (7 , 5.2 × 10 7 , and 1.8 × 10 5 PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.

To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-gamma to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.

The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium Staphylococcus aureus . There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013–5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine if srtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordonii exhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci.

Plasma pharmacokinetics of ST-246, smallpox therapeutic, was evaluated in mice, rabbits, monkeys and dogs following repeat oral administrations by gavage. The dog showed the lowest Tmax of 0.83 h and the monkey, the highest value of 3.25 h. A 2- to 4-fold greater dose-normalized Cmax was observed for the dog compared to the other species. The mouse showed the highest dose-normalized AUC, which was 2-fold greater than that for the rabbit and monkey both of which by approximation, recorded the lowest value. The Cl/F increased across species from 0.05 L/h for mouse to 42.52 L/h for dog. The mouse showed the lowest VD/F of 0.41 L and the monkey, the highest VD/F of 392.95 L. The calculated extraction ratios were 0.104, 0.363, 0.231 and 0.591 for mouse, rabbit, monkey and dog, respectively. The dog showed the lowest terminal half-life of 3.10 h and the monkey, the highest value of 9.94 h. The simple allometric human VD/F and MLP-corrected Cl/F were 2311.51 L and 51.35 L/h, respectively, with calculated human extraction ratio of 0.153 and terminal half-life of 31.20 h. Overall, a species-specific difference was observed for Cl/F with this parameter increasing across species from mouse to dog. The human MLP-corrected Cl/F, terminal half-life, extraction ratios were in close proximity to the observed estimates. In addition, the first-in-humans (FIH) dose of 485 mg, determined from the MLP-corrected allometry Cl/F, was well within the dose range of 400 mg and 600 mg administered in healthy adult human volunteers.

T protein is a trypsin- and pepsin-resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize HindIII digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE6(1-299), coding for the amino-terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy-terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy-terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction-fragment size heterogeneity. Clearly, there is considerable diversity among T-serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.

The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized withEscherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Several linear B-cell epitopes within the three proteins were recognized by sera from the immunized mice. In addition, protein-specific cellular responses were detected in spleens of immunized mice by a gamma interferon enzyme-linked immunospot assay using peptides derived from each protein. Our data suggest that a subunit vaccine incorporating bacterially expressed IMV- and EEV-specific proteins can be effective in stimulating anti-vaccinia virus immune responses and providing protection against lethal virus challenge.

An active area of research in the development of Streptococcus gordonii for use as a bacterial commensal vector involves the identification and utilization of strong promoters for high-level expression of heterologous products. Escherichia coli plasmid vectors containing different streptococcal promoters often fail to become established in E. coli for unknown reasons. Therefore, it is desirable at times to transform S. gordonii, which is naturally competent, with small quantities of nascently ligated DNA without using E. coli first to amplify or screen the product. By comparing the efficiency of two methods used to induce competence in S. gordonii, it was shown that the use of a synthetic competence stimulating peptide substantially enhanced plasmid uptake by S. gordonii. We amplified the amylase-binding protein (abpA) promoter from the S. gordonii genome and, using a synthetic peptide to induce competence, directly introduced plasmid DNA containing this promoter into S. gordonii as an unamplified product of ligation. This plasmid facilitated abundant secretion of a heterologous product by S. gordonii. By assessing the levels of heterologous product secreted by two plasmid constructs, it was possible to evaluate the relative strength of two native promoters.

Streptococcus gordonii shows promise as a live mucosal vaccine vector for immunization against respiratory pathogens. In preparation for clinical trials to evaluate S. gordonii engineered to express group A streptococcal M protein antigens, we characterized the responses of 150 healthy volunteers to combined nasal and oral inoculation with approximately 1.5 × 10 9 CFU of SP204(1-1), an S. gordonii strain not bearing vaccine antigens. SP204(1-1) was selected for resistance to streptomycin and 5-fluoro-2-deoxyuridine to distinguish it from indigenous flora. In two antibiotic treatment studies, we performed serial culturing of nose, mouth, and saliva samples from 120 subjects treated with azithromycin beginning 5 days after inoculation to determine whether SP204(1-1) could be rapidly eliminated should safety concerns arise. A natural history study was performed to assess the time until spontaneous eradication in the remaining 30 subjects, who did not receive the antibiotic and who were monitored with repeated culturing for 14 weeks after inoculation. SP204(1-1) was generally well tolerated. Symptoms reported most often within 5 days of inoculation were nasal congestion (36%), headache (30%), and sore throat (19%). The strain was detected by culturing in 98% of subjects. A single dose of azithromycin eliminated colonization in 95% of subjects; all subjects receiving a 5-day course of an antibiotic showed clearance by day 11. Without the antibiotic, 82% of subjects showed spontaneous eradication of the implanted strain within 7 days, and all showed clearance by 35 days. The results of these clinical trials provide encouragement that the use of S. gordonii as a live mucosal vaccine vector is a feasible strategy.

A novel vaccine (LL-CRR) made from live, nonpathogenicLactococcus lactisthat expresses the conserved C-repeat region (CRR) of M protein fromStreptococcus pyogenesserotype 6 was tested in mice. Nasally vaccinated mice produced CRR-specific salivary immunoglobulin A (IgA) and serum IgG. Subcutaneously vaccinated mice produced CRR-specific serum IgG but not salivary IgA. A combined regimen produced responses similar to the salivary IgA of nasally vaccinated mice and serum IgG of subcutaneously vaccinated mice. Mice vaccinated nasally or with the combined regimen were significantly protected against pharyngeal infection following a nasal challenge withS. pyogenesM serotype 14. Mice vaccinated subcutaneously were not protected against pharyngeal infection. Mice in all three LL-CRR vaccination groups were significantly protected against the lethal effects ofS. pyogenes. Only 1 of 77 challenged mice that were vaccinated with LL-CRR died, whereas 60 of 118 challenged mice that were vaccinated with a control strain or phosphate-buffered saline died. In conclusion, mucosal vaccination with LL-CRR produced CRR-specific salivary IgA and serum IgG, prevented pharyngeal infection withS. pyogenes, and promoted survival.

Several key protein structural attributes were altered in an effort to optimize expression and immunogenicity of a foreign protein (M protein from Streptococcus pyogenes ) exposed on the surface of Streptococcus gordonii commensal bacterial vectors: (i) a shorter N-terminal region, (ii) the addition of a 94-amino-acid spacer, and (iii) the addition of extra C-repeat regions (CRR) from the M6 protein. A decrease in the amount of cell surface M6 was observed upon deletion of 10 or more amino acid residues at the N terminus. On the other hand, reactivity of monoclonal antibody to surface M6 increased with the addition of the spacer adjacent to the proline- and glycine-rich region, and an increase in epitope dosage was obtained by adding another CRR immediately downstream of the original CRR. The results obtained should facilitate the design of improved vaccine candidates using this antigen delivery technology.

The DegP protease, a multifunctional chaperone and protease, has been shown to be essential for virulence in gram-negative pathogens such as Salmonella enterica serovar Typhimurium, Brucella abortus, Yersinia enterocolitica , and Pseudomonas aeruginosa . The function of DegP in pathogenesis appears to be the degradation of damaged proteins that accumulate as a result of the initial host response to infection, which includes the release of reactive oxygen intermediates. Additionally, the DegP protease plays a major role in monitoring and maintaining the Escherichia coli periplasm and influences E. coli pilus biogenesis. We report here the identification of highly homologous enzymes in Streptococcus pyogenes, Streptococcus gordonii, Streptococcus mutans, Staphylococcus aureus , and Enterococcus faecalis . Moreover, the phenotype of an insertionally inactivated degP allele in S. pyogenes is similar to that reported for E. coli , with temperature sensitivity for growth and enhanced sensitivity to reactive oxygen intermediates. Virulence studies in a mouse model of streptococcal infection indicate that a functional DegP protease is required for full virulence. These results suggest DegP as an attractive broad-spectrum target for future anti-infective drug development.

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.

We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli . The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5′-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.

The antigenic relatedness of surface-exposed portions of M protein molecules derived from group A streptococcal isolates representing more than 50 distinct serotypes was examined. The data indicate that the majority of serotypes fall into two major classes. Class I M protein molecules share a surface-exposed, antigenic domain comprising the C repeat region defined for M6 protein. The C repeat region of M6 protein is located adjacent to the COOH-terminal side of the pepsin-susceptible site. In contrast, Class I M proteins display considerably less antigenic relatedness to the B repeat region of M6 protein, which lies immediately NH2-terminal to the pepsin site. Surface-exposed portions of Class II M proteins lack antigenic epitopes that define the Class I molecules. Studies in the 1970s demonstrated that M protein serotypes can be divided into two groups based on both immunoreactivity directed to an unknown surface antigen (termed M-associated protein) and production of serum opacity factor. These two groups closely parallel our current definition of Class I and Class II serotypes. Both classes retain the antiphagocytic property characteristic of M protein, and Class II M proteins share some immunodeterminants with Class I M proteins, although the shared determinants do not appear to be exposed on the streptococcal surface. Nearly all streptococcal serotypes associated with outbreaks of acute rheumatic fever express M protein of a Class I serotype. Thus, the surface-exposed, conserved C repeat domain of Class I serotypes may be a virulence determinant for rheumatic fever.

Fifty-three isolates of Streptococcus uberis from various sources were examined for the presence of a common antigen. Initially, a serum was produced in rabbits which, by using rocket line immunoelectrophoresis, proved to react with identity to all of the S. uberis crude extracts as well as group B and E streptococcal extracts. The antigen(s) responsible for this cross-reactivity was partially purified by Sephacryl S-200 gel chromatography and analyzed by fused rocket immunoelectrophoresis. Further analysis by immunodiffusion showed that probably two antigens in the gel chromatography-consolidated fractions were common to the S. uberis and group B and E isolates, but that one of the antigens present was unique to S. uberis. Trypsin destroyed the immunoreactivity of this antigen. Isolation of this common antigen could possibly alleviate some of the tedium associated with the identification of this organism.

The ability of the group A streptococcus to cause human infection is primarily attributable to the M protein, a molecule located on the surface of the cell wall. This protein enables the organism to resist phagocytic attack by human neutrophils, thus allowing the streptococcus to persist in the infected tissue until such time as antibodies are produced against the M molecule (1). Because type-specific antibodies to the M protein lead to the effective clearance of the invading streptococcus, the ability of the organism to vary the antigenic structure of the M molecule has probably been responsible for the survival of the streptococcus in nature (1,2). Lancefield showed that, though delayed in their appearance, once formed, type-specific opsonic antibodies persist in the host for more than 30 years (2). To date, more than 80 se- rologically distinct M molecules have been identified in addition to a large number of clinical isolates for which typing sera are not yet available ((3), WHO meeting, Tokyo, Japan 1985).