Streptococcal protective antigens (Spa): a new family of type-specific proteins of group A streptococci

Previous studies in our laboratory described a new group A streptococcal protective antigen (Spa) in type 18 streptococci that was distinct from the type 18 M protein. This study was undertaken to identify additional serotypes of group A streptococci that express Spa proteins. PCR techniques were used to identify and clone a new spa gene from type 36 streptococci. The 5′ sequence of spa36 was highly variable compared to spa18, while the 3′ sequence was conserved. Antisera against Spa36 opsonized type 36 streptococci but not type 18 streptococci, indicating that the opsonic Spa epitopes were type-specific. Antisera against the conserved carboxy-terminal half of Spa18 were used to identify Spa or Spa-like proteins expressed on the surface of 25 of 70 different serotypes of GAS. Spa proteins may represent a new family of type-specific surface antigens that function in concert with M proteins to elicit protective immune responses.     

Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein.

Two murine monoclonal antibodies (immunoglobulin M) evoked against human kidney tissue were examined for cross-reactivity with group A streptococci. A glomerulus-specific antibody, PMII.40.H.2, cross-reacted in an enzyme-linked immunosorbent assay with purified pepsin-extracted M proteins from type 6 and 12 streptococci, but not type 1, 3, 5, 19, and 24 M proteins. The cross-reactive monoclonal antibody also opsonized type 6 and 12 streptococci, indicating that it was directed against protective M protein epitopes that were exposed on the surfaces of these organisms. A control antibody, which was tubule specific, did not cross-react with any of the purified M proteins, nor did it opsonize any of the serotypes of streptococci tested. Western immunoblot experiments identified the glomerular cross-reactive antigen as a 43-kilodalton protein. The results demonstrate that M protein of group A streptococci and glomerular antigens of the human kidney possess cross-reactive determinants.     

Function of the fibronectin-binding serum opacity factor of Streptococcus pyogenes in adherence to epithelial cells

The serum opacity factor (SOF) of Streptococcus pyogenes is a serotyping tool and pathogenesis factor. Using SOF-coated latex beads in cell adherence assays and antiserum directed against SOF in S. pyogenes-HEp-2 cell adherence inhibition experiments, we demonstrate SOF involvement in the fibronectin-mediated adherence of S. pyogenes to epithelial cells. SOF exclusively targets the 30-kDa N-terminal region of fibronectin. The interaction revealed association and dissociation constants 1 order of magnitude lower than those of other S. pyogenes fibronectin-binding proteins.     

Expression of protective and cardiac tissue cross-reactive epitopes of type 5 streptococcal M protein in Escherichia coli.

The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.     

Immunogenicity of a 26-valent group A streptococcal vaccine

A multivalent vaccine containing amino-terminal M protein fragments from 26 different serotypes of group A streptococci was constructed by recombinant techniques. The vaccine consisted of four different recombinant proteins that were formulated with alum to contain 400 microg of protein per dose. Rabbits were immunized via the intramuscular route at 0, 4, and 16 weeks. Immune sera were assayed for the presence of type-specific antibodies against the individual recombinant M peptides by enzyme-linked immunosorbent assay and for opsonic antibodies by in vitro opsonization tests and indirect bactericidal tests. The 26-valent vaccine was highly immunogenic and elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. The immune sera were broadly opsonic and were bactericidal against the majority of the 26 different serotypes. Importantly, none of the immune sera cross-reacted with human tissues. Our results indicate that type-specific, protective M protein epitopes can be incorporated into complex, multivalent vaccines designed to elicit broadly protective opsonic antibodies in the absence of tissue-cross-reactive antibodies.     

Virulence properties of type VII Streptococcus agalactiae (group B streptococci) and immunochemical analysis of capsular type polysaccharide.

Strains of a new polysaccharide type of group B streptococci (GBS), type VII, have been isolated from human carriers and invasive infections. Some of these strains bear the protein antigen c or R, as do other GBS serotypes. The capsular type polysaccharide is sialylated and this residue is involved in the immunodeterminant structure. All type VII strains examined were virulent in CD-1 mice; the LD50 after intraperitoneal (i.p.) challenge was 4.57 (SD 0.12) x10(7) cfu for the reference strain and 5.49 (SD 1.5) x10(7) cfu for clinical isolates. A particular feature of this serotype was the ability to induce septic arthritis not only when injected intravenously (i.v.), but also when injected i.p. Rabbit antiserum against the capsular type VII polysaccharide exhibited opsonic activity in a phagocytosis assay and protective activity against infection.     

Homologous and heterologous protection of mice with group A streptococcal M protein vaccines.

Purified streptococcal M proteins precipitated with alum (APM) were used to immunize mice. A trivalent vaccine of serotypes 1, 3, and 12 protected mice against challenges by homologous live streptococci and also conferred protection against serotypes 6 and 14 but not against a strain of group B streptococci. Monovalent APM vaccines afforded homologous protection and restricted heterologous protection. The extent of heterologous protection was a function of serotype combinations and was also dose dependent. Rabbit antisera exhibiting strong opsonic activities were active in vitro and in passive mouse protection only for homologous serotypes. Mouse antisera did not passively transfer protection and were not bactericidal in vitro. It was concluded that homologous and heterologous active mouse protection was most likely a result of shared antigenic determinants of the various M proteins although protection of mice could not be measured as a function of circulating anti-M antibodies.     

Murine monoclonal antibodies reactive with human heart and group A streptococcal membrane antigens.

Ten selected murine hybridoma cell lines that produce monoclonal antibodies against M type 5 Streptococcus pyogenes and human heart antigen were isolated. All of the monoclonal antibodies studied were determined to be the immunoglobulin M isotype. The antibodies were characterized on the basis of their reactions with Triton X-100-extracted whole human heart antigens, sodium dodecyl sulfate-extracted sarcolemmal antigens, and whole streptococci or their membranes. Enzyme-linked immunosorbent assays and Western immunoblotting techniques were used to compare the reactivity of the monoclonal antibodies. All 10 of the antibodies were first selected for their reactivity with Triton X-100-extracted heart antigens and whole group A, M type 5 streptococci. These antibodies were then divided into two categories: strong reactors or weak reactors with human sarcolemmal and streptococcal membranes. Among the strong reactors, two different types of monoclonal antibodies were observed on the basis of their immunobanding patterns with sarcolemmal and streptococcal membranes on Western blots. Monoclonal antibodies that were strong reactors with sarcolemmal and group A streptococcal membrane antigen were directed against a determinant on a family of proteins. The major reactants of sarcolemmal extracts were high-molecular-weight proteins near 200,000. Some monoclonal antibodies demonstrated more specificity for the heart than did others when reacted with separated Triton X-100-extracted tissue antigens from the heart, kidney, and skeletal muscle. One of the monoclonal antibodies that reacted with group A streptococci reacted with a Triton X-100-extracted heart antigen ca. 40,000 daltons in size. None of these monoclonal antibodies opsonized type 5 Streptococcus pyogenes, and in enzyme-linked immunosorbent assays most of the antibodies were found to react to a lesser degree with other groups of streptococci. Monoclonal antibody was used to probe normal and rheumatic sarcolemma for differences in reactivity. Although the rheumatic heart reacted more intensely, no major differences between the immunobanding patterns of normal and rheumatic hearts were observed.     

Unique and common protective epitopes among different serotypes of group A streptococcal M proteins defined with hybridoma antibodies.

A set of four monoclonal antibodies was produced against a highly purified pepsin extract of type 5 streptococcal M protein. Three of the four antibodies cross-reacted with purified M proteins from heterologous serotypes and opsonized the respective heterologous organisms. Our studies suggest that monoclonal antibodies may be useful in identifying subpeptides of various M proteins containing common, protective epitopes that are capable of evoking antibodies that would protect against several different potentially "rheumatogenic" serotypes.     

Opsonization of encapsulated Cryptococcus neoformans by specific anticapsular antibody.

Antisera prepared in rabbits against either whole encapsulated cells of Cryptococcus neoformans or purified cryptococcal polysaccharide were opsonic for the encapsulated yeast. The opsonic activity was removed by absorption with whole cryptococci and was inhibited by free polysaccharide. As little as 0.13 microgram of cryptococcal polysaccharide produced a 50% inhibition of opsonization. Various degrees of neutralization by polysaccharides from the four cryptococcal serotypes suggested that the opsonins were type specific. Fractionation of antiserum on Bio-Gel A-5m (Bio-Rad Laboratories) and diethylaminoethyl cellulose showed that the opsonins were antibodies of the immunoglobulin G class. These opsonizing antibodies did not require heat-labile serum components for optimal phagocytosis of the yeast. Inhibition studies using 2-deoxy-D-glucose demonstrated that ingestion of encapsulated cryptococci opsonized with anticapsular antibody was a 2-deoxy-D-glucose-inhibitable process. This result differed from similar studies with non-encapsulated cryptococci which showed that ingestion of non-encapsulated cryptococci opsonized with normal serum was not inhibited by 2-deoxy-D-glucose.     

Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.

A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.     

Systemic and mucosal immunizations with fibronectin-binding protein FBP54 induce protective immune responses against Streptococcus pyogenes challenge in mice

The purpose of this study was to examine the suitability of fibronectin-binding protein FBP54 as a putative vaccine for Streptococcus pyogenes infections. When the distribution of the fbp54 gene among the clinical isolates representing various M serotypes was tested by PCR and Southern blot assays, it was found that all of the strains possess this gene. Furthermore, a significant increase in immunoglobulin G (IgG) antibody titers against FBP54 was observed in sera from patients with S. pyogenes infections compared with those from healthy volunteers (P < 0.005). Mice were immunized with FBP54 subcutaneously, orally, or nasally. An enzyme-linked immunosorbent assay revealed that antigen-specific IgG antibodies were induced in the sera of immunized mice, while high salivary levels of IgA antibodies were detected after oral and nasal immunizations. Mice subcutaneously or orally immunized with FBP54 survived significantly longer following the challenge with S. pyogenes than did nonimmunized mice (P < 0.001). These results indicate that FBP54 is a promising vaccine for the prevention of S. pyogenes infections.     

Antiphagocytic function of an IgG glycosyl hydrolase from Streptococcus equi subsp. equi and its use as a vaccine component

EndoSe from Streptococcus equi subsp. equi is an enzyme hydrolyzing glycosyl groups on IgG, analogous to EndoS from Streptococcus pyogenes. We here show that the activity of EndoSe leads to an antiphagocytic function and may thus be a contributory factor to immune evasion of S. equi. Despite the damaging effect that EndoSe has on IgG, antibodies against EndoSe can neutralize its function. Antibodies against EndoSe restored the opsonic activity of specific opsonizing antibodies. Mice infected with either S. equi subsp. equi or subsp. zooepidemicus or S. pyogenes could be protected by vaccination with EndoSe. It is speculated that EndoSe could be a suitable vaccine candidate against streptococcal infections.     

Association between the 65-kilodalton heat shock protein, Streptococcus sanguis, and the corresponding antibodies in Behçet''s syndrome.

The etiology of Behcet's syndrome (BS) is unknown, but a number of streptococcal species have been implicated. A hypothesis was postulated that a shared antigen, such as a stress protein, might account for some of these findings. Indeed, a rabbit antiserum against a 65-kDa heat shock protein of Mycobacterium tuberculosis revealed a corresponding 65-kDa band with all six Streptococcus sanguis strains examined and S. pyogenes but not with S. salivarius. By applying a panel of nine monoclonal antibodies to the mycobacterial 65-kDa heat shock protein, an approximately 65-kDa antigen was identified in the uncommon serotypes of S. sanguis ST3 and H.83 and one with a different Mr was identified in KTH-1 and S. pyogenes. Monoclonal antibodies Y1.2, C1.1, II H9, and ML30, which reacted with these streptococci, recognize residues 11 to 27, 88 to 123, 107 to 122, and 276 to 297 of the 65-kDa heat shock protein, respectively, suggesting that these residues are conserved among some uncommon serotypes of S. sanguis and S. pyogenes. Immunoblot analyses of sera from patients with BS for immunoglobulin A (IgA) and IgG antibodies revealed bands of 65 to 70 kDa with the mycobacterial heat shock protein, S. sanguis strains, and S. pyogenes, although these reactivities were also found to a lesser extent in controls. A 65- to 70-kDa band was found more frequently with S. sanguis KTH-2 or KTH-3 and IgA in serum from patients with BS than with serum from controls (P less than 0.02). Antibodies in serum were then studied by a radioimmunoassay, and in patients with BS this revealed significantly raised IgA antibodies to the recombinant 65-kDa mycobacterial heat shock protein and to soluble protein extracts of S. sanguis ST3, KTH-1, KTH-2, and KTH-3. Whereas significant anti-65-kDa heat shock protein and anti-S. sanguis ST3 antibodies were also found in sera from patients with rheumatoid arthritis and recurrent oral ulcers, the anti-S. sanguis KTH-1, KTH-2, and KTH-3 antibodies were confined to BS. The results are consistent with the hypothesis that some of the streptococcal antigens are associated with heat shock or stress proteins, which will need to be formally established by isolating heat shock proteins from streptococci.     

Antigenicity of the M proteins of group A hemolytic streptococci: further evidence for shared determinants among serotypes.

Shared antigenic determinants between M proteins of group A streptococci appear to be widespread among serotypes. This is demonstrated by the ability of purified M proteins to absorb opsonic antibody from a variety of heterologous antisera prepared against whole cells or purified M proteins. This absorption procedure has the capacity to separate passive mouse protecting and passive hemagglutinating antibodies from opsonic antibodies measured in vitro. When whole cells or M proteins are used as heterologous absorbents, immunoglobulin may be recovered from the cells or precipitates. The recovered antibody has most of the opsonic and some of the precipitating qualities of the original unabsorbed serum, but hemagglutinating titers are significantly lower. These data provide additional evidence that shared antigenicity among M proteins can be the result of common antigenic determinants. Arguments are presented that these cross-reactions are not the result of a nonspecific protein fraction associated with purified M proteins.     

Fibrinogen binding and resistance to phagocytosis of Streptococcus sanguis expressing cloned M protein of Streptococcus pyogenes.

The biological properties of Streptococcus pyogenes M protein cloned and expressed in S. sanguis were investigated. The spm-5 gene previously cloned into Escherichia coli was subcloned into the E. coli-S. sanguis shuttle plasmid pVA838 to produce a newly constructed plasmid, pBK100. Cells of S. sanguis transformed with pBK100 expressed 53-, 55-, and 58-kilodalton polypeptides reacting with type 5 M protein antiserum in immunoblots. The M protein was expressed on the surface of S. sanguis cells as shown by the capacity of the intact cells to (i) inhibit the reactivity of anti-type 5 antibodies with purified M protein as demonstrated by enzyme-linked immunosorbent assay; (ii) inhibit the opsonization by M5 antisera of type 5 S. pyogenes; (iii) express M-protein-like fibrils on the surface of the organisms that react with M5 antisera as revealed by immunoelectron microscopy; (iv) bind plasma fibrinogen and, as a consequence, resist phagocytosis by human blood neutrophils; and (v) be rendered susceptible to phagocytosis by opsonic M5 antisera. These results provide additional evidence that streptococcal M proteins bind host proteins as a ploy to evade host defense mechanisms.     

Phagocytosis of murine lymphoma cells by macrophages. II. Differences between opsonic and cytotoxic activity of mice immunized with lymphoma cells

Primary allogeneic antiserum raised in C57B1 mice directed against a DBA/2 lymphoma, L5178Y, was found to contain at least two types of opsonic activity associated with the γ-globulin fraction of the antiserum. One was found at a low level corresponding to cytotoxic activity, binding strongly to L5178Y cells and resistant to treatment with mercaptoethanol. The other, which accounted for most of the opsonic activity, was relatively non-specific for L5178Y cells, was easily eluted from the cells, was not associated with cytotoxicity and was sensitive to mercaptoethanol treatment. Two types of opsonic activity were also found in allogeneic antiserum against the CBA/2 lymphoma, TLX5. Both types of lymphoma antisera opsonized sheep red cells, but anti-sheep red cell serum was highly specific, exhibiting no degree of opsonic cross-reactivity with either type of lymphoma cell.     

Pneumococcal pneumonia and bacteremia model in mice for the analysis of protective antibodies

Pneumococci cause infection by colonizing the nasopharynx and invading the mucosal surfaces. Infection models in mice, where the natural route of infection is mimicked, may be useful to study antibody mediated protection against pneumococcal pneumonia and bacteremia. We have established a pneumococcal pneumonia and bacteremia model in mice and investigated the protective capacity of human antibodies. Intranasal challenge with serotypes 1, 3, 6A and 8 caused lung infection and bacteremia which was lethal. Serotype 6B caused low, but detectable, infection and other serotypes tested were not virulent. Passive immunization with a human IgG preparation i.p. protected mice in a dose dependent manner against bacteremia caused by the virulent serotypes (except serotype 3) and partially or completely cleared pneumococci from the lungs of mice infected with serotypes 1, 6A and 8. Adsorption of antibodies with homologous capsular polysaccharides eliminated protection against disease but adsorption with cell wall polysaccharides (CWPS) did not. Furthermore, a good correlation was observed between protection of sera in vivo and opsonic activity in vitro. The results indicate that the model may be useful to analyse the levels, isotypes, specificity and other characteristics of human antibodies which protect against pneumococcal infection and to evaluate the protective potential of pneumococcal vaccine candidates.     

Multivalent Group A Streptococcal Vaccine Elicits Bactericidal Antibodies against Variant M Subtypes

Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.     

Multivalent group A streptococcal vaccine elicits bactericidal antibodies against variant M subtypes

Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.