The enhancement of the immunogenicity of streptococcal group-A M protein by conjugation with a synthetic polyelectrolyte]

Immunization with the polypeptide fragment of group A streptococcal protein M conjugated with the copolymer of acrylic acid and N-vinylpyrrolidone in complete Freund's adjuvant has been found to lead to a sharp increase in the level of antibodies to the type-specific determinants of protein M, detected in the enzyme immunoassay (EIA). The possibility of the application of such sera to preliminary typing of streptococci in EIA with the use of whole microbial cells as antigens has been shown. The data on high activity of the sera thus obtained in the bactericidal test with streptococci of the homologous type are presented. Recommendations on the use of sera obtained by the above method for highly precise typing of the virulent cultures of group A streptococci in the bactericidal test are given.     

Streptococcus fryi sp. nov., a novel species with Lancefield group M antigens

The taxonomic characteristics of β-hemolytic streptococcal strains that reacted with Lancefield group M antisera were investigated. Group M streptococci have not been proposed as a species to date. Four strains of the group M streptococci isolated from dog were located within the pyogenic group of the genus Streptococcus on 16S rRNA gene-based phylogenetic analysis; the group M strains were located a short distance away from all other members of the group. The homology values of 16S rRNA gene sequences between group M strains and all other streptococci were<95.6%. Group M strains exhibited low levels of DNA-DNA homology to other streptococcal species. Some biochemical traits, such as β-galactosidase activity and acid production from glycogen, could distinguish these group M strains from other closely related species. Thus, these strains are proposed to constitute a new species -Streptococcus fryi sp. nov. The type strain is PAGU 653(T) (=NCTC 10235(T)=JCM 16387(T)).     

Effect of Incubation Temperature on T-Agglutination Typing of Streptococcus pyogenes

The temperature of incubation affected the typability of beta-hemolytic group A streptococci by T-agglutination tests. When strains could not be typed after routine incubation at 30 C, they were incubated at 22 to 25 C, and nearly a 10% increase in typability was achieved. The clinical source of the strains was related to their typability. Incubation at the lower temperature was required for successful typing of higher percentages of strains from the skin and other clinical sources than from the throat. Sixty per cent of the skin strains were represented by six serotypes. Of these, 53% of the strains required incubation at 22 to 25 C before they could be typed.     

Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).     

Sequence of protective epitopes of streptococcal M proteins shared with cardiac sarcolemmal membranes

Synthetic peptide fragments spanning the entire amino acid sequence of pep M5 were used to detect epitopes cross-reactive with heart tissue components other than myosin. Heart-cross-reactive pep M5 antibodies were affinity purified by absorption to and elution from purified sarcolemmal membranes. Only one of the synthetic peptides, SM5(164-197)C, inhibited reactivity of the affinity-purified antibodies with pep M5 by ELISA. SM5(164-197)C linked to KLH evoked both opsonic and heart-cross-reactive antibodies in rabbits. In addition to type 5, the immune sera opsonized M types 6, 18, 19, and 49 streptococci. The antisera reacted strongly with isolated cardiac sarcolemmal membranes by immunofluorescence. In Western blots of cardiac tissue, the anti-SM5(164-197)C reacted with a 40 kDa protein but not with myosin. The reaction was inhibited by pep M5 and SM5(164-197)C but not by any of the other peptides spanning pep M5. The cross-reactive anti-SM5(164-197)C affinity purified on sarcolemmal membranes opsonized types 5, 6, and 19 but not type 24 streptococci. These results indicate that SM5(164-197)C contains heart-cross-reactive, opsonic epitopes that are shared among heterologous serotypes of group A streptococci.     

Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.     

Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.     

Proposal of a new scheme for the serological typing of Enterococcus faecalis strains.

No systematic study on serotyping of Enterococcus faecalis has been reported since 1964 when M.E. Sharpe conducted serotyping of group D streptococcus in U.K. So, we attempted to re-evaluate serotyping of E. faecalis. For this purpose, we received 42 Sharpe's strains and first examined for their biochemical characteristics as E. faecalis. Only 9 of the 42 strains were identified as E. faecalis. We raised rabbit antisera against a large number of E. faecalis strains, including the 9 Sharpe's strains, 2 strains obtained from CDC in U.S.A. and 36 strains isolated from patients hospitalized in different cities of Japan. From the results of cross-agglutination tests and absorption tests performed on these antisera using a large number of E. faecalis strains, we were able to classify 21 distinct serotype strains and to prepare 21 monospecific typing antisera by absorption of the antisera to the type strains with appropriate cross-agglutinating strains. When 832 E. faecalis strains were serotyped with the 21 typing antisera, 638 strains (76.7%) were typable. Thus, we propose a provisional scheme of 21 distinct serovars in E. faecalis.     

Susceptibility of chicken embryos to group A streptococci: Correlation with fibrinogen binding

Abstract One problem in investigating group A streptococcal infections and virulence is the lack of appropriate in vivo models. In this study we introduce the chicken embryo model for determining virulence of Streptococcus pyogenes . We found that M protein positive strains, if administered intravenously, were highly virulent for 12-day-old chicken embryos. The LD₅₀ of the strains tested could be correlated directly with the amount of cell wall exposed M protein, which has been determined by the capacity of streptococci to bind fibrinogen and by the ability of streptococci to survive in fresh normal human blood. The number of colony forming units (cfu) of M⁺ strains necessary to kill 50% of embryonated eggs was significantly lower (<10² cfu) than for M⁻ variants (>10⁴ cfu). Albumin and/or IgG binding to streptococcal cells, which can also take place in proteins of the M protein family which do not bind to fibrinogen, did not show that clear correlation to the virulence in chicken embryos that did fibrinogen binding. Application of anti-streptococcal M protein antisera from chicken and rabbit reduced the lethality of the chicken embryos. In contrast, no correlation was found between lethality of chicken embryos and the in vitro production of erythrogenic toxins by the administered strains. Thus the results indicate that the presence of M-protein with its fibrinogen binding activity on the streptococcal cell surface is necessary for virulence of group A streptococci in the chicken embryo model.     

Genetic characterization of atypical Shigella flexneri isolated in Korea

Three types of serotypically atypical Shigella flexneri strains were isolated from 2007 to 2008 in patients at the Korea National Institute of Health (NIH). These strains were characterized and compared with serologically typical S.flexneri. One type of strain either displayed nonreacting typing or grouping sera, reacting strongly only with polyB antisera, which indicates this strain is S. flexneri (polyB:un). The second type displayed reactions with one of the typing sera (IV) and did not bind any grouping sera (IV:un). The remaining type of strain displayed a plural agglutination pattern, reacted with one typing sera (II), and bound with two grouping sera (II:(3)4,7(8)). Among these atypical strains IV:un and II:(3)4,7(8) strains showed higher multi-antibiotic resistance in ampicillin, streptomycin, and trimethoprim-sulfamethoxazole than typical strains. Furthermore, all II:(3)4,7(8) strains harbored integrons. This study suggests that these multiple antibiotic-resistant atypical S. flexneri are new subserotypes of S.flexneri that await further serological classification.     

Typing of Streptococcus group A isolated from patients and healthy persons

The typing of 80% of 381 streptococcal strains, group A, under study was accomplished with a set of diagnostic anti-T sera obtained from the Sevak Institute (Czechoslovakia). None of the T-types could be related with certainty to the localization of the infective agent in the human body (the pharynx, the skin). Different T-types were shown to circulate in definite regions of the USSR. To enhance the differentiating capacity of T-typing, the enzymatic (lipoproteinase and NADase) activity of the strains was determined, thus permitting the subdivision of the T-types into still smaller groups. The typing of OF+ strains of unknown M-specificity could be carried out by means of the blood sera of healthy persons, containing antibodies to streptococcal lipoproteinase. The conclusion on the expediency of using the determination of lipoproteinase and NADase as an additional marker in the typing of group A streptococci was made.     

Serological Typing of 31 Achromogenic and 40 Melanogenic Pseudomonas aeruginosa Strains

Thirty-one achromogenic and 40 melanogenic Pseudomonas aeruginosa strains were studied with 10 monovalent typing sera (3). Twenty-one of the achromogenic (67.7%) and seven of the melanogenic (17.5%) strains were agglutinated by one of the 10 typing sera. Ten achromogenic and 33 melanogenic strains were not agglutinated by any of the 10 typing sera. As far as this set of antisera is concerned, the typability of achromogenic and melanogenic P. aeruginosa strains appears to be much lower than that of the chromogenic, nonmelanogenic strains of the species reported previously.     

NON-BETA-HEMOLYTIC GROUP M-REACTING STREPTOCOCCI OF HUMAN ORIGIN.

Rifkind, David (National Institutes of Health, Bethesda, Md.) and Roger M. Cole. Non-beta-hemolytic group M-reacting streptococci of human origin. J. Bacteriol. 84:163-168. 1962.-In 8 years, 14 strains of alpha- and gamma-hemolytic streptococci, reacting only with group M antiserum, were isolated from a variety of human sources. Two alpha-hemolytic strains from the blood of endocarditis patients were compared biochemically and immunologically with the two original canine beta-hemolytic strains of Fry (ATCC 9934 and 9935). The human strains do not produce ammonia or ferment glycogen, whereas the animal strains do. The animal strains share two trypsin-labile antigens with the human strains, and one of them (ATCC 9935) also shares at least four trypsin-stable antigens with the human strains. The other strain of Fry (ATCC 9934) appears to lack these trypsin-stable antigens. These results indicate that the human strains correspond to those designated biotype I whereas ATCC 9935 belongs to biotype II and ATCC 9934 to biotype III.The nature of the group M antigen is undefined, but the gel-diffusion methods employed in this work suggest that several antigens may be responsible for precipitates seen as positive reactions to group M antisera when testing is done by the usual methods in tubes.     

The modification of the cell wall proteins in group A streptococci type M 29 under the influence of spermidine contained in the culture medium

The addition of spermidine into growth medium used for the cultivation of group A streptococci, type M 29, leads to changes in the amino acid composition of cell walls and surface proteins isolated by the method of E. H. Beachey et al. The separation of surface proteins into fibrinogen-binding proteins and fibrinogen receptors by affinity chromatography techniques on cellulose with covalently bound fibrinogen indicates that the proportion of these proteins in pepsin extracts obtained from different strains varies. Both spermidine and avirulent strains have similar content of fibrinogen-binding proteins, although these proteins are absent in virulent strains. Different amounts of fibrinogen receptors are extracted from all strains. As shown in the enzyme immunoassay, fibrinogen receptors contain no group-specific polysaccharide A, Fc-receptors and interact with total antiserum to group A streptococci, type M 29 [correction of 28]. Fibrinogen receptors isolated from the strains under study have been found to have similar amino acid composition. On the basis of these results we believe that neither receptor capacity to fibrinogen nor amino acid composition is indicative of the protective properties of protein M.     

Antigenic diversity of IgA receptors in Streptococcus pyogenes

Abstract In a previous study, group A and group B streptococcal IgA receptors were shown to differ serologically, in agreement with their known structural unrelatedness. The present study was undertaken to serologically compare the IgA binding epitopes of group A streptococcal strains representing various serotypes by the use of antisera to this species. It was found that blocking antibodies occurred in antisera to IgA binding but not to non-binding strains and that binding of IgA to a streptococcal strain was generally blocked by antiserum to the homologous type. However, cross-testing of a panel of 11 IgA binding strains, representing various M and T serotypes, with 10 different antisera to group A streptococci, demonstrated that IgA receptors were inhibited to a highly variable degree and that inhibition patterns were unique for each type. Comparing solubilized IgA receptors of various strains in immunoblot experiments, a variation in the molecular mass, between approximately 35 and 45 kDa, emerged. The IgA binding epitopes, analogous to protective sites of streptococcal M-protein, thus exhibited hypervariability which may suggest that IgA binding also plays a key role for evading host immune defence mechanisms.     

Application of immunoprecipitation in agar gel for the serological typing of soybean root-nodules

The results of serological typing of root-nodule homogenates from soybean plants inoculated by mixed bacterial inocula under field and greenhouse conditions are submitted. The inocula were prepared from strains capable of serological differentiation on the basis of their somatic antigens. Individual root-nodule homogenates were typed serologically by immunoprecipitation in agar with antisera against the inoculum strains. This method gave accurate determination of the origin of 605 out of a total of 616 serologically typed root-nodules and of the individual bacterial strains which participated in their formation.     

The Use of Standardized Reference Antigens in the Serogrouping of Streptococci

Streptococci isolated from the dental plaque of five animal species were identified by physiological and serological methods. Twenty-nine strains of streptococci considered to resemble Streptococcus mitior or Strep. bovis on the basis of physiological data reacted with Lancefield group B or group K antisera respectively in tube precipitation tests. Further serological studies with standardized antigens from known serogroup B and K streptococci revealed that only three of these 29 isolates had been serogrouped accurately and carried the appropriate group antigen. Comparisons were made between the reactivity of the antisera produced by Difco and Wellcome Reagents with acid and autoclaved extracts of the strains. It was shown that the accuracy of serogrouping such isolates could be improved if the tests were made in a gel diffusion system that included a reference antigen.     

Comparison of adherence to and penetration of a human laryngeal epithelial cell line by group A streptococci of various M protein types

Clinically isolated group A streptococci (GAS) of different M protein types were studied using aminoglycoside exclusion and [2,8-3H]adenine radiolabeled GAS assays to compare the abilities of different strains to adhere to and internalize within human laryngeal epithelial (HEp-2) cells. GAS isolated from patients with pharyngitis and GAS isolated from patients with more severe disease, such as necrotizing fasciitis, adhered to and penetrated HEp-2 cells equally well. M3, M4, M6, and M12 strains adhered to and were internalized within HEp-2 cells more than M1 strains. M18 GAS producing hyaluronic acid capsules were less adherent and less invasive than the M3, M4, M6, and M12 strains. An M3-producing GAS strain and its M protein-deficient isogenic strain adhered similarly to HEp-2 cells, but the M protein-deficient strain exhibited greater penetration. Preincubation of HEp-2 cells with an N-terminal synthetic M3 peptide did not alter the adherence or penetration by an M3 strain. In summary, this study demonstrates that GAS from invasive and non-invasive disease adhere to and penetrate HEp-2 cells equally well and that multiple strains of GAS with various M protein types have the ability to adhere to and penetrate HEp-2 cells.     

Biochemical and biological properties of the binding of human fibrinogen to M protein in group A streptococci.

Fibrinogen is known to bind to group A streptococci and precipitate with extracts containing streptococcal M protein. We have previously shown that the binding of fibrinogen to M-positive streptococci prevents opsonization by complement and protects that organism from phagocytosis in nonimmune blood. In the present study, we used 3H-labeled fibrinogen, a highly purified peptide fragment of type 24 M protein (pep M24), and anti-pep M sera to show that fibrinogen binds to M-positive streptococci with high affinity (dissociation constants, 1 to 5 nM); occupation of the high-affinity binding sites suffices to protect the organism from phagocytosis; proteolytic treatments that remove M protein from streptococcal cells abolish binding; binding is competitively inhibited by anti-pep M sera; pep M24 precipitates fibrinogen; and binding to type 24 cells is inhibited by pep M24. We conclude that M protein is the cell surface structure principally responsible for binding fibrinogen on the surface of M-positive streptococci and that this binding contributes to the known antiopsonic property of M proteins.     

Sheep histo compatibility antigens: a population level comparison between lymphocyte antigens previously defined in France, England and Scotland, and sheep red cell groups

A comparison test was performed to look for correlations between the three nomenclature systems for sheep histo compatibility antigens which have been previously described in France, England and Scotland. 187 French sheep from a wide variety of breeds were typed for lymphocyte antigens with antisera which detect the OLA, P and ED series of antigens; they were also tested against. 387 uncharacterized French antisera. Six clusters of sera were found which showed correspondence between antigens of at least two of the three nomenclatures; five of these clusters gave high r values of 0.78–0.94. New antisera from French sheep were found which contributed to the above clusters but few additional clusters were noted. No correlation was found between any of the lymphocyte groups of antisera tested and the sheep red cell antigens which were also tested.