Toxic effects of streptococcal M protein on platelets and polymorphonuclear leukocytes in human blood.

Purified M protein isolated from Group A streptococci produced cytotoxic reactions in normal human blood in vitro. In the presence of M antigen, platelets aggregated, fused, and lysed. Polymorphonuclear leukocytes (PMN) surrounded the platelet aggregates, then became highly vacuolated and lysed. In addition, PMN progressively lost their capacity to phagocytose unrelated bacteria and to migrate in glass capillary pipettes. Platelet-PMN reactions were directly proportional to the type-specific precipitin reactivity of each M preparation and could be removed with homologous M antibody, only. Moreover, the reactivity of M protein was abolished by enzymatic digestion with trypsin, but not with lysozyme, strongly suggesting that cell-wall mucopeptide was not involved. Preliminary studies showed that platelet-PMN reactions require heat-stable and heat-labile serum factors, presumably antibody and complement. It is suggested that cytotoxic determinants are uncovered by the extraction and purification process and are intimately associated with the type-specific M determinant, possibly in a molecular complex.

Human immune response to immunization with a structurally defined polypeptide fragment of streptococcal M protein.

We tested the ability of pepsin-extracted, highly purified M protein to induce type-specific immunity in experimental animals and humans. M protein was prepared from limited peptic digests of whole group A type 24 streptococci and was purified to chemical homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, quantitative amino acid analysis, and Edman degradation. For vaccination, the lyophilized M24 protein preparation (pep M24) was precipitated in aluminum hydroxide. When injected into laboratory animals, alum-precipitated pep M24 produced type-specific protective antibodies and was free of non-type-specific immunoreactivity. In man, skin tests with 1-microgram doses of pep M24 were negative in all 37 adults tested. 12 adult human volunteers received two-four subcutaneous injections of 100-200 micrograms of alum-precipitated pep M24 at intervals of at least 2 wk. The immune response to pep M24 was measured by a variety of assays designed to detect (a) type-specific humoral antibodies (opsonophagocytic, long chain, and mouse protection tests); (b) total humoral antibodies (complement fixation and enzyme-linked immunosorbent assay); (c) cellular immunity (skin tests); and (d) heart cross-reactive antibodies (immunofluorescence). Type-specific opsonic antibodies developed in 10 of the 12 vaccinees, and positive delayed-type skin tests developed in 11. Immune sera from two of the vaccinees were effective in mouse-protection tests against challenge with M24 but not M6 streptococci. None of the volunteers developed heart-reactive antibodies or antibodies to non-type-specific M protein antigens. Alum-precipitated pep M24 was well-tolerated in man, and no serious local or systemic reactions were observed. Thus, pep M24 induces type-specific, protective antibodies in doses that are well-tolerated in man.

Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen.

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.

Mediation of cytotoxic effects of streptococcal M protein by nontype-specific antibody in human sera.

The cytotoxic moiety(ies) in highly purified streptococcal M protein has been shown to be distinct from the type-specific M determinant. This nontype-specific M-associated determinant(s) (NTSM) causes humoral and cellular immunotoxic responses in man. NTSM is common to M protein prepared from all streptococcal serotypes studied so far. In this study, immunoabsorbents prepared by entrapping purified M proteins in polyacrylamide gel were employed to identify, separate, and purify antibodies directed against NTSM as well as against the type-specific M (TSM) determinants. We found that anti-NTSM present in human blood mediated cytotoxic platelet and leukocyte reactions in the presence of "M proteins" prepared from groups A, C, and G streptococci. Human sera that produced cytotoxic reactions and fixed complement in the presence of highly purified M protein but that contained no antibody to the homologous TSM determinant were used as a source of anti-NTSM. Anti-NTSM was absorbed with and eluted from M protein-polyacrylamide particles and identified as IgG. Antibodies to NTSM were present in most normal human sera and some primate sera (rhesus monkey and baboons) but not in the sera of other common laboratory animals. Further absorption studies showed NTSM to be a component not only of extractable M protein but also of protoplast membranes and of cell walls of avirulent streptococci that lacked extractable TSM antigen. Preparation of antisera that can distinguish between the type-specific protective moiety and the closely associated immunotoxic components in purified M protein vaccines may help answer whether or not M-associated moieties play a role in pathogenesis of poststreptococcal diseases.

Current issues in the prevention of rheumatic fever.

Variation in strain virulence helps to account for the wide spectrum of group A streptococcal diseases and for their striking epidemiological variation. Recent studies of the genetic control of the expression of the virulence factors of group A streptococci (GAS) are beginning to illuminate such variation. Although still obscure, the pathogenesis of acute rheumatic fever (RF) requires primary infection of the throat by highly virulent GAS strains. Those that have clearly caused RF contain large hyaluronate capsules and extended M-protein molecules. The M molecule contains some epitopes cross-reactive with host tissues, and also has superantigenic properties like the secreted GAS erythrogenic toxins. In settings where ARF has become rare, GAS pharyngitis continues to be quite common but is most often caused by relatively attenuated strains. These, however, may colonize the throat avidly, and often stubbornly. GAS "skin strains" that cause pyoderma (impetigo) are molecularly distinct from "throat strains". Although they may secondarily colonize and infect the throat, the pyoderma strains are generally less virulent and are not rheumatogenic. Some skin strains, however, may cause acute glomerulonephritis. The diagnosis, treatment and prevention of GAS pharyngitis is reviewed in relation to the varying prevalence of RF in different geographical and social settings.

Can we eradicate rheumatic fever in the 21st century?

In the latter half of the 20th century, the clinical importance of variation in the virulence of strains of GAS has been clearly demonstrated. Although still obscure, the pathogenesis of ARF requires immunologically significant infection of the throat by virulent GAS strains. These strains contain large hyaluronate capsules and large M-protein molecules. The latter contain epitopes cross-reactive with host tissues, and also contain superantigenic toxic moieties. In areas where ARF has become rare, GAS pharyngitis continues to be common but is caused predominantly by GAS strains of relatively low virulence. These, however, may colonize the throat avidly and stubbornly. Molecularly distinct pyoderma strains may cause acute glomerulonephritis, but they are not rheumatogenic even though they may secondarily infect the throat. In developing countries with a very high incidence of rheumatic heart disease, identification of the prevalent rheumatogenic GAS strains and development of a multivalent vaccine against them is currently an interesting strategy. Pending vaccine development, intense primary and secondary penicillin prophylaxis should continue to be sharply focused on populations with the highest prevalence of RHD as such measures may often succeed in driving away the most virulent rheumatogenic clones of GAS from their midst.

Rheumatogenic streptococci and autoimmunity.

The uniqueness of the group A streptococcus in initiating a cardiodestructive disease in a limited segment of the human species, regardless of race or ethnic group, makes the quest for a unique host response to a specific streptococcal antigen an intriguing and persisting challenge for clinical investigators, particularly for those investigators interested in autoimmunity. New methodology is making possible more incisive research approaches. The defined streptococcal antigens that turn out to be epitopes identical with host tissues, such as the M protein/cardiac myosin model or the hyaluronate in the capsule of mucoid rheumatogenic strains, offer the opportunity for more incisive clinical investigations. The isolation and cultivation of cardiotoxic T cell clones directed against such epitopes shared by host and parasite may eventually be possible. We may then learn more about whether autoimmunity is indeed a factor in the pathogenesis of rheumatic heart disease.