Antigenicity of the M proteins of group A hemolytic streptococci. 3. Antibody responses and cutaneous hypersensitivity in humans.

Highly purified M proteins were used for determining cutaneous hypersensitivity and type-specific circulating antibodies in normal adults and infants. 80% of 91 adults and 8% of 59 infants exhibited a transient delayed cutaneous reaction to at least two of types 12, 14, and 24 M proteins. Antibodies assayed by passive hemagglutination were observed in 90% of the adults and 13% of the infants. Vaccines of 10 microg of alum-precipitated M protein or 20 microg of the soluble antigen were administered to adults not exhibiting delayed hypersensitivity. Within 2 wk hemagglutination liters increased significantly in 31 of 33 subjects. Preimmunization antibody levels indicated that these responses were probably anamnestic reactions from previous exposures to homologous serotypes of group A streptococci. Sera exhibiting large increments in antibody titers resulting from M protein inoculations also had type-specific bactericidal properties. "Attenuated" M proteins, produced by partial degradation with trypsin induced only minimal cutaneous reactions in hypersensitive adults, but still retained most of the antigenic specificity when assayed in vitro and in vivo. The utility of M protein vaccines for human use is discussed in reference to the low incidence of cutaneous hypersensitivity in infants, the potentials of polyvalent attenuated M protein vaccines and the apparent absence of immune cross-reactivity between pure M proteins and human heart and kidney tissues.

Streptococcal-induced cell-mediated-immune destruction of cardiac myofibers in vitro.

We have demonstrated that T lymphocytes from the spleens of adult guinea pigs sensitized to group A streptococcal antigens are cytotoxic for cultured fetal guinea pig heart cells. Lymphocyte cytotoxicity, measured by 51Cr release from target cells, was stimulated by sensitization in vivo with group A whole cells, cell walls, and purified protoplast membranes emulsified with complete Freund's adjuvant (CFA). Sensitization with group C streptococcal antigens in CFA or CFA alone produced lymphocytes with little or no specific cytotoxic activity. Target cells of cultured fetal skeletal muscle, liver, or skin were relatively refractory to effector cell cytotoxicity. The presence of antigenic determinants on the membranes of cultured myofibers, cross-reacting with group A streptococcal cellular antigens, was confirmed by immunofluorescence. These data are discussed in terms of a model for poststreptococcal rheumatic myocarditis in which cell-mediated autoimmune mechanisms may participate.

Protective study with a group A streptococcal M protein vaccine. Infectivity challenge of human volunteers.

Healthy adult male volunteers were immunized with purified M protein from Group A streptococci. Type 1. The vaccine was administered subcutaneously as an aluminum hydroxide-precipitated antigen in three montly doses. Control subjects received a placebo of the aluminum hydroxide adjuvant. To test the efficacy of the immunization, vaccinees and controls were challenged with a virulent strain of Type 1 streptococci applied to the pharynx. The immunization and challenge of the vaccinated and control subjects (19 men in each group) were carried out as a double blind experiment. All subjects were carefully screened by physical and laboratory examinations before and after the immunization and infectivity schedules. 30-50 days after the last injection, the vaccinees and control subjects were infected with the streptococci. Careful surveillance was maintained to evaluate the extent of acquired streptococcal infection. Throat cultures, leukocytes counts, temperatures, and physical signs and symptoms were monitored daily. All subjects received 1.2 million U of penicillin intramuscularly no later than 6 days after inoculation with the culture. Illness was judged by the appearance of exudative pharyngitis and cervical adenopathy accompanied by a positive throat culture. By these criteria, 9 of the 19 placebo controls, and 1 of 19 vaccinees were ill. No residual illness or clinical complications was observed after the penicillin treatment. It is concluded that the alum-precipitated M protein vaccine afforded protection against an upper respiratory Type 1 streptococcal infection.

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.

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.

Micro complement fixation assay for type-specific group A streptococcal antibody.

A micro complement fixation assay was devised to measure the type-specificity of anti-M antibody. Unabsorbed sera were from rabbits hyperimmunized with heat-killed streptococci and from humans with naturally occurring antibody or immunized with purified M protein vaccines. These sera fixed complement only in the presence of homologous M proteins (serotypes 1, 3, 6, 12, and 14). The complement fixation reaction paralleled the results obtained with the Lancefield bactericidal (opsonic) assay and did not exhibit the cross-reactions frequently seen with type-specific anti-M sera assayed by passive hemagglutination. All serological activity of the antisera resided in the fraction corresponding to 7S globulin eluted from a Sephadex G-200 gel filtration column. Because of the close correlation between micro complement fixation and the bactericidal assay for type-specific antibody, it is proposed that the micro complement fixation procedure, as we have described it, merits further evaluation as a substitute for the bactericidal test of immunity to group A streptococcal infection.

Immune response to phosphorylcholine. IX. Characterization of hybridoma anti-TEPC15 antibodies.

Hybridoma antibodies against the PC-binding T15 BALB/c myeloma protein were raised by cell fusion with anti-T15 A/He immune cells. The idiotype specificity of these monoclonal anti-T15 antibodies was determined with a panel of different myeloma and hybridoma immunoglobulins. Two types of anti-T15 antibodies are seen. One reacts with a number of different IgA myeloma proteins and with serum IgA of certain strains of mice; this reactivity most likely is due to allotypy. The other group consists of anti-T15 antibodies that are specific for the T15 idiotype and are therefore termed anti-idiotypic. The bindings of the anti-idiotype antibodies to T15 were specifically inhibited by T15 (F(ab')2 but not by other PC-binding myeloma proteins of different idiotypes. The relationship of the idiotype-specific anti-T15 antibodies to the PC-binding site of the T15 idiotype was analyzed by hapten inhibition of anti-idiotypic binding and by inhibition of BALB/c anti-PC splenic hemolytic plaque formation. Anti-T15 antibodies, for which the T15 binding is inhibited by PC or PC-BSA, also specifically inhibit anti-PC plaque formation. These antibodies are labeled site and near-site anti-idiotypic antibodies. Site and near-site-specific anti-idiotypic antibodies recognize different idiotopes on the T15 molecules. The possible differential biologic activities of these anti-idiotopes in idiotype network regulation is considered.

Protective studies with a group A streptococcal M protein vaccine. II. Challange of volenteers after local immunization in the upper respiratory tract.

Twenty-one adult volunteers were immunized at monthly intervals with three doses of purified type 1 M protein of group A Streptococcus. The soluble vaccine in buffer was administered by aerosol spray into the nares and oropharynx; 23 control subjects received a buffer placebo in the same manner. Antibody responses were observed in sera and nasal washings of some but not all vaccines. Approximately 30 days after the last dose, all subjects were challenged with homologus streptococci applied by swab to the phayngeal-tonsillar areas. In a double-blind system of evaluation, physical signs and symptoms were followed for assessment of infection. Illness was defined on the basis of a positive throat culture, fever, a twofold increase in white blood cell count over baseline, exudative pharyngitis, and cervical adenopathy. By these criteria four vaccinees and 11 controls were obviously ill. One vaccinee and six controls were questionably ill, fulfilling some but not all of the criteria. sixteen vaccinees and six controls were not ill (P less than 0.001). Positive throat cultures were observed in five vaccines and 19 controls (P less than 0.001). Penicillin was administered five days after challenge. No poststreptoccal sequelae or other complication were observed. Thus local immunization with M protein apparently can prevent both colonization and clinical illness after challenge with homologous streptococci.

Protective studies with group A streptococcal M protein vaccine. III. Challenge of volunteers after systemic or intranasal immunization with Type 3 or Type 12 group A Streptococcus.

Alum-precipitated and soluble, purified M protein vaccines were prepared from type 3 and type 12 group A Streptococcus. Adult volunteers were assigned to one of three groups: group I received placebo by both parenteral and intranasal routes; group 2 received vaccine parenterally (either type 3 or type 12) and placebo intranasally; and group 3 received placebo parenterally and vaccine intranasally (either type 3 or type 12). Subjects were inoculated three times at montly intervals. Thirty to 50 days after the last dose, all subjects were challenged with homologous streptococci applied to the oropharynx. Six subjects (30%) vaccinated subcutaneously had definite illness, three (15%) had probable illness, and 11 (55%) had no illness. In the group vaccinated intranasally, four (14%) had definite illness, two (7%) had probable illness, and 22 (79%) had no illness. Fifteen controls (42%) had definite illness, and 21 (58%) had no illness. The rate of colonization was significantly lower in recipients of intranasal vaccine. Average clinical scores and vaccine side effects were also decreased in subjects vaccinated intranasally. Induced serum antibody as measured by passive hemagglutination was not a reliable predictor of resistance to streptococcal pharyngitis. Penicillin was administered to all subjects five days after challenge. No sequelae of streptococcal infection or other complications occurred. Thus, local immunization with M protein apparently may reduce both colonization and clinical illness after challenge with homologous streptococci.