A B S T R A C T 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 monthly 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.

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.

Epidemiological evidence and laboratory experimentation over the past 20 yr supports the conclusion that immunity to group A streptococcal infection is type-specific. Antibodies directed against the M proteins, the antigens responsible for the specificity of the 50 or more serotypes of group A streptococci, are protective by virtue of their opsonic capacity. This subject has been thoroughly reviewed by Lancefield (1). Recent work has demonstrated that several small doses, e. g. 10/~g, of highly purified M proteins induce type-specific bactericidal antibodies in rabbits (2). The success of these latter experiments has been the impetus for this investigation on the development of a streptococcal vaccine for human use. With highly purified M proteins of types 12, 14, and 24, we have examined the degree of cutaneous hypersensitivity and the level of circulating antibodies in adults and infants. These studies have furnished data on the relative tolerance to M proteins and the possible extent of previous exposure to these serotypes. Secondary bactericidal antibody responses have been induced in adults injected with M protein vaccines without provoking untoward local or systemic reactions. I t is the purpose of this report to ex~mlne the feasibility of type-specific immunization of humans against group A streptococcal infections. Materials and MethodsM Protcin.--Hi~hly purified M proteins were prepared from Group A streptococcal cell walls as previously described (3). A slight modification of the chromatographic procedure eliminated traces of nonspecifie antigens. To a column of carboxymethyl cellulose equilibrated with 0.03 u sodium acetate and containing the M protein sample was added 0.1 ~ sodium acetate buffer at pH 5.5. When the efeuent pH reached 5.5, a linear gradient of 0.1 M potassium phosphate buffer of inc~eAslng pH was applied to the column by adding equal volumes of buffers at pH 6.0, 6.5, and 7.0 to a Technicon "Autograd" mixing chamber for the final elution. Minor fractions of nonspedfic protein emerged prior and subsequent to the elution of the main peak of M protein near pH 6.

The M proteins are the antigens conferring serological specificity to the 45 or 50 types of group A streptococci. IMnore importantly, these proteins play a predominant role in virulence by rendering the streptococci refractory to phagocytosis in the absence of type-specific antibody. Owing to unusual stability at low pH and high temperature,' the M proteins are best obtained from whole cells or streptococcal cell walls by solubilization at pH 2 and 1000. Recent studies have shown that the M proteins may be solubilized from cell walls with a muralytic enzyme released during bacteriophage lysis of group C streptococci. Materials and Methods.-Streptococci and antisera: The group A streptococci, types 12, 14, and 24, culture methods, immunodiffusion techniques, and methods for the preparation of adsorbed and unadsorbed typing sera have been previously described.5 6Reagents: Crude lytic enzyme of the group C streptococcal bacteriophage system was prepared as previously described.6 The crude enzyme was concentrated from the cell lysate by precipitation at 00 in 70% ammonium sulfate. One ,ug of enzyme per ml decreased the optical density of a suspension of whole streptococci from 0.20 to 0.10 in 30 min. Buffered saline containing 0.01 M potassium phosphate pH 7.0 was used as the antigen diluent for serological procedures and Sephadex chromatography.Preparation of M proteins: Streptococci harvested after growth for 18 hr in enriched Difco Todd-Hewitt broth5 were washed and ruptured with glass beads in a Gifford-Wood Eppenbach Micro-Mill according to the method of Markowitz and Lange.7 For smaller amounts of cells the Braun MSK homogenizer was used to obtain cell walls according to the method of Bleiweis et al.8The following is a typical protocol using 130 gm of wet cells broken in the Eppenbach mill. After removal of the glass beads with a coarse sintered glass funnel, the debris composed of cell walls and membranes was washed twice in 2-liter volumes of buffered saline by centrifugation at 9,000 X g for 30 min. All operations were carried out at 0-5'. The cell walls containing the M protein were separated from the membranes by sedimenting the walls at 5,000 X g for 30 min in 2 liters of buffered saline; this step was repeated three times. At each washing the walls were evenly resuspended with a Waring Blendor; octyl alcohol was added to prevent foaming. After the

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.