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 g 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.Group A streptococcal pharyngitis is one of the most common bacterial infections in school age children. In addition, invasive streptococcal infections afflict thousands of children and adults each year, often resulting in death or significant morbidity (37). Although the incidence of acute rheumatic fever (ARF), a nonsuppurative sequela of streptococcal pharyngitis, has declined in developed countries, the disease is rampant in developing countries (40). Efforts to develop a vaccine that would prevent group A streptococcal infections have been ongoing for more than 8 decades (22,28). New molecular techniques (8) and a better understanding of the biology of group A streptococci (11) have allowed the previous obstacles associated with vaccine development to be overcome.Previous studies have shown that the surface M protein is the major virulence determinant and the major protective antigen of group A streptococci (29). The type specificity of each M protein, of which more than 100 are now known, is largely determined by the epitopes located in the amino-terminal 40 to 50 amino acid residues (3,8,14,27). These regions of M proteins have been shown to evoke antibodies with the greatest bactericidal (protective) activity and are least likely to crossreact with human tissues (2,14,20). Thus, our approach has been to combine small amino-terminal M protein peptides to make multivalent vaccines that would elicit opsonic antibodies against epidemiologically important serotypes of group A streptococci (12,21).In the present study, we constructed a 26-valent M proteinbased vaccine by recombinant technology. The vaccine is composed of four different fusion proteins that contain six or seven M protein fragments linked in tandem. Each component protein of the vaccine was designed to serve as its own carrier, thus obviating the need for unrelated proteins. ...

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.

The influence of valence and heavy chain on antibody activity was investigated using transfectoma-derived, class-switched IgG1 and IgM human monoclonal antibodies (MAbs) reactive with the bacterial pathogens Escherichia coli K1 and group B Streptococcus species. IgG-IgM pairs were compared in vitro for antigen binding and opsonic activities and in vivo for protective efficacy in neonatal rats. For the anti-E. coli pair, the IgM MAb was 1000-fold more potent in all assay formats. Importantly, the 50% protection dose (PD50) of the IgM MAb was 10-20 ng/rat, while 100 micrograms of the IgG MAb was only minimally protective. For the group B streptococcal MAbs, the IgM was 100- and 4500-fold more potent in binding and opsonization assays, respectively. However, while 20 micrograms of IgM protected neonatal rats, 100 micrograms of IgG MAb was partly protective. These experiments demonstrate the utility of recombinant DNA technology for creating a panel of antibodies that may aid in selecting potential immunotherapeutic candidates.

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261-1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.Group A streptococci (GAS) are major human pathogens that cause a wide variety of illnesses, ranging from uncomplicated pharyngitis and pyoderma to life-threatening infections such as necrotizing fasciitis and toxic shock syndrome (23). The virulence of GAS is determined in part by their ability to resist opsonization by complement and phagocytic killing by neutrophils (14). It has been known for many years that these virulence characteristics are mediated in large part by the M protein on the surface of the organisms (16). In addition, the M proteins contain protective epitopes that elicit opsonic antibodies that promote phagocytic killing in the immune host (16). Previous studies have shown that insertional inactivation of the emm gene in some GAS serotypes results in an avirulent phenotype (7,19).In a recent study of type 18 streptococci, we found that inactivation of the emm 18 gene had only a minor effect on the ability of the mutant to grow in human blood and on the 50% lethal dose (LD 50 ) in mice compared to the M-positive parent strain (10). We used the M-negative mutant to identify a new surface protein, streptococcal protective antigen (Spa), that was distinct from M protein and contained protective epitopes which elicited bactericidal antibodies (10). The present study was undertaken to determine whether Spa functions as a virulence determinant of type 18 streptococci. Using a series of M-negative and Spa-negative mutants, we show that both of these surface proteins contribute to the virulence of type 18 streptococci. In addition, we have cloned and sequenced the complete spa gene, which allowed a direct comparison of its structure to that of emm genes. MATERIALS AND METHODSBacterial strains. The parent type 18 streptococcal strain 87-282 (designated 18-282) and its M-ne...

Inhibition of rat liver microsomal glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) by orthophosphate and organophosphate esters was examined at pH 6.0 and 7.5 with and without enzyme pretreatment with 0.2% (w/v) deoxycholate. Inhibition by orthophosphate and monoethyl phosphate was competitive with respect to glucose-6-P while inhibition by mono- and di-phenyl phosphate was of the mixed type. Monoalkyl phosphates were more effective inhibitors than the analogous di- and tri-alkyl phosphates and deoxycholate potentiated the inhibitory effects. Mono- and di-phenyl phosphates were more effective inhibitors than triphenyl phosphate, and deoxycholate decreased these inhibitory effects. The results are interpreted in terms of inhibitor and deoxycholate interactions with the enzyme.