On the basis of epidemiological data demonstrating that the majority of cases of pharyngitis, necrotizing fasciitis, and other invasive streptococcal infections are caused by a limited number of serotypes, this 26-valent vaccine could have significant impact on the overall burden of streptococcal disease.
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. ...
Two chimeric receptors were constructed by transposing the coding regions for the putative N-terminal domains of the human calcitonin (hCTR) and glucagon (hGGR) receptors. These receptors were stably expressed as glycosylated proteins with molecular masses of 80 kDa for the calcitonin receptor N-terminus chimera (NtCTr) and 65 kDa for the glucagon receptor N-terminus chimera (NtGGr). The NtCTr chimera binds salmon calcitonin (sCT) with an apparent Kd of 12 nM relative to 0.3 nM for the native hCTR. However, this chimera does not mediate a cAMP response even with a transfectant expressing 1.8 x 10(6) cell surface receptors. Stable transfectants expressing the NtGGr chimera show no detectable binding of 125I-sCT or 125I-human glucagon. Surprisingly, adenylate cyclase is activated through the NtGGr chimera by sCT, pCT, and hCT with half-maximal activation at 2.2 +/- 0.6, 5.8 +/- 2.1, and 810 +/- 151 nM, respectively, and the maximum response is similar to that induced by 25 microM forskolin. The rank-order of competition for sCT binding to the NtCTr chimera is similar to the hCTR (sCT > pCT > hCT), but the concentrations required for half-maximal competition are 100- to > 2000-fold higher. In addition, salmon calcitonin binds with a much more rapid on-rate and off-rate to the NtCTr chimera relative to the hCTR which binds hormone irreversibly. Cross-linking of 125I-sCT to the NtCTr chimera with bis(sulfosuccinimidyl) suberate is much greater than to the hCTR, suggesting unique conformations for the two receptor-hormone complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
High affinity binding was characterized for a number of salmon calcitonin (sCT) analogs to a chimeric receptor (NtCTr) constructed by splicing the N-terminal domain of the human CT receptor onto the C-terminal, transmembrane loop region of the receptor for glucagon. Another chimeric receptor (NtGGr) with the N-terminal domain of the glucagon receptor spliced onto the C-terminal regions of the CT receptor shows no high affinity binding of sCT. Nevertheless, sCT and a number of analogs of the hormone are able to elevate cAMP levels in cells transfected with NtGGr. The least helical analog, des-1-amino-[Ala1,7,Gly8]des-Leu19-sCT, is one of the most active in this regard. Two hormone analogs with modifications in the amino-terminal region, des-Ser2-sCT and [Gly2,3,4,5,6]sCT, show reduced or no activity, respectively, for elevating cAMP in cells expressing the NtGGr. In addition, a 15-fold excess of the peptide sCT-(8-32) antagonizes sCT activation of this receptor. In contrast, these calcitonin analogs exhibited a different rank order for binding to the NtCTr receptor. In fact, des-Ser2-sCT and [Gly8]-des-Leu19-sCT along with the native hormone had the highest helical content as well as the highest binding affinities to the NtCTr receptor. These studies suggest that the helical portion of the hormone within residues 8-22 of sCT is the principal determinant for binding to the receptor N-terminus. Residues 2-6 of sCT interact with the receptor transmembrane loop region and are critical for activation of adenylate cyclase; however, residues 8-32, including Leu16, are responsible for most of the hormone interaction with the transmembrane loop region. Thus, unique requirements exist for CT interaction at the receptor N-terminus relative to the receptor transmembrane loop region, yet there is significant overlap in the hormone determinants that facilitate these interactions.
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