Shigellosis, caused by Shigella species (gram negative), is a prominent, and the most infectious, diarrheal disease. Shigella flexneri, the species responsible for the highest mortality rate, is endemic in most developing countries (23). The 13 serotypes of S. flexneri, with the exception of serotype 6, result from structural modifications of the O-antigen polysaccharide (PS), the outer portion of the lipopolysaccharide (LPS) on the bacterial surface, which is both an essential virulence factor and a protective antigen (10). The basic O-antigen PS repeat, common to the 12 serotypes, is referred to as serotype Y. A murine immunoglobulin G3 (IgG3) monoclonal antibody, SYA/J6, specific for the PS O antigen of the S. flexneri Y LPS, was developed by Bundle (7) and Carlin and coworkers (8). The O-antigen Y PS is a linear heteropolymer with a tetrasaccha- -(13] and is recognized by the monoclonal antibody SYA/J6 (7, 9). The oligosaccharides of S. flexneri serotype Y have been well studied by nuclear magnetic resonance techniques, which have provided a three-dimensional model of the determinant in solution (4,30,31,32) and the identity of the biological repeating unit (9).Glycoconjugate vaccines to prevent shigellosis, focused on oligosaccharide analogues related to Shigella flexneri 2a and other serotypes, have been synthesized and evaluated (3,10,29,38). An interesting approach to vaccine design is the use of molecules that mimic the immunogenic element of interest (11,20,28). Carbohydrate-mimetic peptides have potential as surrogate ligands for traditional carbohydrate vaccines, providing more discriminating immune responses (19,20,28). However, there are few examples of immunological responses with peptide-based PS mimics (18,20,28). Therefore, the requirements for cross-reactivity are not fully understood and are certainly not predictable, because of the limited data set available.In order to further exploit this principle, a carbohydratemimetic peptide of the S. flexneri Y O-antigen PS, MDWNM HAA, cross-reactive with the anti-S. flexneri Y O-PS monoclonal antibody, SYA/J6, was identified by phage library screening (15). The structures of complexes of the antibody SYA/J6 Fab fragment with synthetic deoxytrisaccharide and pentasaccharide ligands, related to the S. flexneri Y O antigen, and with the carbohydrate-mimetic peptide have been determined by X-ray crystallography (17,33,34,35). The structure of the Fab complex with MDWNMHAA revealed differences, and few similarities, with respect to the oligosaccharide complexes (34), providing the first evidence that the modes of binding of the pentasaccharide and octapeptide differ considerably and that few aspects of structural mimicry exist (34).
Abstract:The immunogenicity of a peptide-protein conjugate developed by linking a peptide mimic DRPVPY of the Group A Streptococcus cell-wall polysaccharide (GAS-CWPS), to tetanus toxoid (TT) was examined. BALB/c mice were immunized three times subcutaneously following homologous or heterologous prime/boost strategies at 4-or 6-week intervals in two different experiments. DRPVPY-TT, CWPS-TT, heat-killed, pepsin-treated GAS bacteria (with exposed polysaccharide) and TT, were used as immunogens with alum as adjuvant. Antibody titers were determined by ELISA with GAS bacteria (with exposed polysaccharide) and DRPVPY-linked to bovine serum albumin (BSA, DRPVPY-BSA) as solid phase antigens. All mice primed with DRPVPY-TT developed high IgG antipeptide and anti-GAS titers. The binding of polyclonal anti-peptide antibodies to GAS could be inhibited by purified CWPS, synthetic oligosaccharides corresponding to CWPS, DRPVPY-BSA, DRPVPY and DRPVP, as assessed by competitive-inhibition ELISA. Anti-oligosaccharide titers were also obtained upon titration of anti-peptide sera with synthetic oligosaccharide-BSA conjugates. All mice primed with CWPS-TT and mice primed and boosted with GAS developed IgG anti-peptide titers. These data demonstrate conclusively the cross-reactivity of the immune responses and support the hypothesis of antigenic mimicry of the GAS-CWPS by the hexapeptide DRPVPY. However, mice boosted with DRPVPY-TT, after 6-8 weeks, showed a decrease in IgG anti-GAS titers, but an increase in IgG anti-peptide titers, suggesting carrier-induced suppression of the response to polysaccharide. Strategies are outlined for further refinement of a DRPVPY conjugate as a surrogate of the cell-wall polysaccharide for use in vaccines against Group A Streptococcus.
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