Abstract
Actinobacillus actinomycetemcomitans serotype b has been implicated as a causative agent of localized juvenile periodontitis. The serotype b-specific antigen (SPA) of this organism is known to be a polymer consisting of L-rhamnose and D-fucose. In this study, we investigated the immunogenicity of SPA and SPA-protein conjugate in mice. SPA was extracted from whole cells of A. actinomycetemcomitans strain Y 4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA was activated with cyanogen bromide, and amino groups were added with adipic acid dihydrazide. The purified reaction product was coupled to bovine serum albumin (SPA-BSA) by using 1-ethyl-3 (3-dimetylaminopropyl)-carbodiimide or was biotinylayted in order to detect antibody. BALB/c mice were immunized intranasally with SPA or SPA-BSA (10μg) with chorela toxin B subunit (CTB) (5μg). After 28 and 35 days, the mice were intranasally immunized with SPA or SPA-BSA (10μg). Other groups were subcutaneously immunized with SPA or SPA-BSA (10μg) in complete Freund's adjuvant. After 28 days, the mice were immunized with the same antigen in incomplete Freund's adjuvant. After 42 days, pirocarpine-stimulated saliva and serum samples were collected. Antibodies to SPA were measured by an avidin-biotin based enzyme-linked immunosorbent assay. Biotinylated SPA were immobilized on avidin-coated microtiter plates. Subcutaneous immunization of BALB/c mice with SPA did not induce any antibody responses to SPA. However subcutaneous immunization of BALB/c mice with SPA-BSA induced high serum IgM and serum IgG. Intranasal immunization of BALB/c mice with SPA and CTB did not induce any antibody responses to SPA. On the other hand, the immunization with SPA-BSA and CTB induced high serum IgM, serum IgG and salivary IgA responses to SPA. Serum IgM responses remained high level for 12 weeks after secondary immunization. Serum IgG responses increased with increasing time for 12 weeks after secondary immunization. Maximal salivary IgA responses to SPA were detected 4 weeks after secondary immunization and decreased with the elapse of time. These results suggest that the SPA-protein conjugate may be useful for developing a vaccine against periodontitis.
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