Immunological protection against dental caries can been achieved by immunization with Streptococcus mutans or purified protein antigens (Ag) including Ag l/ll, administered by parental or oral routes that elicit circulating or salivary antibodies, respectively. However, oral immunization to stimulate the common mucosal immune system to produce antibodies of the secretory immunoglobulin A (S-lgA) isotype has hitherto been an inefficient process with most antigens, especially non-viable bacteria or purified proteins that are not readily taken up by the gut- associated lymphoid tissues. As cholera toxin is an exceptionally immunogenic protein by both parental and oral routes by virtue of the affinity of its B subunit (CTB) for cell surface GM1 ganglioside, the ability of conjugates of Ag l/ll and CTB to induce salivary lgA antibodies will be investigated in mice, as a well-established experimental model for immunological studies. Ag l/ll and CTB will be coupled by means of heterobifunctional cross-linking agents, mainly N-succinimidyl 3-(2- pyridyldithio)propionate, at various reagent substitution and coupling ratios, to obtain conjugates that preserve the antigenicity of Ag l/ll and the receptor-binding activity of CTB. These will be administered to mice by gastric intubation in various dose schedules to investigate isotype- specific antibody responses in serum, saliva, intestinal fluid, and other secretions by enzyme-linked immunosorbent assay, and the induction of cells secreting antibodies in spleen, salivary glands, mesenteric lymph nodes, and other mucosal tissues by enzyme-linked immunospot assay. The induction of antigen-specific T helper cells in the same tissues will be investigated by their ability to secrete lymphokines that indicate their subset and that are important in the stimulation of lgA production by B cells. Further experiments will address the duration of salivary antibody responses after oral immunization with Ag l/ll-CTB conjugates, and the establishment of immunological memory in salivary glands and other mucosal immune tissues, by in vitro culture techniques, and adoptive transfer of putative memory B and T cells to naive syngeneic recipients. Induction of protective immunity to dental caries by oral immunization with Ag l/ll-CTB conjugates will be investigated in already established germ-free rat and rhesus monkey models. Finally, the same approach to enhancement of mucosal antibody responses by coupling antigens to CTB will be applied to a 'T-independent' carbohydrate antigen, the cell wall polysaccharide of S. mutans, which is usually poorly immunogenic. This will be coupled to CTB by a variety of established chemical procedures, and the conjugates will be evaluated for the preservation of antigenic and receptor-binding activities in vitro, and the induction of salivary and other mucosal immune responses in vivo, after oral administration. The results obtained are expected to be of value for immunological protection not only against dental caries, but also against other oral diseases, and many other infections that arise at or invade through the mucosal surfaces.
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