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In these studies we analyzed the adjuvant effect of cholera holotoxin or cholera toxin (CT) B subunit on the B cell response to mucosal antigens. Purified Peyer's patch B cells obtained from mice at varying periods of time after oral administration of inactivated influenza virus, with or without a CT preparation, were stimulated in vitro in the absence or presence of various lymphokines. Responses were measured by an antigen- and isotype-specific ELISPOT assay. In this system cultures containing a combination of lymphokines [interleukin 5 (IL 5), interferon-gamma (IFN-gamma), IL 4] gave comparable responses to those containing T cells from immunized mice or supernatant of concanavalin A-stimulated T cells and therefore were assumed to express optimum or near optimum B cell responses. Administration of a CT preparation along with influenza virus increased the number of B cells producing anti-influenza antibodies of both the IgM and IgA isotypes, with the effect on the IgA response at least threefold greater than the effect on the IgM response. These results thus indicate that CT preparations enhance the memory B cells response in Peyer's patches and, in addition, suggest that CT enhances isotype switching. In this antigen-specific B cell system IL 4 augmented responses in cultures containing IL 5 but not IFN-gamma; in addition, IL 5 and IFN-gamma acted in an additive fashion. Thus, these findings suggest that the effects of IL 5 and IFN-gamma are at least in part, mediated via different cellular differentiation pathways.

Intragastric inoculation with whole-virion vaccine of inactivated influenza virus resulted in production of hemagglutinin (HA)-specific immunoglobulin A (IgA) and IgG both in lung lavage fluids and in serum samples of mice. HA-specific IgA was the predominant isotypic antibody secreted in the lung lavage fluids (average IgA/IgG ratio, 13:1), whereas HA-specific IgG was the major antibody class in serum (average IgA/IgG ratio, 0.3:1). These responses were similar to the antibody responses stimulated by intranasal infection with live influenza virus. In vitro cultures of lymphoid cells from lungs and Peyer's patches, but not from spleens, in the presence of homologous antigen, from mice vaccinated intragastrically synthesized mostly HA-specific IgA. Mice immunized parenterally with inactivated influenza virus produced only IgG in lung lavage fluids and sera. Cultures of lymphoid cells from their spleens, but not their lungs, synthesized HA-specific IgG upon antigenic stimulation in vitro; neither synthesized IgA. These in vitro cell culture results, as well as the inverse relationship of IgA/IgG ratios in lung lavage fluids and sera, demonstrated that the IgA antibody in lung lavage fluids was actively synthesized locally in the lungs of intragastrically immunized mice. This finding was consistent with the migratory distribution of antigen-primed lymphoid cells from Peyer's patches to distant lymphoid tissue such as lung. Intragastric vaccination conferred protection against intranasal challenge with a lethal dose of virulent virus.

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SUMMARYTwo split influenza virus vaccines administered intragastrically induced lower titres of haemagglutinin (HA)-specific antibodies in pulmonary secretions than whole virus vaccine or a third split virus vaccine. IgA antibody was the predominant HA-specific Ig class. HA-specific IgA titres decayed substantially within 2 weeks following booster immunization, but persisted for at least another 3.5 months. In contrast, HA-specific IgG was maintained at low titres throughout the 4 month study period. When the total vaccine antigenic mass was administered as one dose or as equally divided doses spread over several days, pulmonary antibody responses were comparable. Mice immunized intragastrically with whole virus vaccine were completely protected against intranasal challenge with a homologous virulent virus of the H3 subtype. Partial protection was obtained when the vaccine used for immunization was a distantly related, antigenically variant strain of the same subtype, but no protection was obtained with a monovalent vaccine of an influenza A subtype different to the challenge virus. These characteristics of the response to intragastric immunization against influenza are consistent with features of a useful vaccine.