Studies on the IgA-independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.