Induction of Local Secretory IgA and Multifunctional CD4⁺ T-helper Cells Following Intranasal Immunization with a H5N1 Whole Inactivated Influenza Virus Vaccine in BALB/c Mice.

Avian influenza subunit vaccines have been shown to be poorly immunogenic, leading to the re-evaluation of the immunogenic and dose-sparing potential of whole virus vaccines. In this study, we investigated the immune responses after one or two doses of intramuscular or intranasal whole inactivated influenza H5N1 virus vaccine in BALB/c mice. Serum samples and nasal washings were collected weekly post-vaccination and analysed using enzyme-linked immunosorbent assay (ELISA). Sera were also analysed by the haemagglutination inhibition (HI) assay. Antibody-secreting cells were measured in lymphocytes from spleen and bone marrow via enzyme-linked immunospot (ELISPOT). Splenocytes were stimulated in vitro, and T-helper profiles were measured through multiplex bead assay in the supernatants, or intracellularly by multiparametric flow cytometry. Both vaccine routes induced high HI titres following the second immunization (intramuscular = 370, intranasal = 230). Moreover, the intramuscular group showed significantly higher levels of serum IgG (P < 0.01), IgG1 (P < 0.01) and IgG2a (P < 0.01) following the second vaccine dose, while the intranasal group exhibited significantly higher levels of serum IgA (P < 0.05) and local IgA (P < 0.01) in the nasal washings. Also, IgA antibody-secreting cells were found in significantly higher numbers in the intranasal group in both the spleen (P < 0.01) and the bone marrow (P < 0.01). Moreover, Th1 (TNF-α, IL-2, IFN-γ) and Th2 (IL-4, IL-5, IL-10) cytokines were expressed by both groups, yet only the intranasal group expressed the Th17 marker IL-17. As the intranasal vaccines induce local IgA and are easily administered, we suggest the intranasally administered whole virus vaccine as a promising candidate for a pandemic H5N1 vaccine.