Systemic and Mucosal Immune Responses Induced by Adenoviral-Vectored Consensus H5 Influenza A Vaccines in Mice and Swine

Background/Objectives: The continued evolution and cross-species transmission of clade 2.3.4.4b H5Nx highly pathogenic avian influenza (HPAI) viruses underscores the need for broadly protective vaccines in swine, a key intermediary host. This study aimed to evaluate systemic and mucosal immune responses elicited by adenoviral-vectored (Ad) vaccines encoding a centralized consensus hemagglutinin antigen (H5CC) in mice and swine. Methods: We constructed H5CC-based vaccines that were delivered using replication-defective (Ad5 and Ad6) and replication-competent (Ad28 and Ad48) human adenoviral vectors. Using a serotype-switched prime-boost strategy, vaccines were delivered intramuscularly (IM) or intranasally (IN) in mice and swine. We determined humoral, mucosal, and cell-mediated immune responses by hemagglutination inhibition (HI), microneutralization assay (MNA), ELISA, and IFN- ELISpot. Protective efficacy was evaluated by lethal H5N1 challenge in mice. Results: All vaccines strategies and routes induced significant levels of anti-H5 immunity. However, Ad5/Ad6 IM immunization elicited strong systemic IgG and MNA titers and robust T cell responses. IN delivery with Ad5/Ad6 induced superior mucosal IgA levels in lungs and nasal secretion. In swine, Ad5/Ad6 IM conferred the highest MNA titer and T cell responses, while the IN route enhanced mucosal IgA. The Ad28/Ad48 vaccines induced immunity in a similar pattern as compared to the Ad5/Ad6 strategy, but to a slightly lesser degree, in general. The commercial H1/H3 swine influenza vaccine failed to elicit cross-protective immunity. All H5CC vaccinated mice survived lethal H5N1 challenge without weight loss. Conclusions: Adenoviral-vectored H5CC vaccines elicit broad, cross-clade immunity with route-dependent immune profiles. IM vaccination is optimal for systemic and cellular responses, while IN delivery enhances mucosal immunity. These findings support the advancement of adenoviral platforms for influenza control in swine and pandemic preparedness.