Sublingual Immunization with Adenovirus F Protein-based Vaccines Stimulates Protective Immunity Against Botulinum Neurotoxin A Intoxication

Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the β-trefoil domain of C-terminus heavy chain (Hcβtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcβtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcβtre-Ad2F alone also induced elevated antibody production in contrast to Hcβtre alone. Plasma from Hcβtre + CT- and Hcβtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcβtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcβtre + CT-immunized mice, which only showed ∼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.