Avian influenza virus vaccines containing Toll-like receptors 2 and 5 ligand adjuvants promote protective immune responses in chickens.

Vaccination remains a useful means for the control of avian influenza viruses (AIV) in chickens. Current vaccines can protect chickens from morbidity and mortality. However, they do not eliminate virus shedding into the environment. Therefore, novel measures must be considered in order to enhance the immunogenicity of AIV vaccines, such as through the administration of immunostimulatory compounds. One such group of compounds is Toll-like receptor (TLR) ligands, such as bacterial flagellin, as well as synthetic lipopeptides such as Pam3CSK4. The objective of the present study was to assess the adjuvant potential of TLR2 and TLR5 ligands flagellin and Pam3 respectively. Chickens were vaccinated twice with an inactivated H4N6 AIV vaccine, 14 days apart. Antibody-mediated responses were assessed in sera and lacrimal secretions, while cell-mediated immune response was assessed by stimulating splenocytes from vaccinated chickens in vitro with the vaccine antigen. To evaluate vaccine efficacy, chickens were challenged with the H4N6 virus, and virus shedding was assessed on day 7 post-challenge. The results suggest that both ligands significantly enhanced antigen-specific IgY antibodies, while only the Pam3 adjuvant induced greater IgM and IgA antibody levels. Chickens receiving the flagellin adjuvant had significantly higher IgY responses, as well as significantly higher hemagglutination-inhibition antibody titers compared to the no adjuvant control. With respect to cell-mediated responses, splenocytes isolated from chickens that received either TLR ligand adjuvant proliferated in response to an in vitro stimulation with vaccine antigens. Lastly, chickens receiving vaccines containing either flagellin or Pam3 adjuvants were partially protected from an experimental AIV challenge and shed significantly less virus compared to controls. Future studies may be aimed at examining the efficacy of Pam3 and flagellin adjuvants for highly pathogenic AIV strains.