A Comparison of Toll-Like Receptor 5 and 21 Ligands as Adjuvants for a Formaldehyde Inactivated H9N2 Avian Influenza Virus Vaccine in Chickens

Low pathogenic avian influenza virus (AIV) infection in chickens can result in economic losses and has impacts on human health. Poultry vaccination is a tool that can be used to decrease infection and transmission of AIVs. Prior research has demonstrated that Toll-like receptor (TLR) ligands can act as vaccine adjuvants and their addition to inactivated AIV vaccines can enhance immune responses elicited in chickens. The objective of this study was to compare the adjuvant capabilities of TLR5 ligand (flagellin) and TLR21 ligand (CpG ODN 2007) administered either alone or in combination with an intramuscular formaldehyde inactivated H9N2 whole virus vaccine in chickens. Along with the inactivated virus, chickens were administered either a single dose of CpG ODN 2007 (2 or 10 μg), flagellin (0.4 or 2 μg), or a combination of both ligands. An additional group received AddaVax™, an oil emulsion style adjuvant. Chickens were vaccinated twice and serum and lachrymal samples were collected weekly following the primary vaccination, and antibody-mediated immune responses were quantified. Results showed that vaccines containing CpG ODN 2007 induce significantly greater systemic and lachrymal antibody responses than vaccines containing flagellin or AddaVax. Combinations of flagellin and CpG ODN 2007 did not demonstrate inhibitory, additive, or synergistic effects on systemic or lachrymal antibody-mediated immune responses. Additionally, for both flagellin and CpG ODN 2007, a fivefold higher dose of each did not induce significantly higher antibody-mediated immune responses compared with the lesser dose. Future studies should examine the induction of cell-mediated immune responses when flagellin, CpG ODN 2007, or other TLR ligands are administered either alone or combined as adjuvants for inactivated H9N2 AIV vaccines.