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.

Induction of immune response in chickens primed in ovo with an inactivated H9N2 avian influenza virus vaccine.

OBJECTIVE: Infection of chickens with low pathogenic avian influenza virus, such as H9N2 virus, culminates in decreased egg production and increased mortality and morbidity if co-infection with other respiratory pathogens occurs. We have previously observed the induction of antibody- and cell-mediated immune responses after intramuscular administration of an H9N2 beta-propiolactone inactivated virus vaccine to chickens. Given the fact that in ovo vaccination represents a practical option for vaccination against H9N2 AIV in chickens, in the current study, we set out to characterize immune responses in chickens against a beta-propiolactone inactivated H9N2 virus vaccine after primary vaccination in ovo on embryonic day 18, and secondary intramuscular vaccination on day 14 post-hatch. We also included the Toll-like receptor 21 ligand, CpG ODN 2007, and an oil emulsion adjuvant, AddaVax™, as adjuvants for the vaccines. RESULTS: Antibody-mediated immune responses were observed after administering the secondary intramuscular vaccine. Cell-mediated immune responses were observed in chickens that received the beta-propiolactone inactivated H9N2 virus combined with AddaVax™. Our results demonstrate that adaptive immune responses can be induced in chickens after a primary in ovo vaccination and secondary intramuscular vaccination.

Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens.

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.