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.

Effects of ligands for Toll-like receptors 3, 4, and 21 as adjuvants on the immunogenicity of an avian influenza vaccine in chickens.

Avian influenza viruses (AIV) are of great concern to the worldwide community as well as the poultry industry. Although existing vaccines are successful in limiting the spread of the virus, these vaccines do not eliminate virus shedding into the environment. As a result, it is of great importance to enhance the efficacy of existing AIV vaccines. Therefore, the objective of the present study was to utilize the immunostimulatory Toll-like receptor ligands poly I:C, lipopolysaccharide (LPS), and CpG DNA motifs, either alone or in combination with each other, as adjuvants to enhance the immunogenicity of an inactivated AIV vaccine. Chickens were vaccinated twice, 14 days apart. Antibody-mediated responses were assessed by collected sera and lacrimal secretions, while cell-mediated immunity was assessed by stimulating splenocytes from vaccinated chickens in vitro with the vaccine antigen. The results suggest that CpG alone served as the best single-ligand adjuvant compared to poly I:C or LPS, as it significantly enhanced antibody-mediated responses, as determined by enzyme-linked immunosorbant assay. Furthermore, upon combining CpG with poly I:C, a robust antibody-mediated and cell-mediated immune response was elicited, resulting in an enhanced hemagglutination inhibition titer and splenocyte proliferation respectively. Future studies may be aimed at assessing the efficacy of the poly I:C and CpG combination adjuvant in protecting against AIV infection.