Systemic immune responses to an inactivated, whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens

Vaccine. 2015 Jul 31;33(32):3947-52. doi: 10.1016/j.vaccine.2015.06.043. Epub 2015 Jun 16.


Commercial vaccines against avian influenza viruses (AIV) in chickens consist mainly of inactivated AIV, requiring parenteral administration and co-delivery of an adjuvant. Limitations in T helper 1 or T helper 2 biased responses generated by these vaccines emphasize the need for alternative, more efficacious adjuvants. The Toll-like receptor (TLR) 21 ligand, CpG oligodeoxynucleotides (ODN), has been established as immunomodulatory in chickens. Therefore, the objective of this study was to investigate the adjuvant potential of high (20μg) and low (2μg) doses of CpG ODN 2007 (CpG 2007) and CpG ODN 1826 (CpG 1826) when administered to chickens with a formalin-inactivated H9N2 AIV. Antibody responses in sera were evaluated in 90 specific pathogen free (SPF) chickens after intramuscular administration of vaccine formulations at 7 and 21 days post-hatch. Antibody responses were assessed based on haemagglutination inhibition (HI) and virus neutralization (VN) assays; virus-specific IgM and IgY antibody responses were evaluated by ELISA. The results suggest that the vaccine formulation containing low dose CpG 2007 was significantly more effective at generating neutralizing (both HI and VN) responses than formulations with high or low doses of CpG 1826 or high dose CpG 2007. Neutralizing responses elicited by low dose CpG 2007 significantly exceeded those generated by a squalene-based adjuvanted vaccine formulation during peak responses. A significantly higher IgM response was elicited by the formulation containing low dose CpG 2007 compared to high and low doses of 1826. Although the low dose of CpG 2007 elicited a higher IgY response than CpG 1826, the difference was not statistically significant. In conclusion, 2μg of CpG 2007 is potentially promising as a vaccine adjuvant when delivered intramuscularly with inactivated H9N2 virus to chickens. Future studies may be directed at determining the mucosal antibody responses to the same vaccine formulations.

Keywords: Antibody response; Chickens; CpG oligodeoxynucleotides; ELISA; H9N2 subtype; Haemagglutination inhibition; Low pathogenic avian influenzavirus; Toll-like receptor 21; Virus neutralization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adjuvants, Immunologic / administration & dosage*
  • Animals
  • Antibodies, Neutralizing / blood*
  • Antibodies, Viral / blood*
  • Chickens
  • Enzyme-Linked Immunosorbent Assay
  • Hemagglutination Inhibition Tests
  • Immunoglobulin M / blood
  • Immunoglobulins / blood
  • Influenza A Virus, H9N2 Subtype / immunology*
  • Influenza Vaccines / administration & dosage
  • Influenza Vaccines / immunology*
  • Influenza in Birds / immunology
  • Influenza in Birds / prevention & control*
  • Influenza in Birds / virology
  • Injections, Intramuscular
  • Neutralization Tests
  • Oligodeoxyribonucleotides / administration & dosage*
  • Treatment Outcome
  • Vaccines, Inactivated / administration & dosage
  • Vaccines, Inactivated / immunology


  • Adjuvants, Immunologic
  • Antibodies, Neutralizing
  • Antibodies, Viral
  • CPG-oligonucleotide
  • IgY
  • Immunoglobulin M
  • Immunoglobulins
  • Influenza Vaccines
  • Oligodeoxyribonucleotides
  • Vaccines, Inactivated