H9N2 avian influenza virus-like particle vaccine provides protective immunity and a strategy for the differentiation of infected from vaccinated animals

Abstract

In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.

Fig. 1

Mean serum HI titers (log 2) induced in SPF chickens after a single dose of vaccination. Sixty 6-week-old SPF chickens (10 chickens per group) were intramuscularly immunized with influenza H9 HA VLPs (2, 5, 10, or 20 μg total protein) or inactivated whole viral vaccine (0.5 ml dose) commercially manufactured HI titers against the homologous antigen [A/chicken/Korea/310/2001(H9N2)] were determined at 3 weeks after vaccination. ISA70: an adjuvant; control: ISA70 adjuvant only without vaccine. ***p < 0.001 by ANOVA with Tukey–Kramer post-test compared to other groups.

Fig. 2

NP serum antibody levels induced by VLP vaccine, inactivated whole virus vaccine, or AIV infection by NP ELISA. SPF chickens were immunized with 20 μg of VLP antigen with ISA70 adjuvant (A) or whole virus inactivated vaccine (B). Antibody levels were determined by NP-cELISA at 3 weeks post vaccination. AIV infected (C) sera were collected at 2 weeks post H9N2 virus challenge. Each bar represents the NP specific antibody value of each chicken.