Influenza virus-like particles harboring H9N2 HA and NA proteins induce a protective immune response in chicken

Abstract

Background: Avian influenza viruses represent a growing threat of an influenza pandemic. The co-circulation of multiple H9N2 genotypes over the past decade has been replaced by one predominant genotype-G57 genotype, which displays a changed antigenicity and improved adaptability in chickens. Effective H9N2 subtype avian influenza virus vaccines for poultry are urgently needed.

Objective: In this study, we constructed H9N2 subtype avian influenza virus-like particle (VLP) and evaluated its protective efficacy in specific pathogen-free (SPF) chickens to lay the foundation for developing an effective vaccine against influenza viruses.

Methods: Expression of influenza proteins in VLPs was confirmed by Western blot, hemagglutination inhibition (HI), and neuraminidase inhibition (NI). The morphology was observed by electron microscopy. A group of 15 three-week-old SPF chickens was divided into three subgroups of five chickens immunized with VLP, commercial vaccine, and PBS. Challenge study was performed to evaluate efficacy of VLP vaccine.

Results and conclusions: The hemagglutinin (HA) and neuraminidase (NA) proteins were co-expressed in the infected cells, self-assembled, and were released into the culture medium in the form of VLPs of diameter ~80 nm. The VLPs exhibited some functional characteristics of a full influenza virus, including hemagglutination and neuraminidase activity. In SPF chickens, the VLPs elicited serum antibodies specific for H9N2 and induced a higher HI titer (as detected by a homologous antigen) than did a commercial H9N2 vaccine (A/chicken/Shanghai/F/1998). Viral shedding from VLP vaccine subgroup was reduced compared with commercial vaccine subgroup and control subgroup.

Keywords: H9N2; chickens; vaccine; virus-like particles.

Figure 1

Preparation of H9N2 subtype virus‐like particle (VLPs). (A) Detection of the hemagglutinin (HA) and neuraminidase proteins within the VLPs using SDS‐PAGE and Western blotting. The influenza proteins are indicated by arrows. M: molecular weight standards. (B) VLPs harvested 72 h after transfection of Sf9 cells. The hemagglutination test was performed by exposing chicken erythrocytes to VLPs. The arrow indicates the HA titer

Figure 2

(A) Electron micrograph of a negatively stained H9N2 virus‐like particle (VLP). (B) A neuraminidase (NA) assay using either VLPs or H9N2 shows that both featured an hemagglutinin titer of 2⁶. The supernatant of Sf9 cells was used as a negative control. Neuraminidase activity was expressed as unit/L

Figure 3

Virus titers in H9N2‐challenged chickens previously inoculated with either virus‐like particles, a commercial vaccine (A/chicken/Shanghai/F/1998 strain), or PBS. Each group of five chickens was vaccinated intramuscularly twice (separated by 14 d), then challenged intranasally with 10⁶ EID ₅₀ H9N2. Tracheal and cloacal swabs were collected from each chicken to determine the extent of replication of the influenza virus, at 1, 2, 3, 5, and 7 d postchallenge (dpc), respectively. The virus shedding was quantified by the 50% egg infectious dose (EID ₅₀), and EID ₅₀ was determined by the Reed and Muench method. No virus was detectable in the cloacal swabs. Virus was present in the tracheal swabs of both nonvaccinated and vaccinated chickens at 1, 2, 3, and 5 dpc. No virus was detected in all tracheal swabs at 7 dpc. (Note: Data marked *differentiated significantly, P < .05)