Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses

Abstract

Both H5N1 and H7N9 subtype avian influenza viruses cause enormous economic losses and pose considerable threats to public health. Bivalent vaccines against both two subtypes are more effective in control of H5N1 and H7N9 viruses in poultry and novel egg-independent vaccines are needed. Herein, H5 and H7 virus like particle (VLP) were generated in a baculovirus expression system and a bivalent H5+H7 VLP vaccine candidate was prepared by combining these two antigens. Single immunization of the bivalent VLP or commercial inactivated vaccines elicited effective antibody immune responses, including hemagglutination inhibition, virus neutralizing and HA-specific IgG antibodies. All vaccinated birds survived lethal challenge with highly pathogenic H5N1 and H7N9 viruses. Furthermore, the bivalent VLP significantly reduced viral shedding and virus replication in chickens, which was comparable to that observed for the commercial inactivated vaccine. However, the bivalent VLP was better than the commercial vaccine in terms of alleviating pulmonary lesions caused by H7N9 virus infection in chickens. Therefore, our study suggests that the bivalent H5+H7 VLP vaccine candidate can serve as a critical alternative for the traditional egg-based inactivated vaccines against H5N1 and H7N9 avian influenza virus infection in poultry.

Keywords: H5N1 subtype; H7N9 subtype; avian influenza virus; baculovirus expression system; bivalent vaccine; chickens; virus-like particle.

Figure 1

Generation and characterization of H5 and H7 VLP. (A) Recombinant baculovirus (rBVs) rBac-H5, rBac-H7, rBac-NA, and rBac-M1 were successfully identified using immunofluorescence assay (IFA) by HA, NA, and M1-specific antibody in Sf9 cells. (B) To generate H5 VLP, Sf9 suspension cells were co-infected with rBac-H5, rBac-NA, and rBac-M1 at a MOI ratio of 1:1:1. To generate H7 VLP, Sf9 suspension cells were co-infected with rBac-H7, rBac-NA, and rBac-M1 at the same MOI ratio. At 72 h p.i., cell supernatant was harvested for collection of the H5 and H7 VLP. A transmission electron microscope (EM) was used for observation of the morphology and size of the H5 and H7 VLP.

Figure 2

The bivalent H5+H7 VLP vaccine induces efficient HI and VN antibodies. (A) To assess the immunogenicity of the vaccine candidate, chickens were intramuscularly (i.m.) immunized with 15 μg of the bivalent H5+H7 VLP or 0.3 mL of the commercial trivalent (H5: Re-11; H5: Re-12; H7: Re-3) vaccine. (B) HI titers at Week 2 and 3 p.v. against H5N1 TT3 virus. (C) HI titers at Week 2 and 3 p.v. against H7N9 GD15 virus. (D) VN antibody against 100 TCID₅₀ of H5N1 TT3 virus. (E) VN antibody against 100 TCID₅₀ of H7N9 GD15 virus. The detection limit was below 4 log₂ for the HI assay and below 10 for the VN assay.

Figure 3

The bivalent H5+H7 VLP vaccine stimulates strong IgG antibody. Chickens were i.m. immunized with 15 μg of the bivalent H5+H7 VLP or 0.3 mL of the commercial trivalent (H5: Re-11; H5: Re-12; H7: Re-3) vaccine. The IgG antibody at Week 3 p.v. was determined by ELISA. (A) IgG antibody using purified HA protein of the TT3 virus as coated antigen. (B) IgG antibody using purified HA protein of the A/Chicken/Anhui/AH13/2013 (AH13) virus as coated antigen. (C) Area under the curve (AUC) of the IgG antibody using purified HA protein of the TT3 virus as coated antigen. (D) AUC of the IgG antibody using purified HA protein of the AH13 virus as coated antigen.

Figure 4

The bivalent H5+H7 VLP vaccine confers good protection against the lethal H5N1 virus. Chickens were i.m. immunized with 15 μg of the bivalent H5+H7 VLP or 0.3 mL of the commercial trivalent (H5: Re-11; H5: Re-12; H7: Re-3) vaccine. (A) Survival rates of the birds after challenged with 10^6.0 EID₅₀ of the H5N1 TT3 virus. (B) Virus titration in laryngotracheal swabs on Day 2 and 4 p.c. (C) Virus titration in cloacal swabs on Day 2 and 4 p.c. Viral titers in the heart (D), cecum (E), lung (F), and spleen (G) of the birds on Day 3 and 5 p.c. Viral titers were determined by measuring EID₅₀. The detection limit was below 10^1.0 EID₅₀/0.1 mL for swabs and 10^1.48 EID₅₀/g for organs.

Figure 5

The bivalent H5+H7 VLP vaccine confers good protection against the lethal H7N9 virus. Chickens were i.m. immunized with 15 μg of the bivalent H5+H7 VLP or 0.3 mL of the commercial trivalent (H5: Re-11; H5: Re-12; H7: Re-3) vaccine. (A) Survival rates of the birds after challenged with 10^6.0 EID₅₀ of the H7N9 GD15 virus. (B) Virus titration in laryngotracheal swabs on Day 2 and 4 p.c. (C) Virus titration in cloacal swabs on Day 2 and 4 p.c. Viral titers in the heart (D), cecum (E), lung (F), and spleen (G) of the birds on Day 3 and 5 p.c. Viral titers were determined by measuring EID₅₀. The detection limit was below 10^1.0 EID₅₀/0.1mL for swabs and 10^1.48 EID₅₀/g for organs.

Figure 6

The bivalent H5+H7 VLP vaccine inhibits lung pathological changes after the lethal H5N1 virus challenge. (A) Hematoxylin and eosin (H & E) staining results of the birds' lung on Day 2 and 4 post-H5N1 virus challenge. Black triangle indicates dilatation in the pulmonary chamber or bronchial; white triangle arrow stands for infiltration of fiber cells, detached epithelial cell mucus, and erythrocytes in the parabrochus. (B) Scores of the overall histopathologic changes in the bird's lung post-H5N1 virus challenge. The specific information concerning the scoring criteria was listed in the Materials and Method section.

Figure 7

The bivalent H5+H7 VLP vaccine alleviates lung injury after the lethal H7N9 virus challenge. (A) H & E staining results of the bird's lung on Day 2 and 4 post-H7N9 virus challenge. Black triangle indicates dilatation in the pulmonary chamber or bronchial; white triangle arrow stands for infiltration of fiber cells, detached epithelial cell mucus, and erythrocytes in the parabrochus; quad star means lymphocytes infiltration in the lung chamber. (B) Scores of the overall histopathologic changes in the bird's lung post-H7N9 virus challenge. The specific information concerning the scoring criteria was listed in the Materials and Method section.