A Model H5N2 Vaccine Strain for Dual Protection Against H5N1 and H9N2 Avian Influenza Viruses

Abstract

Background/Objective: Highly pathogenic (HP) H5Nx and low-pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose global threats to the poultry industry and public health, highlighting the critical need for a dual-protective vaccine. Methods: In this study, we generated a model PR8-derived recombinant H5N2 vaccine strain with hemagglutinin (HA) and neuraminidase (NA) genes from clade 2.3.2.1c H5N1 and Y439-like H9N2 viruses, respectively. To enhance the immunogenicity of the recombinant H5N2 vaccine strain, N-glycans of the HA2 subunit, NA, and M2e were modified. Additionally, we replaced M2e with avian M2e to enhance the antigenic homogeneity of AIVs for better protection. We also replaced PR8 PB2 with 01310 PB2, which is the PB2 gene derived from an LP H9N2 avian influenza virus, to eliminate pathogenicity in mammals. The productivity of the model vaccine strain (rvH5N2-aM2e-vPB2) in embryonated chicken eggs (ECEs), its potential risk of mammalian infection, and the immunogenicity associated with different inactivation methods (formaldehyde (F/A) vs. binary ethyleneimine (BEI)) were evaluated. Results: The rvH5N2-aM2e-vPB2 strain demonstrated high productivity in ECEs and exhibited complete inhibition of replication in mammalian cells. Furthermore, compared with using F/A inactivation, inactivation using BEI significantly enhanced the immune response, particularly against NA. This enhancement resulted in increased virus neutralization titers, supporting its efficacy for dual protection against H5Nx and H9N2 avian influenza viruses. Furthermore, we demonstrated that M2e-specific immune responses, difficult to induce with inactivated vaccines, can be effectively elicited with live vaccines, suggesting a strategy to enhance M2e immunogenicity in whole influenza virus vaccines. Conclusions: Finally, the successful development of the model rH5N2 vaccine strain is described; this strain provides dual protection, has potential applicability in regions where avian influenza is endemic, and can be used to promote the development of versatile H5N2 recombinant vaccines for effective avian influenza control.

Keywords: H9N2 avian influenza virus; N-glycosylation; NA immunity; binary ethylenimine inactivation; dual protection; highly pathogenic avian influenza virus.

Figure 1

Comparison of the replication efficiencies of the recombinant viruses in MDCK cells. MDCK cells were inoculated with recombinant H5N1 or H5N2 virus at an MOI of 0.001. After 1 h of incubation, the inoculum was replaced with fresh medium, and the supernatant was obtained at each time point (0, 24, 48, and 72 h). The viral titer was measured as the TCID₅₀/mL in the MDCK cells, and the results are presented as the means ± SDs of triplicate experiments. Statistical significance was analyzed by two-way ANOVA. The asterisk represents a significant difference between rH5N1 and the other groups (p < 0.001).

Figure 2

Comparison of the serum HI titers induced by recombinant virus vaccines inactivated by formaldehyde (F/A) or binary ethylenimine (BEI). Comparison of the HI titers at 1–4 weeks postvaccination. The serum samples were collected from SPF chickens (n = 5). (A) HI antibody responses against rH5N1 induced by vaccines inactivated with F/A or BEI. (B) HI antibody responses against 01310 (H9N2) induced by vaccines inactivated with F/A or BEI. The HI antibody response induced by the F/A-inactivated vaccine is represented by solid bars, while the response induced by the BEI-inactivated vaccine is represented by open bars. The data are presented as the means ± SD. Statistical significance was analyzed by two-way ANOVA and is denoted by asterisks (* p <0.01).

Figure 3

Comparison of the serum VN titers induced by recombinant virus vaccines inactivated by formaldehyde (F/A) or binary ethylenimine (BEI). Serum samples (n = 5) collected at 2, 3, and 4 weeks post-vaccination were utilized to conduct virus neutralization (VN) tests. (A) VN antibody responses against rH5N1 induced by vaccines inactivated with either F/A or BEI. (B) VN antibody responses against 01310 (H9N2) induced by vaccines inactivated with either F/A or BEI. We distinguished between the two vaccine groups in the bar graphs; the vaccines inactivated with F/A are represented by solid bars, whereas those inactivated with BEI are depicted by open bars. The data are presented as the means ± SD. Statistical significance was analyzed by two-way ANOVA and is denoted by asterisks (* p < 0.01).

Figure 4

Comparison of the serum NI titers induced by recombinant virus vaccines inactivated by formaldehyde (F/A) or binary ethylenimine (BEI). To compare the immunogenicity against NA, a neuraminidase inhibition (NI) assay was performed using the serum samples collected at week 3 post vaccination. The NA activity of the virus alone was set as 100%, and the relative reduction in the NA activity due to the serum was expressed as a percentage of the NA inhibition. (A) NA inhibition curves for each serum sample against rH5N1. (B) NA inhibition curves for each serum sample against 01310 (H9N2). The curves for the vaccine groups inactivated with F/A are shown as dashed lines, and those for the BEI-inactivated vaccine groups are shown as solid lines. The 50% inhibitory concentration (IC₅₀) against (C) rH5N1 or (D) 01310 (H9N2) is represented as the serum dilution titer that achieved 50% inhibition of NA activity. IC₅₀ values for the F/A-inactivated vaccine groups are shown as solid bars, and those for the BEI-inactivated vaccine groups are shown as open bars, presented as mean ± SD. Statistical significance was analyzed via one-way ANOVA, and the results are denoted by asterisks (* p < 0.01).

Figure 5

Comparison of the anti-M2e antibody levels induced by inactivated and live recombinant virus vaccines. Three weeks after inactivated vaccine administration, the serum samples collected from SPF chickens were used to evaluate the antibody responses against two distinct peptides, PR8 M2e and M2e (Av) (avian M2e), via ELISA. (A,B) IgG responses against M2e from vaccines inactivated with formaldehyde (F/A). (C,D) IgG responses against M2e from vaccines inactivated with BEI. (E,F) Antibody responses to M2e in mouse sera collected after inoculation with live viruses. Six-week-old female BALB/c mice (n = 5) were inoculated with 10⁴ EID₅₀ of two live viruses (rH5N2 and rvH5N2-aM2e) or a negative control (PBS). Two weeks post-inoculation, we evaluated the IgG responses against PR8 M2e and M2e (Av) (avian M2e) via ELISA. The data are presented as the means ± SD. Statistical significance was analyzed by two-way ANOVA and is denoted by asterisks (* p < 0.001. ns, not significant). The black asterisks indicate significant differences between the two vaccines, whereas the blue and light blue asterisks represent significant differences between the vaccines and the negative control.

Figure 6

Evaluation of the weight changes, survival rates, and lung viral titers in mice inoculated with rH5N2 and challenged with multiple viruses. Five 6-week-old female BALB/c mice inoculated with 10⁴ EID₅₀ of two live viruses (rH5N2, rvH5N2-aM2e) or PBS (negative) were intranasally challenged with 10⁶ EID₅₀ of the SNU50-5 (A/wild duck/Korea/SNU50-5/2009 (H5N1)), PR8 (A/Puerto Rico/8/1934 (H1N1)), or PR8-M (Av) (PR8 virus with avian M2e) virus at 2 weeks after inoculation. The genome composition of each challenge virus is illustrated, with the corresponding graph displayed at the top. The color coding represents the origin of each genome segment: blue indicates segments derived from the PR8 virus, while orange represents segments derived from the SNU50-5 virus, including the avian M2e sequence. Notably, the SNU50-5 virus incorporates the avian M2e sequence. Body weight changes (A–C) and survival rates (D–F) were monitored for 2 weeks after being challenged. Three days post-challenge, the mice (n = 3) were sacrificed, and the lung viral titer (G–I) was determined. The lung viral titers are presented as the means ± SD and were analyzed via one-way ANOVA (* p < 0.05. ns, not significant).