DNA Vaccine Encoding a Modified Hemagglutinin Trimer of Avian Influenza A Virus H5N8 Protects Mice from Viral Challenge

Abstract

The development of a safe and effective vaccine against avian influenza A virus (AIV) H5N8 is relevant due to the widespread distribution of this virus in the bird population and the existing potential risk of human infection, which can lead to significant public health concerns. Here, we developed an experimental pVAX-H5 DNA vaccine encoding a modified trimer of AIV H5N8 hemagglutinin. Immunization of BALB/c mice with pVAX-H5 using jet injection elicited high titer antibody response (the average titer in ELISA was 1 × 10⁵), and generated a high level of neutralizing antibodies against H5N8 and T-cell response, as determined by ELISpot analysis. Both liquid and lyophilized forms of pVAX-H5 DNA vaccine provided 100% protection of immunized mice against lethal challenge with influenza A virus A/turkey/Stavropol/320-01/2020 (H5N8). The results obtained indicate that pVAX-H5 has good opportunities as a vaccine candidate against the influenza A virus (H5N8).

Keywords: DNA vaccines; immunogenicity; influenza A (H5N8) virus; jet injection.

Figure 1

Design of the modified HA amino acid sequence. (a) Scheme for designing the amino acid sequence of influenza A virus (H5N8) hemagglutinin. (b) Modeling the structure of influenza A virus (H5N8) hemagglutinin. The different structural elements are shown in different colors: HA1 subunit (light brown), HA2 subunit (blue), linker (rose) and T4 fibritin trimerization domain (grey).

Figure 2

Expression of the hemagglutinin gene. (a) Electropherogram of RT-PCR products in 1% agarose gel: lane 1—1 kb DNA ladder (SibEnzyme); lane 2—use of total RNA pVAX-H5 as a template; lane 3—use of total RNA from pure cells as a template; (b) analysis of HA/H5 protein production in HEK293 cells by Western blotting using serum of a ferret immunized with influenza A (H5N8) virus: Lane 1—lysate of untransfected HEK293 cells; Lane 2—culture medium collected from untransfected HEK293 cells; Lane 3—molecular weight marker Precision Plus Protein Dual Color Standards (Bio-rad, Hercules, CA, USA); Lane 4—lysate of HEK293 cells transfected with plasmid pVAX-H5; Lane 5—culture medium collected from HEK293 cells transfected with the pVAX-H5 plasmid; Lane 6—recombinant hemagglutinin HA/H5 of influenza A virus (H5N8) (K+) [43].

Figure 3

Analysis of humoral and cellular response to DNA vaccine pVAX-H5. (a) Scheme of mouse immunization. (b) Reciprocal titer (dilution) of hemagglutinin-specific antibodies detected in immune sera using ELISA. (c) The neutralization activity of immune sera against influenza virus A/turkey/Stavropol/320-01/2020 (H5N8). Reciprocal titer values are provided in the plot. (d) ELISpot results. Number of splenocytes expressing IFN-γ in response to stimulation with a pool of specific peptides, determined by ELISpot. Each bar represents the average number of IFN-γ spot-forming cells (SFCs) per million splenocytes stimulated. (e) The representative images of ELISpot wells (top row: splenocytes not stimulated with peptides; bottom row: splenocytes stimulated with peptide pool or a mitogen). pVAX-H5—a group of animals immunized with the liquid form of the pVAX-H5 DNA vaccine; pVAX—group of animals immunized with the pVAX1 vector; intact—a group of animals that have not been subjected to any manipulation. In panels (b–d), data are provided as median with range. Significance was calculated using non-parametric one-way Kruskal–Wallis analysis of variance with correction for multiple comparisons and Dunn’s statistical hypothesis test (** p < 0.01, *** p < 0.001).

Figure 4

Immunogenic and protective properties of DNA vaccine pVAX-H5. (a) Scheme of mice immunization and virus challenge. (b) ELISA results. Reciprocal titer values are provided in the plot. Data are provided as median with range. Significance was calculated using non-parametric one-way Kruskal–Wallis analysis of variance with correction for multiple comparisons and Dunn’s statistical hypothesis test (** p < 0.01, ns: no significance). (c) Survival curves of immunized animals after infection with the influenza virus strain A/Astrakhan/3212/2020 (H5N8). Survival function modeling was performed using the Kaplan–Meier multiplier estimator, and comparison of survival with the control group was performed using the Mantel–Cox test. pVAX-H5—a group of animals immunized with the liquid form of the pVAX-H5 DNA vaccine; pVAX-H5, lyoph., sucrose—a group of animals immunized with a lyophilized form of DNA vaccine pVAX-H5; inactivated H5N8—a group of animals immunized with the inactivated H5N8 virus; pVAX—group of animals immunized with the pVAX1 vector; intact—a group of animals that have not been subjected to any manipulation.