DNA Vaccine Encoding a Modified Hemagglutinin of Influenza A/H5N8 Virus Protects Mice from Infection with a Lethal Dose of Influenza A/H5N1 Virus

1. Introduction

Influenza A and B viruses cause seasonal epidemics, but only influenza A viruses have caused pandemics in the past with high mortality and economic costs [1,2]. The wide host range provides influenza A viruses with a high probability of genetic reassortment, resulting in zoonotic viruses that can spread to the human population. In particular, highly pathogenic avian influenza (HPAI) H5 viruses also have pandemic potential [3,4,5,6,7,8,9,10,11]. According to WHO, HPAI H5 viruses have caused 904 human infections, including 464 deaths [12]. In 2020, a new H5N1 virus of clade 2.3.4.4b emerged as a result of reassortment, which by now has become the most common variant of the highly pathogenic H5 influenza virus [13]. HPAIV H5N1 clade 2.3.4.4b have demonstrated an enhanced ability to cross species barriers and infect several mammalian species, including humans [14,15]. The host range of the virus has recently been expanded to include ruminants, particularly dairy cattle in the United States [16,17]. In addition, several cases of HPAIV H5N1 infection in humans have also been reported among dairy farm workers [18,19,20,21]. This is believed to be the first confirmed case of mammal-to-human transmission of HPAIV H5N1. In addition, it has been shown that a few amino acid substitutions (e.g., substitutions of Gln for Leu at position 226 in hemagglutinin [22]) are sufficient for A/H5 viruses to become airborne between mammals [23].

The continuous emergence of new influenza virus strains increases the need for the development of new vaccines and for improved vaccine technologies that provide an effective immune response and can be optimized for rapid and scalable production [11,24]. Currently, FDA-approved and licensed whole-virus and live attenuated human influenza A virus vaccines are mainly produced using chicken embryos, and the manufacturing process can take up to 9 months [25]. Nucleic acid-based vaccines (DNA and mRNA vaccines) are one of the promising approaches to vaccine development. These vaccines are non-infectious and have an ability to induce humoral and cellular immune responses. The production of such vaccines is uniform, rapid and scalable [26,27]. These advantages make them suitable for the development of vaccines against influenza, including highly pathogenic avian influenza with pandemic potential [28,29,30,31,32,33,34,35,36,37,38]. One of the disadvantages of DNA vaccines is low immunogenicity, which can be improved by the delivery method. Jet injection with customized nozzles is a promising method that provides high immunogenicity with minimal side effects [38,39,40,41,42,43].

Thus, given the high incidence of infection and mortality in mammals [44,45,46,47,48], as well as the high variability of circulating HPAI H5 virus strains, it is necessary to conduct studies aimed at investigating the activity of existing and emerging vaccines against different HPAI H5 strains of clade 2.3.4.4b [49]. In the study [50], it was demonstrated that licensed H5N1 vaccines derived from HPAI H5N1 virus strains (A/Vietnam, clade 1 and A/Indonesia, clade 2.1) held in reserve for a pandemic in the United States were able to induce cross-neutralizing antibodies against HPAI clade 2.3.4.4b A/Astrakhan/3212/2020 (H5N8) virus. Previously, we developed DNA vaccine pVAX-H5 and investigated its immunogenicity against homologous strain A/Astrakhan/3212/2020 (H5N8) at a dose of 100 μg [48]. This study presents the results of the dose-dependent effect of DNA vaccine pVAX-H5 in the range of 1 to 200 μg and the cross-reactivity of sera of animals immunized with 100 µg of DNA vaccine in microneutralization assay against strains A/chicken/Astrakhan/321-05/2020 (H5N8), A/chicken/Komi/24-4V/2023 (H5N1), A/chicken/Khabarovsk/24-1V/2022 (H5N1) and A/chicken/Voronezh/193-1V/2024 (H5N1). The protective properties of the DNA vaccine against homologous strain A/Astrakhan/3212/2020 (H5N8) and heterologous strain A/chicken/Khabarovsk/24-1V/2022 (H5N1) were also demonstrated.

2. Materials and Methods

2.1. Strains of Viruses, Bacteria, Cell Cultures

Influenza A/Astrakhan/3212/2020 (H5N8), A/chicken/Astrakhan/321-05/2020 (H5N8), A/chicken/Khabarovsk/24-1V/2022 (H5N1), A/chicken/Voronezh/193-1V/2024 (H5N1), A/turkey/Stavropol/320-01/2020 (H5N8), A/chicken/Komi/24-4V/2023 (H5N1) viruses (FBRI SRC VB «Vector», Rospotrebnadzor) were used. Antigenic characterization of the viruses according to hemagglutination inhibition (HI) assay is presented in Table 1.

Plasmid DNA was obtained using E. coli Stbl3 strain (F′ proA+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 ∆(mrr-hsdRMS-mcrBC)).

Microneutralization assay was performed using the Madin-Darby canine kidney (MDCK) cell culture (Cell Culture Collection of FBRI SRC VB «Vector», Rospotrebnadzor).

2.2. Development of DNA Vaccine pVAX-H5

The design, development and production of the DNA vaccine pVAX-H5 is described in our previous work [51].

2.3. Experimental Design

The Guide for the Care and Use of Laboratory Animals was used for animal experiments. The Animal Care and Use Committee (IACUC) at the State Research Center of Virology and Biotechnology “Vector” established the protocols for work with animals (BEC Protocol No. 1 of 03/21/2023).

Female BALB/c mice (16-18 g) were used for immunization. Immunization preparations were dissolved in 50 μL of physiological solution and were injected into the area of the left hind paw by jet injection using a Comfort-IN injector (Australia) with individual nozzles. A detailed description of the immunization protocol using jet injection is described in our previous work [51].

The experiment consisted of two parts. The first part of the experiment was to assess the humoral response to the different doses of the experimental DNA vaccine. 7 groups (6 animals in each group) were involved in the study. Groups 1-5 were injected with 1, 10, 50, 100 and 200 μg of pVAX-H5 solution in saline, respectively; Group 6 was injected intramuscularly with 50 μg of recombinant hemagglutinin in complex with incomplete Freund's adjuvant as a positive control; Group 7 included the intact animals. Immunization was performed twice with an interval of 21 days. 14 days after the second immunization, blood was taken from the retrobulbar sinus of the animal’s eye to analyse the humoral response. Animals were taken out of the experiment by the cervical dislocation method.

The second part of the experiment was to assess the protective properties of the experimental DNA vaccine. 4 groups (10 animals in each group) were involved in the study. Groups 1-2 were injected with 100 μg of pVAX-H5 solution in saline; Groups 3-4 were injected with 100 μg of pVAX1 in saline. Immunization was performed twice with an interval of 21 days. 14 days after the second immunization, blood was taken from the retro-orbital sinus.

2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was performed on the immunosorbent of recombinant hemagglutinin of influenza virus A/turkey/Stavropol/320-01/2020 (H5N8) (FBRI SRC VB «Vector») [52]. A detailed description of the ELISA protocol is described in our previous work [51].

2.5. HI and Microneutralization Assays

The HI assay was carried out as described in [52]. Standardized influenza virus was prepared by diluting each virus strain to a final HA titer of 4 HA units/25 µl. The animal sera were treated with Receptor-Destroying Enzyme (RDE) and heat-inactivated prior to the assay. Then 2-fold dilutions of serum were mixed with 25 µl of standardized influenza virus (each well contained a final volume of 50 µl). The mixture was incubated at room temperature for 30 minutes. Then 50 µl of 0.5% turkey erythrocyte suspension was added to each well. The assay was incubated at 4-8°C until hemagglutination occurred in the control.

Microneutralization assay was performed as described in [52]. Briefly, each standardized virus contained 100 TCID₅₀/100 µL. Two-fold dilutions of serum (200 µL) were mixed with 200 µL of standardized influenza virus, the suspensions were incubated for 1 h at 37°C, 5% CO2. Non-immune mouse sera were used as the negative controls. Ferret reference sera (SRC VB Vector, Koltsovo, Russia) were used as the positive controls. Then, 200 µL of the suspension was added to the wells of culture plates with MDCK-SIAT1 cells. After 60 minutes, the inoculum was removed and the cells were washed with culture medium. The cells were cultured for 3 days in Opti-MEM I medium with 1 μg/mL TPCK-trypsin (Sigma-Aldrich) at 37°C, 5% CO2. After that, cells were stained with crystal violet solution, washed with water and analyzed using an Agilent BioTek Cytation 5 multi-mode cell visualization reader (Thermo Fisher Scientific). All analysis were repeated in three replicates. The titer was taken as the serum dilution at which 50% of the cells survived. All negative controls had less than 5% surviving cells.

2.6. Challenge Studies

All challenge studies were carried out in accordance with the requirements of SanPiN3.3686-21 “Sanitary and Epidemiologic Requirements for the Prevention of Infectious Diseases” and Directive 2010/63/EC of the European Parliament and of the Council of the European Union of September 22, 2010 on the protection of animals used for research purposes.

Fourteen days after the second immunization, mice were intranasally infected with influenza A/Astrakhan/3212/2020 (H5N8) and A/chicken/Khabarovsk/24-1V/2022 (H5N1) viruses at a dose of 50 MLD50. Infection was carried out under anesthesia using a mixture of tiletamine and zolazepam and xylazine hydrochloride. After infection, the mice were monitored daily for 14 days, recording any manifestation of clinical symptoms of the disease, such as dishevelment, decreased body temperature, weight loss, neurological disorders and cases of death. If severe conditions that could lead to the death of the animal occurred, such as loss of more than 20% of initial weight or lethargy, they were euthanized by cervical dislocation. The remaining mice were humanely killed in the same manner at the end of the experiment.

2.7. Statistical Analysis

GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis of the data obtained. Quantitative data are provided as median with range and analyzed using nonparametric tests. The Mann-Whitney test was used in case of two independent groups. Kaplan-Meier multiplier estimator was used to construct the survival curve, and the Mantel-Cox test was used to compare the survival rates of the experimental and control groups.

3. Results

DNA vaccine pVAX-H5 encoding a modified hemagglutinin of influenza virus A/turkey/Stavropol/320-01/2020 (H5N8) was previously developed and its immunogenic properties were investigated [51]. In this study, the first step was to analyze the immunogenicity of different doses of DNA vaccine pVAX-H5. BALB/c mice were immunized with 1, 10, 50, 100 or 200 μg of pVAX-H5 twice at 3-week intervals using jet injection (Figure 1a). At 2 weeks after the second immunization, serum samples were obtained and analyzed by ELISA. All doses of vaccine pVAX-H5 induced the formation of specific antibodies to recombinant HA of influenza virus A/turkey/Stavropol/320-01/2020 (H5N8) (Figure 1b). The mean median titers for different doses were: 1 μg - 1:12150, 10 μg - 1:36450, 50 μg - 1:36450, 100 μg - 1:72900, and 200 μg - 1:36450. All vaccine doses also induced the formation of neutralizing antibodies against the corresponding A/turkey/Stavropol/320-01/2020 (H5N8) virus strain (Figure 1c). Based on these data, a dose of 100 μg of pVAX-H5 DNA vaccine was selected for further studies.

Sera of animals immunized with 100 μg of pVAX-H5 were also analyzed in the microneutralization assay against three strains of A/H5 clade 2.3.4.4b (Table 2). All viruses were isolated from sectional material of birds during outbreaks of highly pathogenic influenza in Russia in 2022-2024, including A/chicken/Komi/24-4V/2023 (H5N1), A/chicken/Khabarovsk/24-1V/2022 (H5N1), and A/chicken/Voronezh/193-1V/2024 (H5N1). Antigenic properties of the viruses are presented in Table 1.

As shown in the data presented in Table 2, the DNA vaccine pVAX-H5 based on the hemagglutinin of A/turkey/Stavropol/320-01/2020 (H5N8) strain induced the production of antibodies that neutralized both the homologous strain A/Astrakhan/3212/2020 (H5N8) and the other strains used in the experiment. The sera reacted well with all viruses, with titers against A/chicken/Komi/24-4V/2023 (H5N1) and A/chicken/Voronezh/193-1V/2024 (H5N1) ranging from 160 to 1280. The lowest neutralizing antibody titers were detected against influenza virus A/chicken/Khabarovsk/24-1V/2022 (H5N1) (40-80). This is possibly due to the lower antigenic similarity of this virus to the vaccine strain (see Table 1). Differences in amino acid sequence are presented in Table 3. It should be noted that the HA in the DNA vaccine pVAX-H5 does not contain cytoplasmic and transmembrane domains.

Next, the protective effect of pVAX-H5 (100 μg) was investigated upon infection with both homologous and heterologous HPAI H5 clade 2.3.4.4b viruses. BALB/c mice were immunized according to the scheme shown in Figure 2a. Before the second immunization and two weeks after the second immunization, animal serum samples were obtained for ELISA analysis, and the results showed that after the first immunization there was a slight rise in the level of specific antibodies, with a median titer of 1:4050. After the second immunization, the median titer reached 1:109356, consistent with the previous experiment (Figure 2b). Mice were then infected with a lethal dose of homologous virus A/Astrakhan/3212/2020 (H5N8) or heterologous virus A/chicken/Khabarovsk/24-1V/2022 (H5N1) (Figure 2a). All immunized animals survived after infection with A/Astrakhan/3212/2020 (H5N8) virus and showed no clinical signs of disease, whereas intact animals died or were euthanized due to severe (>20%) weight loss or neurological symptoms by day 10 post-infection (Figure 2c). When infected with a lethal dose of heterologous A/chicken/Khabarovsk/24-1V/2022 (H5N1) virus, all vaccinated animals also survived, while all intact animals, died or were euthanized by day 10 post-infection (Figure 2c). These results indicate that the DNA vaccine effectively induces cross-protective immunity in vivo.

4. Discussion

Global outbreaks of HPAI H5 viruses in wild birds and infection of other animals create the conditions for the virus to evolve in mammals, which could eventually lead to a virus strain capable of sustained human-to-human transmission, potentially resulting in a pandemic. This potential cross-species transmission requires continuous monitoring of avian influenza viruses and vaccine development [24,53,54,55]. A promising area for influenza vaccine development is nucleic acid-based vaccines: DNA and mRNA vaccines. These types of vaccines do not require the use of chicken embryos or cell cultures for production [26,56]. Determination of the nucleotide sequence of an actual virus strain is sufficient to initiate the development of a new vaccine [24].

In the development of nucleic acid-based influenza vaccines, it is important for an effective immune response to ensure that the antigen, in our case hemagglutinin, is presented in a prefusion conformation. It has been previously shown that stabilizing substitutions in pH switch regions provide a closed conformation of the HA proteolysis site and prevent protein cleavage into subunits, which enhances the expression, quality and stability of HA of influenza A and B viruses [57,58]. Therefore, in the development of DNA vaccine, we modified HA by making stabilizing amino acid substitutions in the pH-switch region and added the trimerizing domain of phage T4. As a result, a DNA vaccine encoding the secreted HA A/turkey/Stavropol/320-01/2020 (H5N8) was developed and its immunogenic properties were investigated against the homologous strain A/Astrakhan/3212/2020 (H5N8) 2.3.4.4b [51], which was recommended by WHO as a vaccine strain [59]. The resulting DNA vaccine pVAX-H5 at a dose of 100 μg was found to induce the formation of neutralizing antibodies and provide protection against the homologous strain [51]. However, antigenic drift and antigenic shifts result in changing influenza virus variants causing outbreaks, and therefore it is important to determine whether existing and developing vaccines retain protective effects against new viral isolates [8]. For example, between 2019 and 2022, the major HPAI virus subtype causing global epizootics has changed from H5N8 to H5N1 [24,60].

This study was aimed to investigate the dose-dependent effect and cross-reactivity of the DNA vaccine pVAX-H5. According to the comparative immunogenicity results, high levels of specific antibodies were observed starting from a dose of 50 μg (Figure 1). The highest titer of specific antibodies to recombinant HA was in the group receiving 100 μg. Notably, the higher dose of pVAX-H5 (200 µg) did not provide an increase in antibody titer compared to 100 µg (data are not significantly different, but there is a tendency to decrease in the case of 200 µg). The observed effect may be related to the fact that foreign DNA has adjuvant properties, activating innate immunity, which in turn may negatively affect specific immunogenicity [61,62]. In the case of mRNA vaccines, dsRNA impurities have been shown to activate innate immunity, leading to a significant decrease in the immunogenicity of mRNA vaccines [63,64,65,66]. Therefore, dose escalation may be inappropriate or even harmful in the case of DNA vaccines administered by jet injection. For a more detailed understanding of the immune response to higher doses of pVAX-H5, doses in a wider range, e.g. up to 1 mg, and vaccination in other animal models should be investigated in the future.

Analysis of sera of animals immunized with 100 µg of pVAX-H5 in the microneutralization assay against heterologous H5N1 strains showed that the sera had neutralizing activity against these strains. The lowest neutralizing activity was observed against A/chicken/Khabarovsk/24-1V/2022 (H5N1). This strain also showed the lowest titers in HI assay (Table 1) and has the highest number of amino acid substitutions in HA relative to the vaccine strain (Table 2). It is worth noting that there are no clear criteria for a protective titer for the microneutralization assay, as in HI assay [67]. Thus, some researchers use a titer ≥80 as an endpoint for the efficacy of H5N1 avian influenza vaccines [68], while others consider a seroprotection threshold of 1:20 to be acceptable [69].

Next, we tested the protective effect of the DNA vaccine against both homologous and heterologous strains. It was of particular interest to determine if pVAX-H5 would provide protection against infection with a lethal dose of A/chicken/Khabarovsk/24-1V/2022 (H5N1), since against this strain the neutralizing titers were the lowest. It was shown that all animals immunized with 100 μg of pVAX-H5 survived, whereas all animals receiving the control plasmid pVAX1 died when infected with both the homologous H5N8 and heterologous H5N1 strains (Figure 2c). It has also been previously shown that mRNA vaccines encoding HA clade 2.3.4.4b in vitro provide antibody formation that binds to different strains of H5 subtype virus and protects mice from infection with clade 2.3.2.1a virus [70,71]. In this study, we did not analyze the specific T-cell response. However, in past work [51], we showed its formation in response to the administration of 100 μg of pVAX-H5 in similar settings. Therefore, we hypothesize that in this case, in addition to neutralizing antibodies, T-cell immunity was involved in the formation of protective immunity. Recent studies by other groups have also shown that T-cell immunity induced by mRNA-based vaccine contributes to protection against H5 virus infection [32,70,72].

Thus, the data obtained indicate that the DNA vaccine encoding a modified hemagglutinin of influenza A/H5N8 virus is capable of protecting mice from infection with a lethal dose of influenza A/H5N1 virus. The findings support the feasibility of the DNA vaccine development for the pandemic preparedness against HPAI viruses, which pose the increasing threat to human health.