In this study, we characterized recombinant hemagglutinin (HA) of influenza A (H5N8) virus produced in Chinese hamster ovary cells (CHO-K1s). Immunochemical analysis showed that the recombinant hemagglutinin was recognized by the serum of ferrets infected with influenza A (H5N8) virus, indicating that its antigenic properties were retained. Two groups of Balb/c mice were immunized with intramuscular injection of recombinant hemagglutinin or propiolactone inactivated A/Astrakhan/3212/2020 (H5N8) influenza virus. The results demonstrated that both immunogens induced a specific antibody response as determined by ELISA. Virus neutralization assay revealed that sera of immunized animals were able to neutralize A/turkey/Stavropol/320-01/2020 (H5N8) influenza virus—the average neutralizing titer was 2560. Immunization with both recombinant HA/H5 hemagglutinin and inactivated virus gave 100% protection against lethal H5N8 virus challenge. This study shows that recombinant HA (H5N8) protein may be a useful antigen candidate for developing subunit vaccines against influenza A (H5N8) virus with suitable immunogenicity and protective efficacy.

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).

Highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4b viruses are widespread in wild and domestic birds, causing severe economic damage to the global poultry industry. Moreover, viruses of this clade are known to cause infections in mammals, posing a potential pandemic threat. Due to the ongoing evolution and change in the dominant strains of H5 clade 2.3.4.4b, it is important to investigate the cross-reactivity of vaccines in use and under development against clade 2.3.4.4b viruses. In this study, the immunogenicity of the previously developed DNA vaccine encoding a modified hemagglutinin of the influenza A/turkey/Stavropol/320-01/2020 (H5N8) virus, administered by jet injection at doses of 1, 10, 50, 100, and 200 μg, was investigated. The highest titer of specific to recombinant hemagglutinin antibodies was detected in the group of animals injected with 100 µg of DNA vaccine. The cross-reactivity study of sera of animals immunized with 100 µg of DNA vaccine in a microneutralization assay against the strains A/chicken/Astrakhan/321-05/2020 (H5N8), A/chicken/Komi/24-4V/2023 (H5N1), and A/chicken/Khabarovsk/24-1V/2022 (H5N1) showed the formation of cross-neutralizing antibodies. Moreover, the study of protective properties showed that the DNA vaccine protected animals from mortality after infection with A/chicken/Khabarovsk/24-1V/2022 (H5N1) virus.

Highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4b viruses are widespread in wild and domestic birds, causing severe economic damage to the global poultry industry. Moreover, viruses of this clade can adapt to mammals, posing a potential pandemic threat. With the constant evolution and change of dominant strains of H5 clade 2.3.4.4b, it is important to investigate the cross-reactivity of available and developing vaccine preparations. In this study, the immuno-genicity of the previously developed DNA vaccine encoding a modified hemagglutinin of influ-enza A/turkey/Stavropol/320-01/2020 (H5N8) virus, administered by jet injection at doses of 1, 10, 50, 100 and 200 μg, was investigated. The highest titer of specific to recombinant hemagglutinin antibodies was detected in the group of animals injected with 100 µg of DNA vaccine. The cross-reactivity study 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) showed the formation of cross-neutralizing antibodies. Moreover, the study of protective properties showed that the DNA vaccine prevented morbidity and mortality of animals after infection with both homologous strain A/Astrakhan/3212/2020 (H5N8) and heterologous strain A/chicken/Khabarovsk/24-1V/2022 (H5N1).

Background: Avian influenza is a critical zoonotic infection threatening both the poultry industry and global public health. While traditional intramuscular vaccines elicit systemic immunity, they often fail to provide robust local protection at mucosal surfaces. There is thus significant interest in the development of mucosal avian influenza vaccines administered via the intranasal route. However, in humans, this approach is significantly hampered by the availability of safe and effective adjuvants. Methods: This study investigated the immunogenicity of a modified recombinant influenza A/H5 hemagglutinin (rHA/H5-modif) formulated with Novochizol, a novel chitosan-derived delivery system, administered intranasally to laboratory animals. Results: Our results demonstrate that mucosal immunization with the rHA/H5-modif/Novochizol complex induces potent humoral (IgG and IgA) and cell-mediated immune responses. Crucially, the formulation provided 100% survival in mice following a lethal challenge with highly pathogenic avian influenza A/H5. Conclusions: These findings position the rHA/H5-modif/Novochizol complex as a promising candidate for next-generation mucosal vaccines, in particular against highly pathogenic avian influenza A/H5 subtype.