Andrei S. Gudymo

INTRODUCTION. The highly pathogenic avian influenza virus subtype H5Nx is rapidly evolving and infecting various species of wild and domestic mammals, including cattle, highlighting its pandemic potential for humans. To combat the pandemic threat, it is essential to develop suitable vaccine platforms, including mRNA vaccines, the efficacy of which largely depends on the delivery system. This study evaluates the immunogenicity and protective efficacy of the mRNA-H5 vaccine, delivered via lipid nanoparticles (LNPs) and jet injection (JI). AIM. The aim of the study was to evaluate the immunogenic and protective properties of the experimental mRNA-H5 vaccine in a ferret model. MATERIALS AND METHODS. Ferrets were immunized twice with the mRNA-H5 vaccine at a dose of 30 μg formulated with LNPs and at a dose of 100 μg administered by JI. Sera from immunized ferrets were analyzed using an enzyme-linked immunosorbent assay (ELISA) against the recombinant HA/H5 protein, an enzyme-linked immunospot (ELISpot) assay using a pool of specific peptides, and a virus neutralization assay in MDCK-SIAT1 cell culture using the influenza virus strain A/turkey/Stavropol/320-01/2020 (H5N8). Immunized ferrets were intranasally challenged with 5 LD ₅₀ of the influenza virus strain A/dalmatian pelican/Kalmykia/ 330-10V/2023 (H5N1), and their status was recorded for 14 days after challenge. RESULTS. Using ELISA, it was established that specific antibody titers were determined in groups of animals immunized with the mRNA-H5 vaccine administered by JI or formulated with LNPs; the average titers in the groups were 1:180,000 and 1:4,000,000, respectively. Using the virus neutralization assay, sera from animals immunized with the mRNA-H5 vaccine administered by JI or formulated with LNPs had virus-neutralizing activity; the average 50% neutralizing titers were 1:250 and 1:1500, respectively. The ELISpot assay showed that in groups of ferrets immunized with the mRNA-H5 vaccine, specific cellular immunity was induced. Immunization with the mRNA-H5 vaccine provided 100% protection against mortality in ferrets challenged with 5 LD ₅₀ of the influenza virus strain A/dalmatian pelican/Kalmykia/330-10V/2023 (H5N1). CONCLUSIONS. This study demonstrated the effective induction of virus-specific humoral and cellular immune responses in ferrets immunized with the mRNA-H5 vaccine administered by JI or formulated with LNPs.

Introduction. Immunohistochemical (IHC) analysis is one of the main methods for detecting and visualizing the protein determinants of the virus in organs. The nanoscale structures of viruses complicate the visualization of particle topography, which prevents a more detailed understanding of the pathogenesis of viral infections. Unfortunately, general IHC protocols are not universal and often their application in special cases is impossible. Optimizing the coloring methodology can significantly save research time. Based on this, this paper describes a step-by-step methodology for IHC staining of paraffin sections of animal organs infected with the SARS-CoV-2 virus. The purpose of the study. Optimization of the chromogenic IHC staining protocol when working with infected material, using the example of SARS-CoV-2. Materials and methods. IHC analysis was performed on paraffin sections of lung tissue of ferrets (Mustela putorius furo) infected with Omicron and Delta strains of SARS-CoV-2. Results. During the study, it was demonstrated that the optimal exposure time for samples with hydrogen peroxide was at least 30 minutes. The use of BSA before the application of primary antibodies also contributed to a decrease in background staining, while the optimal concentration of the reagent was at least 1%. It is noteworthy that the addition of BSA to the primary antibody solution also contributed to a decrease in the non-specific signal. In the context of the current work, dilution of antibodies applied to S-protein by 1000 times was optimal to minimize the proportion of non-specific tissue staining. Conclusion. In the work on optimizing the IHC protocol for paraffin sections, the main aspects affecting the staining quality were demonstrated and described. When working with material containing SARS-CoV-2, options for eliminating methodological mistakes were proposed.

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.

Avian influenza is a critical zoonotic infection threatening both the poultry industry and global public health. While traditional intramuscular vaccines elicit systemic im-munity, 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. This study investigated the immunogenic-ity 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. 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. These findings position the rHA/H5-modif/Novochizol complex as a promising candidate for next-generation mucosal vaccines, in particular against highly pathogenic avian influen-za A/H5 subtype.

Background/Objectives: The development of a vaccine against highly pathogenic avian influenza viruses subtype A/H5 is an urgent task due to concerns about its pandemic potential. Methods: In this study, we have developed an experimental mRNA vaccine, mRNA-H5, encoding a modified hemagglutinin trimer of influenza virus A/turkey/Stavropol/320-01/2020 (H5N8). BALB/c mice were immunized with the mRNA-H5 vaccine using lipid nanoparticles (LNPs) and needle-free jet injection (JI). Subsequently, the immune response to vaccine was assessed using ELISA, microneutralization assay, and ICS methods, and a challenge study was conducted. Results: mRNA-H5 was shown to effectively stimulate specific humoral and T-cell immune responses. Moreover, mRNA-H5 delivered by LNPs and JI provided 100% protection of immunized mice against lethal challenge with homologous and heterologous strains of avian influenza virus (A/Astrakhan/3212/2020 (H5N8) and A/chicken/Magadan/14-7V/2022 (H5N1), respectively). Conclusions: The present results indicate that JI can be considered as an alternative to LNPs for mRNA delivery, and according to the literature, JI is safer than delivery using LNP. mRNA-H5 has potential as a vaccine against infection with highly pathogenic avian influenza A/H5 viruses with pandemic potential.

The development of a vaccine against highly pathogenic avian influenza A/H5 viruses is an urgent task due to concerns about its pandemic potential. Here, we have developed an experimental mRNA vaccine mRNA-H5, encoding a modified hemagglutinin trimer of influenza virus A/turkey/Stavropol/320-01/2020 (H5N8). Immunization of BALB/c mice with the obtained mRNA-H5 vaccine using lipid nanoparticles (LNPs) and needle-free jet injection (JI) has been shown to effectively stimulate specific humoral and T-cell immune responses. Moreover, mRNA-H5 delivered by LNPs and JI provided 100% protectivity of immunized mice against lethal challenge with homologous and heterologous strains of avian influenza virus (A/Astrakhan/3212/2020 (H5N8) and A/chicken/Magadan/14-7V/2022 (H5N1), respectively). The present results indicate that needle-free jet injection can be considered as an alternative to LNPs for mRNA delivery. mRNA-H5 is a promising candidate as a vaccine against highly pathogenic avian influenza A/H5 viruses with pandemic potential.

The 2023–2024 flu season in Russia was dominated by influenza A(H3N2) viruses. In this study, we isolated seasonal influenza viruses from human respiratory specimens, analyzed their genetic and antigenic properties, and assessed their susceptibility to neuraminidase inhibitors. In total, we isolated 207 influenza virus isolates. Of them, 95.2% were subtyped as A(H3N2), 1.9% as A(H1N1)pdm09, and 2.9% as B/Victoria influenza viruses. The hemagglutinin sequences of the A(H3N2) isolates showed that they belonged to several subclades within the 2a.3a.1 genetic group, with the J2 subclade being predominant. Despite their genetic diversity, all A(H3N2) strains tested using a hemagglutination inhibition assay were antigenically similar to the egg-propagated vaccine strain A/Darwin/09/2021(H3N2). The B/Victoria virus isolates belonged to the C.5 and C.5.7 subclades of the genetic group V1A.3a.2 and were antigenically similar to cell- and egg-propagated variants of the vaccine strain B/Austria/1359417/2021. All of the A(H1N1)pdm09 isolates belonged to the 6B.1A.5a.2a genetic clade and were well-recognized by a ferret antiserum raised against a cell-propagated A/Wisconsin/67/2022(H1N1pdm09)-like reference strain. All of the tested isolates were susceptible to oseltamivir and zanamivir, including two A(H1N1)pdm09 strains with an NA-S247N substitution. Seroprevalence analysis showed that 60%, 54%, and 46% of the human blood samples tested were seropositive for the A(H3N2), A(H1N1)pdm09 and B/Victoria antigens from the 2022–2023 vaccine.

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.