Ksenia Ivanovna Ivanova

INTRODUCTION. The low physicochemical stability of mRNA and the dependence of current mRNA vaccines on the successful encapsulation into lipid nanoparticles and adherence to mandatory cold chain requirements limit their storage and transportation. A promising approach is the lyophilization of nonencapsulated mRNA with its subsequent delivery by jet injection. However, the preservation of its structure and functional activity during long-term storage has been insufficiently studied. AIM. To evaluate the structural and functional stability of lyophilized nonencapsulated mRNA during long-term storage and its immunogenicity as a component of a trivalent mRNA vaccine against seasonal influenza administered to mice by jet injection. MATERIALS AND METHODS. mRNA encoding green fluorescent protein (GFP) was lyophilized with various cryoprotectants and stored at −20, 4, and 20 °C. Its functional activity was determined by GFP expression after transfection of HEK293 cells, and its integrity was analyzed by microchip electrophoresis. The immunogenicity of the lyophilized and nonlyophilized mRNA-Vector-Flu vaccine was evaluated in female inbred BALB/c mice after two jet immunizations by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HAI) assay, virus neutralization assay, and interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay. RESULTS. Based on the results of a preliminary screening, a 10% trehalose formulation was selected for further studies because it preserved the functional activity of GFP mRNA at the level closest to that of the control preparation. The lyophilized GFP mRNA retained its functional activity after 12 months of storage at all tested temperatures; no statistically significant differences were found compared with the mRNA synthesized immediately before use ( p >0.05). The major mRNA fraction was preserved, although minor fractions of partially degraded mRNA were detected after 6–12 months, particularly at 20 °C. After 3 months of storage of the vaccine at 4 °C, the geometric mean antibody titers against hemagglutinin subtype 1 (H1), hemagglutinin subtype 3 (H3), and influenza B hemagglutinin (HB) were 1:382,000, 1:628,000, and 1:273,000, respectively, compared with 1:710,000, 1:628,000, and 1:328,000 in the nonlyophilized vaccine group. HAI titers were 1:37–1:80, virus-neutralizing antibody titers were 1:50–1:90; and the numbers of IFN-γ-secreting cells were 440 and 500 spot-forming cells (SFC) per 1×10 ⁶ splenocytes. No significant between-group differences were identified. CONCLUSIONS. Lyophilization with 10% trehalose preserved the functional activity of nonencapsulated mRNA for 12 months despite signs of partial degradation at 20 °C. After 3 months of storage at 4 °C, the trivalent vaccine retained its ability to induce humoral and T-cell immune responses in mice. These findings support the potential of combining lyophilization and jet injection for the development of stable mRNA vaccines.

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.

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.

Introduction. Influenza A virus is one of the major global infectious threats. Along with seasonal epidemics, zoonotic influenza viruses (subtypes А(H5Nх), А(H7Nх), А(H9N2)) pose an increased risk, requiring constant monitoring of herd immunity. The aim of the work was to study human blood sera against influenza viruses, both vaccine strains and virus strains with pandemic potential. Materials and methods. Blood sera from healthy donors collected in different regions of the Russian Federation were tested in the hemagglutination inhibition (HI) assay with vaccine strains of influenza A and B viruses. Blood sera from individuals involved in poultry farming were tested in the hemagglutination inhibition and virus neutralization assays with avian influenza viruses. Results. From 2021 to 2025, only a few samples were positivein the HI assay with A(H5Nx) influenza viruses, and approximately 1% of samples were positive in HI assay with A(H9N2) virus. All samples positive in the HI assay with A(H5) viruses were negative in the neutralization assay, and approximately 85% of samples positive in the HI assay with A(H9N2) virus contained virus-neutralizing antibodies. Conclusion. By 2023–2024, humoral immunity to seasonal influenza, following its decline during the COVID-19 pandemic, was at levels typical of pre-pandemic seasons and generally met the recommendations of Rospotrebnadzor and the World Health Organization. Currently, influenza viruses with pandemic potential, such as subtypes A(H5N1) and A(H9N2), are not circulating in the human population in the Russian Federation. However, insufficient herd immunity to seasonal influenza viruses poses additional risks for the emergence and spread of new virus variants.

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.

In 2025, high epizootic activity of highly pathogenic avian influenza virus (HPAIV) persisted globally, as evidenced by more than 7,000 outbreaks reported in poultry and wild birds across 75 countries, resulting in mortality or culling of birds exceeding 98 million specimens. The majority of outbreaks worldwide were associated with HPAIV A(H5N1) clade 2.3.4.4b, which has remained the predominant lineage in global circulation since 2021. In addition to the dominant A(H5N1) clade 2.3.4.4b subtype, circulation of other highly pathogenic subtypes was observed, including A(H5N5), A(H5N2), A(H5N8), and A(H5N9), as well as A(H7), indicating ongoing evolution of avian influenza viruses in natural and anthropogenic ecosystems. In the United States and several other countries, HPAIV A(H5N1) caused numerous infections in wild and domestic mammals as well as in humans. In 2025, the world’s first human case of A(H5N5) influenza was documented in the United States. A total of 74 zoonotic HPAIV human infections were reported globally in 2025, including 32 cases caused by A(H5) viruses of various clades (2.3.4.4b, 2.3.2.1e, 2.3.2.1a), reflecting a continuing increase in interspecies transmission that poses a potential public health threat. In the Russian Federation, high epizootic activity of HPAIV persisted throughout 2025, with outbreaks caused by A(H5N1) clade 2.3.4.4b viruses reported in both wild and domestic birds. Notably, in the Russian Far East, circulation of an A(H5N1) virus carrying mammalian-adaptive mutations associated with increased virulence in mammals was detected. Additionally, amino acid substitutions in antigenic sites were identified in several A(H5N1) isolates. Despite ongoing virus evolution, no substantial alterations in the antigenic properties of HPAIV A(H5N1) strains circulating in the Russian Federation have been observed to date.

Background: Seasonal influenza remains a significant public health problem, and the constant antigenic drift of viruses requires regular vaccine updates. mRNA vaccines offer a promising platform for the development of new, effective influenza vaccines. Administration of the naked mRNA vaccine using a needle-free jet injection system further enhances its safety, reduces cost, and eliminates the need for lipid nanoparticles, which are traditionally used for mRNA delivery. Lyophilization of naked mRNA allows for long-term storage at +4 °C. Methods: We designed and produced an mRNA vaccine against seasonal influenza, designated mRNA-Vector-Flu, encoding the hemagglutinin (HA) of the A/Wisconsin/67/2022(H1N1)pdm09, A/Darwin/9/2021(H3N2), and B/Austria/1359417/2021 strains. The vaccine was lyophilized and stored for 1 month in a refrigerator (+4 °C). A comparative immunogenicity study was conducted between synthesized immediately before use prepared and lyophilized naked mRNA-Vector-Flu. The preparations were administered to BALB/c mice using a jet needleless injection twice, 3 weeks apart. Immunogenicity was assessed on day 35 of the study. Results: A comparative immunogenicity study of naked mRNA-Vector-Flu demonstrated that both the synthesized immediately before use prepared formulation and the lyophilized form, stored at +4 °C for a month, induced similar levels of virus-specific antibodies and generated a pronounced T-cell immune response. Conclusions: Delivery of the naked mRNA vaccine using a needle-free jet injection ensures a high-level immune response, which improves its safety, reduces its cost, and eliminates the need for lipid nanoparticles traditionally used for mRNA delivery. At the same time, lyophilization of the naked mRNA vaccine preserves its biological activity and ensures its storage for at least a month at +4 °C temperatures. Our results demonstrate that our proposed approach can be considered a promising direction for the development and improvement of the mRNA vaccine platform.

Background: Seasonal influenza remains a significant public health problem, and the constant antigenic drift of viruses requires regular vaccine updates. mRNA vaccines offer a promising platform for the development of new, effective influenza vaccines. Administration of the naked mRNA vaccine using a needle-free jet injection system further enhances its safety, reduces cost, and eliminates the need for lipid nanoparticles, which are traditionally used for mRNA delivery. Lyophilization of naked mRNA allows for longterm storage at +4°C. Methods: We designed and produced an mRNA vaccine against seasonal influenza, designated mRNA-Vector-Flu, encoding the hemagglutinin (HA) of the A/Wisconsin/67/2022(H1N1)pdm09, A/Darwin/9/2021(H3N2), and B/Austria/1359417/2021 strains. The vaccine was lyophilized and stored for 1 month in a refrigerator (+4°C). A comparative immunogenicity study was conducted between synthesized immediately before use prepared and lyophilized naked mRNA-Vector-Flu. The preparations were administered to BALB/c mice using a jet needleless injection twice, 3 weeks apart. Immunogenicity was assessed on day 35 of the study. Results: A comparative immunogenicity study of naked mRNA-Vector-Flu demonstrated that both the synthesized immediately before use prepared formulation and the lyophilized form, stored at +4°C for a month, induced similar levels of virus-specific antibodies and generated a pro-nounced T-cell immune response. Conclusions: Delivery of the naked mRNA vaccine us-ing a needle-free jet injection ensures a high-level immune response, which improves its safety, reduces its cost, and eliminates the need for lipid nanoparticles traditionally used for mRNA delivery. At the same time, lyophilization of the naked mRNA vaccine pre-serves its biological activity and ensures its long-term storage at above-zero temperatures. Our results demonstrate that our proposed approach can be considered a promising di-rection for the development and improvement of the mRNA vaccine platform.

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.