Sergey V. Sharabrin

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. Despite the effectiveness of existing COVID-19 vaccines, the emergence of new SARS-CoV-2 variants necessitates the development of immunogens capable of eliciting a broad, cross-reactive immune response. A promising approach is the development of mRNA vaccines that induce both humoral and T-cell responses, which could provide protection against viral variants that evade neutralizing antibodies. AIM. This study aimed to investigate the immunogenicity of an experimental two-component mRNA vaccine encoding an artificial multi-epitope T-cell immunogen and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, to demonstrate the potential of this vaccine to elicit a broad, cross-reactive response required for effective protection against highly variable viruses. MATERIALS AND METHODS. The mRNAs were synthesized in vitro : mRNA-RBD encoded the RBD, and mRNA-BSI encoded the multi-epitope T-cell immunogen. The vaccine mRNA was encapsulated in lipid nanoparticles (LNPs); particle size was assessed using dynamic light scattering. BALB/c mice (24 females) were divided into 4 groups of 6 animals each. For immunization, 10 μg of mRNA-LNP (either alone or as a mixture of the two constructs) was administered intramuscularly twice, 3 weeks apart. Humoral response was evaluated by titers of virus-specific and neutralizing antibodies; T-cell response was assessed by ELISpot (spot-forming units, SFUs), sandwich ELISA, intracellular cytokine staining, and flow cytometry. RESULTS. The LNPs encapsulating the vaccine mRNA had a size of 115.4–123.7 nm and a low polydispersity index (0.165–0.211). The two-component vaccine (mRNA-RBD + mRNA-BSI) induced high titers of RBD-specific antibodies (1:131,322) and neutralizing antibodies against the Wuhan-Hu-1 strain (1:3,620) and the Omicron BA.5.2 strain (1:718). The T-cell response in the two-component vaccine group was characterized by high IFN-γ production: the number of IFN-γ-producing lymphocytes and the IFN-γ concentration in splenocyte culture were 660 SFUs/million cells (ELISpot) and 990 pg/mL (ELISA), respectively; both significantly exceeded the corresponding values in the mRNA-RBD group (343 SFUs/million and 60 pg/mL, respectively). Intracellular staining revealed an increased proportion of cytokine-producing CD4 ⁺ and CD8 ⁺ T cells (IFN-γ, IL-2, TNF-α) in all immunized groups. CONCLUSIONS. The combination of B-cell and T-cell immunogens in a two-component mRNA vaccine enhances the humoral response and promotes a broad T-cell response, ensuring effective recognition of emerging SARS-CoV-2 variants. The data support the potential of this platform for the development of vaccines against highly variable viral infections.

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: 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.

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.

Seasonal influenza is an acute respiratory illness caused by the influenza A and B viruses that circulate worldwide. Due to high variability, new strains of the virus emerge every year. Therefore, vaccine formulation has to be revised every year. The advantages of mRNA vaccines are that they can be produced quickly and without preliminary adaptation of the vaccine strain to chicken embryos. Here, the results of developing and studying the mRNA-C3-H1 vaccine encoding the hemagglutinin (HA) of the influenza A(H1N1)pdm09 virus are presented. The design and production of a DNA-template for the synthesis of mature HA mRNA in one step were described. The obtained mRNA was purified from double-stranded RNA impurities using a method based on the use of cellulose powder. The efficacy of the vaccine was assessed on BALB/c mice. The mice were immunized with a “naked” mRNA vaccine using a needle-free jet injector. According to the ELISA results, the average antibody titer in the serum of immunized animals was 4.6 × 10⁵. Sera of immunized animals neutralized the mouse-adapted influenza A/California/04/09 (H1N1) MA8 virus with an average titer of 6 × 10². As shown by the ELISpot method, the developed mRNA vaccine induced a T-cell immune response in mice. After stimulation of splenocytes with specific peptides, the average number of T-lymphocytes secreting IFN-γ was 236 per 10⁶ cells. Immunization with the mRNA vaccine was shown to protect mice from infection with a lethal dose of the influenza A/California/04/09 (H1N1) MA8 virus. Thus, the developed experimental mRNA-C3-H1 vaccine is immunogenic and prevents morbidity and mortality in mice after infection with a homologous strain of the influenza virus.

Background: Although mRNA vaccines encapsulated in lipid nanoparticles (LNPs) have demonstrated a safety profile with minimal serious adverse events in clinical trials, there is opportunity to further reduce mRNA reactogenicity. The development of naked mRNA vaccines could improve vaccine tolerability. Naked nucleic acid delivery using the jet injection method may be a solution. Methods: In the first part of the study, the optimal conditions providing low traumatization and high expression of the model mRNA-GFP molecule in the tissues of laboratory animals were determined. Then, we used the selected protocol to immunize BALB/c mice with mRNA-RBD encoding the SARS-CoV-2 receptor-binding domain (RBD). It was demonstrated that mice vaccinated with naked mRNA-RBD developed a high level of specific antibodies with virus-neutralizing activity. The vaccine also induced a strong RBD-specific T-cell response and reduced the viral load in the lungs of the animals after infection with the SARS-CoV-2 virus. The level of immune response in mice immunized with mRNA-RBD using a spring-loaded jet injector was comparable to that in animals immunized with mRNA-RBD encapsulated in LNPs. Results: In this study, the efficacy of an inexpensive, simple, and safe method of mRNA delivery using a spring-loaded jet injector was evaluated and validated. Conclusions: Our findings suggest that the jet injection method may be a possible alternative to LNPs for delivering mRNA vaccines against SARS-CoV-2 infection.

The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these