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.

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.

Avian influenza A virus subtype H5 is widespread in the bird population, and there is a risk of its transmission to humans, therefore, it is necessary to develop an effective vaccine to prevent virus spread.   The aim of the work was to develop prototypes of DNA vaccines encoding modified variants of the hemagglutinin trimer of highly pathogenic avian influenza A virus (H5N8) and its fragment (stem) and to study their immunogenic properties.   Material and methods. DNA constructs were designed, produced and purified, their expression was analyzed in eukaryotic cells using reverse transcription PCR and Western blot analysis. Laboratory animals (Syrian hamsters) were immunized with experimental DNA vaccines using the jet injection method, the antibody titer in their sera was determined using ELISA. The sera were also tested in the virus neutralization reaction in MDCK cell culture.   Results and discussion. Expression of target genes in experimental DNA vaccines was confirmed at the transcription and translation levels. Immunogenicity analysis showed that immunization of Syrian hamsters with pVAX-H5 and pVAX-H5 delT4 constructs encoding hemagglutinin ectodomain variants with and without the fibritin trimerizing domain of bacteriophage T4 using jet injection resulted in the formation of specific antibodies (average titer 1:3×104 and 1:5×103, respectively) with virus-neutralizing activity (average 50 % neutralizing titer 1:40). At the same time, specific antibodies were not detected in the group of animals immunized with the DNA vaccine encoding the hemagglutinin stalk.   Conclusions. In the course of the work, DNA vaccines encoding variants of the hemagglutinin trimer of the influenza virus subtype A (H5N8) and its fragment (stem) were designed and obtained. A comparison of the immunogenicity of experimental DNA vaccines was conducted and the most promising variant of the design – pVAX-H5, encoding a modified trimer of the hemagglutinin of the influenza virus with a trimerizing domain of the fibritin of the bacteriophage T4 – was established.

Introduction. During the COVID-19 pandemic, the development of preventive vaccines, including those based on new platforms, became extremely relevant. One such platform is vaccines, which combine, for example, DNA and protein components into a single vaccine. The aim of this study was to investigate the immunogenicity of a DNA vaccine encoding a polyepitopic T-cell immunogen of the SARS-CoV-2 virus, combined with the recombinant RBD protein (the receptor-binding domain of the SARS-CoV-2 virus S protein, Wuhan-Hu-1 strain) conjugated to a polycationic carrier – polyglucin-spermidine (PGS), and to assess the contribution of individual components to the development of an immune response in BALB/c mice. Materials and methods. To create the DNA vaccine (pBSI-COV-Ub), we used a strategy of designing an artificial polyepitope immunogen consisting of conserved immunodominant fragments of various structural proteins of the SARS-CoV-2 virus, containing a large number of T-lymphocyte epitopes: helper and cytotoxic. The recombinant RBD protein was conjugated with the polycation PGS, and upon mixing it with DNA, it formed the vaccine complex CCV–BSI, whose immunogenic properties were investigated in this work. Results. Immunization of BALB/c mice with the CCV–BSI combined construct resulted in the induction of high antibody titers with neutralizing activity against live SARS-CoV-2 virus, as well as the formation of a virus-specific T-cell response, as demonstrated by ELISA, neutralization assay and ELISpot. It has been shown that the protein component contributes to the humoral immune response, while DNA contributes to the cellular immune response. Administration of the recombinant RBD protein led to the induction of only antibodies, administration of the DNA vaccine led to the induction of only a T-cell response, and administration of the combined preparation led to the induction of both a humoral immune response and specific T cells. Conclusion. The unique combination of DNA and protein within a single vaccine construct allows for overcoming the limitations of each of these vaccine types and leads to the induction of both arms of immunity. The protein component can be replaced according to the current viral strain, and a universal T-cell immunogen can provide a response to a wide range of circulating variants. This platform can be further used to develop vaccines against various highly variable viruses.

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.