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/Objectives: In recent years, researchers have directed considerable attention toward the activation of the T-cell response in the development of vaccines against viral infections, including the tick-borne encephalitis virus (TBEV). A particularly promising approach to developing effective and safe T-cell vaccines involves the use of artificial multi-epitope immunogens. The selection of spacers that link epitopes within a construct can have a significant effect on the immunogenicity of multi-epitope constructs. The objective of this study was to design multi-epitope TBEV immunogens using various spacers and to evaluate their immunogenicity. Methods: The present study involved the design of three multi-epitope immunogens, which were developed based on T-cell epitopes from TBEV proteins. The AG1-ub construct contained optimized alanine spacers, the AG2-ub construct contained GPGPG spacers, and the AG4-ub construct contained no spacers. Three DNA vaccines encoding the designed immunogens were subsequently produced. To assess the immunogenicity of the constructs, BALB/c mice were immunized with the designed DNA vaccines via electroporation. Results: The ELISpot assay demonstrated that DNA vaccines encoding AG1-ub and AG4-ub induced a significant number of IFN-γ-producing cells. The DNA vaccine encoding AG2-ub, a multi-epitope with GPGPG spacers, exhibited low immunogenicity, potentially attributable to inadequate processing or misfolding of the AG2-ub protein, thereby affecting the molecule’s structure. Conclusions: The data provided in this study on the effect of spacers on the immunogenicity of multi-epitope constructs can be used to optimize the design of vaccine candidates. However, further research is needed to assess their clinical potential.

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.

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.

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.

In recent years, significant progress has been made in the field of drug development, particularly due to the use of computer modeling methods. One of the key stages in the development of new antiviral drugs is testing the efficacy of promising candidates in in vitro experiments using target viruses. The application of new technologies for conducting primary screening with pseudotyped viruses simplifies research, increases its efficiency and ensures the biosafety of the conducted studies. The aim of this review is to analyze previous studies that have demonstrated the successful use of pseudovirus technology for the search of new chemotherapeutic agents against a range of RNA-containing viruses. The analysis involved the literature presented in the PubMed, Scopus, Elsevier, and Google Scholar databases as of March 1, 2025. For the search, the following keywords were used: pseudovirus, virus inhibition, antiviral drugs, RNA viruses. Pseudotyped viruses are recombinant viral particles that have the core proteins of one virus and the surface proteins of another, studied virus. The advantages of pseudovirus technology are its safety, high level of reproducibility of results, and the possibility of standardization. The lentivirus-based system was one of the first to be developed and remains one of the most in-demand. Using pseudoviruses, candidate molecules for infections caused by RNA-containing viruses, such as HIV-1, hepatitis C virus, tick-borne encephalitis virus, avian influenza viruses, and SARS-CoV-2, have been selected and studied. Most of the selected drugs act at the initial stage of the virus entry into the target cell. The examples provided illustrate the significant contribution of pseudovirus technology in dealing with serious socially significant diseases caused by RNA-containing viruses.

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.

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.

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).

The tick-borne encephalitis (TBE) virus is highly pathogenic and can affect the central nervous system, leading to severe chronic effects or death. The only effective measure to combat TBE is vaccine prophylaxis. Vaccines that are currently used for mass immunization are based on inactivated TBE virus, they provide a protective immune response, but such vaccines require multiple administrations. A possible reason for short-term immunity is an incomplete functional T-cell response to these types of vaccines. The aim of this review is to analyze the literature on the role of the T-cell immune response in protecting the body from tick-borne encephalitis, its importance for vaccine development, and to consider approaches to the development of new TBE vaccines based on different platforms. When preparing the review, we analyzed the literature presented in scientific databases — PubMed, Scopus, Elsevier, Google Scholar as of April 2024. The following keywords were used for the search: vaccine, tick-borne encephalitis virus, T-cell immune response, flaviviruses. A several publications have demonstrated that T-cell responses following natural infection with TBE virus and after vaccination with inactivated virus are different. During viral infection, both Th1- and Th2-type CD4+ T cells and CD8+ T cells are activated and play an important role in the elimination of viral infection. After vaccination, the only Th2-type CD4+ T-cell response predominates, which may be the reason for the short-lived immune response. To date, a number of different types of experimental TBE vaccines are being studied, such as live-attenuated vaccines, viral vector vaccines, subunit vaccines, virus-like particles, DNA and mRNA vaccines, and polyepitope immunogens. In our opinion, the most promising in terms of T-cell response activation are vaccines based on T-cell polyepitope immunogens delivered in the form of DNA or mRNA.

We studied a needle-free jet injection delivery of an experimental mRNA vaccine encoding the receptor-binding domain of the SARS-CoV-2 S protein (mRNA-RBD). Immunization of BALB/c mice with mRNA-RBD by a needle-free jet injector induced high levels of antibodies with virus-neutralizing activity and a virus-specific T-cell response. The immune response was low in the group of mice that received intramuscular injection of mRNA-RBD. The effectiveness of this simple and safe method of mRNA delivering has been demonstrated. Thus, jet injection of mRNA vaccine can be a good alternative to lipid nanoparticles.

The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.

The vaccinia virus (VACV) has been used for prophylactic immunization against smallpox for many decades. However, the VACV-based vaccine had been highly reactogenic. Therefore, after the eradication of smallpox, the World Health Organization in 1980 recommended that vaccination against this infection be discontinued. As a result, there has been a rise in the occurrence of orthopoxvirus infections in humans in recent years, with the most severe being the 2022 monkeypox epidemic that reached all continents. Thus, it is crucial to address the pressing matter of developing safe and highly immunogenic vaccines for new generations to combat orthopoxvirus infections. In a previous study, we created a LAD strain by modifying the LIVP (L) VACV strain, which is used as a first-generation smallpox vaccine in Russia. This modification involved introducing mutations in the A34R gene to enhance extracellular virion production and deleting the A35R gene to counteract the antibody response to the viral infection. In this study, a strain LADA was created with an additional deletion in the DNA of the LAD strain ati gene. This ati gene directs the production of a major non-virion immunogen. The findings indicate that the LADA VACV variant exhibits lower levels of reactogenicity in BALB/c mice during intranasal infection, as compared to the original L strain. Following intradermal immunization with a 105 PFU dose, both the LAD and LADA strains were found to induce a significantly enhanced cellular immune response in mice when compared to the L strain. At the same time, the highest level of virus-specific IFN-γ producing cells for the LAD variant was detected on the 7th day post-immunization (dpi), whereas for LADA, it was observed on 14 dpi. The LAD and LADA strains induced significantly elevated levels of VACV-specific IgG compared to the original L strain, particularly between 28 and 56 dpi. The vaccinated mice were intranasally infected with the cowpox virus at a dose of 460 LD50 to assess the protective immunity at 62 dpi. The LADA virus conferred complete protection to mice, with the LAD strain providing 70% protection and the parent strain L offering protection to only 60% of the animals.

Plasmid DNA-based vaccines are attracting considerable interest because of their potential as a platform technology that can be used for a variety of purposes from prevention to therapy. The COVID-19 pandemic has stimulated the development of this platform. The DNA vaccine against COVID-19, developed by Zydus Cadila, was the world’s first DNA vaccine approved for human vaccination. However, the problem of low immunogenicity of DNA vaccines has not yet been completely solved. This article will describe the authors’ experience in creating plasmid DNA-based vaccines against COVID-19, including the design of target antigens, artificial polyepitope T-cell immunogens, delivery of the resulting plasmid constructs using polycationic biodegradable polymers, and producing artificial self-assembled particles incorporating the recombinant protein and DNA vaccine.

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.

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).

The mouse paramyxovirus Sendai, which is capable of limited replication in human bronchial epithelial cells without causing disease, is well suited for the development of vector-based intranasal vaccines against respiratory infections, including SARS-CoV-2. Using the Moscow strain of the Sendai virus, we developed a vaccine construct, Sen-Sdelta(M), which expresses the full-length spike (S) protein of the SARS-CoV-2 delta variant. A single intranasal delivery of Sen-Sdelta(M) to Syrian hamsters and BALB/c mice induced high titers of virus-neutralizing antibodies specific to the SARS-CoV-2 delta variant. A significant T-cell response, as determined by IFN-γ ELISpot and ICS methods, was also demonstrated in the mouse model. Mice and hamsters vaccinated with Sen-Sdelta(M) were well protected against SARS-CoV-2 challenge. The viral load in the lungs and nasal turbinates, measured by RT-qPCR and TCID50 assay, decreased dramatically in vaccinated groups. The most prominent effect was revealed in a highly sensitive hamster model, where no tissue samples contained detectable levels of infectious SARS-CoV-2. These results indicate that Sen-Sdelta(M) is a promising candidate as a single-dose intranasal vaccine against SARS-CoV-2, including variants of concern.

Introduction. Obtaining stabilized recombinant HIV-1 Env trimers that have a close to the native conformation is one of the directions in the field of development of vaccines against HIV-1. The aim of the study was to obtain and characterize the stabilized trimer Env of HIV-1 SOSIP.664 based on the circulating genetic variant of the recombinant form CRF63_02A6. Materials and methods. Bioinformatics resources were used to design the trimer Env gene based on the HIV-1 recombinant genetic variant CRF63_02A6. The designed gene was synthesized and cloned as part of an integration plasmid vector. A stable producer of trimer Env was obtained by transfection of the CHO-K1 cell line using the developed plasmid vector. Purification of the protein complex was performed using affinity chromatography and gel filtration. Antigenic properties of trimer Env were studied using immunochemical analysis using broadly neutralizing HIV-1 monoclonal antibodies (bnAbs). Results. A new variant of the stabilized trimer Env of the surface glycoprotein of the recombinant form CRF63_02A6 HIV-1 SOSIP.664 was designed, including additional stabilizing modifications. Based on the CHO-K1 cell line, a stable producer was obtained and a purification protocol for the designed trimer Env was developed. It was found that the trimer Env CRF63_02A6 SOSIP.664 is effectively recognized by bnAbs 2G12, VRC01 and PGT126. Conclusion. The obtained results indicate the prospects for further study of the structural features of the trimer Env CRF63_02A6 SOSIP.664, as well as its immunogenicity and the possibility of using it as a vaccine antigen.

INTRODUCTION:Nucleic acids represent a promising platform for creating vaccines. One disadvantage of this approach is its relatively low immunogenicity. Electroporation (EP) is an effective way to increase the DNA vaccines immunogenicity. However, due to the different configurations of devices used for EP, EP protocols optimization is required not only to enhance immunogenicity, but also to ensure greater safety and tolerability of the EP procedure.AREA COVERED:An data analysis for recent years on the DNA vaccines delivery against viral and parasitic infections using EP was carried out. The study of various EP physical characteristics, such as frequency, pulse duration, pulse interval, should be considered along with the immunogenic construct design and the site of delivery of the vaccine, through the study of the immunogenic and protective characteristics of the latter.EXPERT OPINION:Future research should focus on regulating the humoral and cellular response required for protection against infectious agents by modifying the EP protocol. Significant efforts will be directed to establishing the possibility of redirecting the immune response toward the Th1 or Th2 response by changing the EP physical parameters. It will allow for an individual selective approach during EP, depending on the pathogen type of an infectious disease.

During the COVID-19 pandemic, the development of prophylactic vaccines, including those based on new platforms, became highly relevant. One such platform is the creation of vaccines combining DNA and protein components in one construct. For the creation of DNA vaccine, we chose the full-length spike protein (S) of the SARS-CoV-2 virus and used the recombinant receptor-binding domain (RBD) of the S protein produced in CHO-K1 cells as a protein component. The immunogenicity of the developed combined vaccine and its individual components was compared and the contribution of each component to the induction of the immune response was analyzed. The combined DNA/protein vaccine possesses the advantages of both underlying approaches and is capable of inducing both humoral (similar to subunit vaccines) and cellular (similar to DNA vaccines) immunity.