Inna Dolzhikova

Filoviruses cause highly lethal infectious diseases with hemorrhagic symptoms and extremely high fatality rate (up to 90%). Although two vaccines against Ebola virus (EBOV) are licensed for application in endemic areas, vaccines against other filoviruses (SUDV, BDBV, MARV) are still at pre-clinical or clinical stages. The development of vaccines requires protective efficacy studies using animal models of infection. However, animal studies using wild-type filoviruses require maximum biosafety level (BSL-4), which hinders filoviral vaccine advancement. In this study, we developed a surrogate animal model of EBOV-, SUDV-, BDBV- and MARV- GP-mediated entry using Ifnar1-knockout (-/-) mice and recombinant vesicular stomatitis viruses (rVSVs), carrying a genetic sequence of filoviral glycoprotein (GP) and pseudotyped with correspondent GP. We showed that rVSV-EBOV-GP was 100% lethal for Ifnar1(-/-) mice after inoculation by multiple routes, and its active propagation was within 24 h post-infection. We performed dose-dependent lethality assessment in small groups to estimate median lethal doses for rVSV-EBOV-GP, rVSV-SUDV-GP, rVSV-BDBV-GP, rVSV-MARV-GP in Ifnar1-knockout mice and showed high viral load in liver and immune cell reservoirs. The developed models contribute to safe and effective research of filoviral vaccines and therapeutic antibodies under BSL-2 conditions.

Background/Objectives: Epitope-based mRNA vaccines represent a promising strategy for eliciting protective T-cell immunity against SARS-CoV-2 and as well as for non-infectious mRNA-based vaccines. However, how the structural architecture of vaccine constructs (including epitope arrangement, linker composition, signal peptide presence, and the combination of MHC class I and II epitopes) shapes the quality of T-cell responses remains poorly understood. Methods: Ten tandem minigene mRNA constructs (Cons1–10) encoding different combinations of MHC class I and class II epitopes from SARS-CoV-2 proteins (S, N, M, ORF3a) were designed, encapsulated in lipid nanoparticles, and administered to C57BL/6 mice. Immunogenicity was assessed by cytokine profiling (IFN-γ, IL-2, IL-4, IL-10) and T-cell proliferation assays. Protective efficacy was evaluated in K18-hACE2 transgenic mice challenged with SARS-CoV-2. Results: Constructs lacking a signal peptide and enriched in MHC class I-restricted epitopes induced robust Th1 responses and strong CD8⁺ T-cell proliferation, achieving up to 66% survival following lethal challenge. In contrast, constructs associated with elevated IL-10 and IL-4 production conferred limited protection (11–33%), consistent with functional skewing towards regulatory or Th2-associated immune profiles. Conclusions: These findings establish a direct link between construct design parameters and T-cell polarization quality, and provide a rational framework for next-generation epitope-based mRNA vaccine development.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus causing severe human disease and representing a significant public health threat. The absence of licensed vaccines and specific antiviral therapies highlights the need for well-characterized experimental models suitable for studying viral pathogenicity and evaluating medical countermeasures. In this study, we established and characterized an integrated in vitro and in vivo platform for CCHFV research. Virus propagation was optimized by comparing Vero E6 and SW-13 cell lines. SW-13 cells supported efficient CCHFV replication resulting in pronounced virus-induced cytopathic effects, whereas no detectable cytopathic effects were observed in Vero E6 cells. Virus titers determined by cytopathic effect based endpoint dilution and antigen ELISA coincided, supporting the use of SW-13 cells for virus stock preparation. Using virus stocks generated in SW-13 cells, interferon-deficient mice were infected with different doses of CCHFV to assess dose-dependent pathogenicity. Infection resulted in severe disease and high mortality across all tested doses. Based on survival analysis, a dose of 10 TCID50; was selected for further characterization, which revealed progressive weight loss and high viral loads in peripheral organs. Overall, this study establishes a robust IFN knockout mouse model suitable for studying CCHFV pathogenicity, comparing viral genovariants, and evaluating vaccines, antiviral compounds, and neutralizing antibodies.

Background/Objectives: Flaviviruses, including West Nile virus (WNV), pose global health challenges due to their worldwide distribution, pathogenicity, and lack of effective treatments or vaccines. Today, WNV is considered the most important causative agent of viral encephalitis worldwide. This study investigated the different forms of the main WNV antigen—the preM/E protein—in the context of its immunogenic and protective properties. Methods: The recombinant adenovirus type 2 (rAd2) vectors expressing different forms of the WNV preM/E genes were obtained using standard molecular biology techniques. Immunogenicity in mice was assessed by enzyme-linked immunosorbent assay (ELISA) and virus neutralization assay. Immunological efficacy was evaluated in a mouse viral challenge model. Results: The rAd2 vector expressing the West Nile virus preM/E gene with mutations in the fusion loop exhibited robust immunogenicity when administered intramuscularly either once or in a homologous prime-boost regimen. This antigen form, as part of an adenoviral vector, protected mice from death in viral challenge experiments, providing 100% survival following WNV challenge. Conclusions: We believe that a vaccination strategy involving a recombinant adenoviral vector based on human adenovirus type 2 and the WNV antigen represented by the preM/E gene with mutations in the fusion loop may be a promising approach for combating West Nile virus infection.

Introduction. In 2022, the World Health Organization declared monkeypox a public health emergency. The monkeypox virus (MPV) is part of the Orthopoxvirus genus within the Poxviridae family. During replication, orthopoxviruses produce two distinct forms of viral particles: the extracellular enveloped virion (EEV), released via exocytosis, and the intracellular mature virion (IMV), released through cell lysis. These forms differ in surface proteins composition, influencing their immunogenicity and infectivity. Aim. To evaluate the immunogenic and protective activity of nine surface antigens of vaccinia virus. Materials and methods . Recombinant human adenoviruses type 2 (rAd2) carrying surface antigens of vaccinia virus were obtained using homologous recombination in bacteria, followed by adenoviral particle assembly in HEK293 cells. The immunogenic and protective properties of these adenoviruses were tested in BALB/c mice. The presence of antibodies to the vaccinia virus was assessed using ELISA, and survival rates were evaluated in a lethal infection model after intranasal challenge with the vaccinia virus strain Western Reserve. Results. The most immunogenic and protective antigens of the vaccinia virus within rAd2 were glycoprotein B5 of the EEV and membrane-associated protein H3 of the IMV, both showing 100% protective efficacy after intranasal immunization. Conclusion. Using a panel of recombinant adenoviruses carrying genes of vaccinia virus surface proteins, it was shown that optimal protection is achieved using a combination of enveloped and mature virion antigens. This method could be used for development of new multivalent preparations against various viral infections.

The Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2), a causative agent of the COVID-19 disease, has been constantly evolving since its first identification. Mutations that are embedded in the viral genomic RNA affect the properties of the virus and lead to the emergence of new variants. During the COVID-19 pandemic, the World Health Organization has identified more than ten variants of the SARS-CoV-2 virus. Five of these—Alpha, Beta, Gamma, Delta, and Omicron—were classified as variants of concern (VOCs), as they caused significant outbreaks of the disease. Additionally, two progeny variants of Omicron, designated JN.1 and KS.1, are still causing new waves of infections. Due to the emergence of various SARS-CoV-2 variants, in some cases, it has become important to identify a particular variant in a sample. Here, we have developed a multiplexed probe-based real-time PCR system for the identification of SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, Omicron B.1.1.529/BA.1, and Omicron BA.2), as well as modern Omicron variants JN.1 and KS.1. The sensitivity and specificity of the PCR system have been tested using isolated viral genomes and RNA preparations from human nasopharyngeal swabs. The system allows for rapid identification of coronavirus variants in the cryopreserved and fresh samples.

Lassa hemorrhagic fever is an acute human infectious disease with high mortality rate and pandemic potential. To date, there are no approved drugs for the specific treatment or prevention of Lassa hemorrhagic fever in the world. The aim of this study was to develop and evaluate the immunobiological properties and preclinical safety of a candidate vaccine for the prevention of Lassa hemorrhagic fever (LHF) based on recombinant human adenoviral vectors. Standard genetic engineering techniques, molecular biology techniques, virological methods, and animal testing procedures were used in the course of the study. A combined vector candidate vaccine for the prevention of Lassa hemorrhagic fever has been designed and characterized. The vaccine is composed of two components for heterologous immunization in a prime-boost regimen. Both components are based on recombinant replication-defective adenovirus vectors. The first component of the vaccine is a recombinant human adenovirus type 26; the second component is a recombinant human adenovirus type 5. Both recombinant vectors contain the codon-optimized sequence of Lassa virus glycoprotein. Two experimental batches of the candidate vaccine were produced under GMP-conditions and analyzed. The results of studies in compliance with appropriate specifications for viral vector vaccines are provided. In preclinical studies in mice, antigen-specific IgG response was detected after immunization with two vaccine components, either separately, or in a prime-boost regimen. The time dynamics of the IgG response was also studied on 42, 77, 119 and 147 days after immunization. At the same time, despite achieving 100% seroconversion, no virus neutralizing antibodies were detected in any of the samples collected from immunized mice. A biodistribution study showed that 24 hours following intramuscular injection of the vaccine components, the DNA of adenovirus vectors was detected only at the site of injection and in regional lymph nodes. Based on preclinical safety assessments (including acute toxicity, chronic toxicity, immunotoxicity, allergenic properties, reproductive toxicity), no contraindications were found for initiation of the candidate Lassa vaccine clinical trials. Taken together, the results demonstrate that the candidate vaccine for prevention of Lassa hemorrhagic fever based on recombinant adenovirus vectors types 26 and 5 is a promising drug for specific immunoprophylaxis.

Background: The use of adenoviral vector vaccines is an effective way to induce an antiviral immune response. Most vaccines used worldwide are based on human adenovirus type 5 (Ad5). Pre-existing natural immunity to Ad5 is a limitation to the frequent use of Ad5-based vectors in gene therapy and vaccination. In the present study, we assessed the seroprevalence and cross-reactivity of neutralizing antibodies to six adenovirus types: Ad1, Ad2, Ad5, Ad19, Ad26, and Ad37. Methods: The study used immunological and virological research methods. Adenovirus vectors encoding the enhanced green fluorescent protein (EGFP) were developed by the N.F. Gamaleya National Research Center for Epidemiology and Microbiology. For seroprevalence and cross-neutralation study we used a panel of sera from 101 volunteers. Results: The conducted correlation analysis showed a strong relationship between seropositivity and the geometric mean titer of neutralizing antibodies to adenovirus. An increase in antibody titers indicates the formation of a humoral immune response and population immunity. We determined that there is no cross-reactivity between NtAbs to Ad1, Ad2, Ad5 and Ad26. Conclusions: Based on the presented data, we assume that Ad1 or Ad2 should be considered the most promising alternative candidates for the development of vaccine vectors.

Introduction. Many different vaccines for the prevention of COVID-19 have received emergency use approval in the shortest possible time. Due to the high rate of variability of the pathogen, in this study we analyzed the variability of the structural proteins of the SARS-CoV-2 virus and compared their protective potential in protecting animals from COVID-19. The aim of the study was to compare the protective potential of the SARS-CoV-2 structural proteins in protecting animals from COVID-19. Materials and methods. The SARS-CoV-2 virus was used in the study. Transgenic mice B6.Cg-Tg(K18-ACE2)2Prlmn/J (F1) were used as model animals. Recombinant adenoviral vectors rAd5-S, rAd5-N, rAd5-M were used for immunization of animals. Various genetic, virological and immunological methods, as well as methods of working with animals, were used in the study. Results. The largest number of amino acid substitutions in the structural proteins of different SARS-CoV-2 variants was detected in glycoprotein S, the smallest — in nucleoprotein N. In the COVID-19 animal model, it was shown that only the use of glycoprotein S as a vaccine antigen allows to form protective immunity that protects 100% of animals from a lethal infection caused by the SARS-CoV-2 virus, while the use of protein N protects 50% of animals from a lethal infection, and protein M does not have a protective potential. Conclusion. The data obtained, as well as the analysis of the epidemiological efficacy of various mRNA and vector vaccines, demonstrate that the use of the SARS-CoV-2 glycoprotein S as an antigen allows to form the highest level of protection. Due to the constant change in circulating variants of the SARS-CoV-2 virus, the decrease in the effectiveness of the vaccines with the original antigen composition against new variants of the virus and the continuing high incidence of COVID-19, it is necessary to continuously monitor the effectiveness of vaccines against new variants of the virus and promptly update the antigen composition of vaccines when a decrease in effectiveness is detected.