Olga V. Zubkova

Adenovirus vector vaccines became an important class of vaccines following their approval for the prevention of Ebola virus fever and COVID-19. Quality dat

Introduction. The spread of tick-borne encephalitis virus (Orthoflavivirus encephalitidis) continues. Existing vaccines have several limitations. Research new vaccine platforms will enable the development of more effective strategies of disease prevention. Materials and methods. The constructs for producing vaccine preparations were obtained using the methods described earlier. Study of the effectiveness of candidate vaccines based on chimeric yellow fever virus, adenovirus serotype 25 and mRNA encoding a fragment of the tick-borne encephalitis viral polyprotein corresponding to the PrM and E regions, immunogenicity and protective efficacy was performed using BALB/c mice. The neutralizing activity assessment of animal sera and mice challenge were performed using the tick-borne encephalitis virus strain Sofjin-Chumakov. Results. It was established that immunization of animals with mRNA encoding the PrM and E regions of the viral polyprotein of tick-borne encephalitis virus resulted in the induction of a higher level of neutralizing antibodies compared to the other vaccine platforms studied in the immunization schedule used. In addition, this platform provided 100% protection of animals from the lethal infection caused by the tick-borne encephalitis virus. Conclusion. The study demonstrated that delivery of the nucleotide sequence encoding the PrM and E regions of the tick-borne encephalitis virus polyprotein within mRNA elicits a pronounced humoral immune response during animal immunization. These results demonstrate the fundamental feasibility of effectively expressing the same tick-borne encephalitis virus antigen in various vaccine platforms, each with its own advantages and limitations.

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. Adenovirus infection occurs globally in the form of sporadic cases and isolated outbreaks. Human adenovirus type 55 (HAdV-55), endemic in China and South Korea, causes acute respiratory viral infections (ARVI) of varying severity, both among the civilian population and in military units in different countries of the world. Genomic research facilitates reliable identification of HAdV-55. The aim of this study was to identify HAdV isolated in Moscow in 2022, as well as to conduct whole-genome sequencing and comparative genomic research. Materials and methods. HAdV-55 was isolated from a sample of a patient hospitalized with pneumonia and analyzed using restriction fragment length polymorphism analysis and whole-genome sequencing. Bioinformatics comparative analysis was performed on a sample of sequences of 83 isolates. Results. The whole-genome sequencing of first isolated in Russia HAdV-55 was conducted. The sequence of isolate SCV3008:Ad55 was deposited in GenBank (Accession Number PQ641625). Unique mutations in the SCV3008:Ad55 genome were identified, one of which resulted in a conservative T29A substitution in the penton that did not affect its functions. Phylogenetic analysis showed clustering of SCV3008:Ad55 with isolates of clade II, which included representatives of 7 countries on different continents, indicating a wide distribution of HAdV-55. Isolates from endemic regions of China and South Korea formed separate clades. The study of microsatellite length polymorphism in untranslated regions of the genome became an additional tool for distinguishing closely related genomes. Conclusion. The obtained genomic information laid the foundation for further monitoring for HAdV-55 in Russia and demonstrated the informativeness and significance of whole-genome studies for monitoring adenoviruses. The development and implementation of genotyping methods aimed at detecting HAdV-55 and other clinically relevant genotypes will significantly improve the effectiveness of the diagnosis of adenovirus infections with the threat of developing bronchopneumonia.

Despite the widespread accessibility of vaccines and antivirals, seasonal influenza virus epidemics continue to pose a threat to public health. In this study, we constructed a recombinant replication-deficient simian adenovirus type 25 vector carrying the full-length hemagglutinin (HA) of the H1N1 influenza virus, named rSAd25-H1. Both systemic and mucosal humoral immune responses, as well as the protective efficacy, were assessed in mice immunized via the intramuscular (IM) or intranasal (IN) route. A single-dose IM or IN administration of rSAd25-H1 elicited a robust systemic IgG antibody response, including hemagglutination inhibition antibodies. As expected, only IN immunization was able to induce IgA production in serum and respiratory mucosa. Notably, a single dose of rSAd25-H1 at the highest dose (10¹⁰ viral particles) conferred complete protection against lethal homologous H1N1 challenge in mice despite the route of administration. These findings demonstrate the potential of simian adenovirus type 25-based vectors as a promising candidate for intranasal vaccine development targeting respiratory pathogens.

Background: Organoids are a valuable model for studying hereditary diseases such as cystic fibrosis (CF). Recombinant adenoviral (rAdV) and adeno-associated viral (rAAV) vectors are promising tools for CF gene therapy and genome editing. Objective: This study aims to determine the most efficient viral vector (rAdV5, rAAV serotypes 5, 6 and 9) and transduction protocol for delivering transgenes to lung organoids (LOs), providing a foundation for future CF gene therapy development. Methods: Three transduction protocols were used taking into account the specificities of LOs’ cultivation in specific matrices, both with and without organoid extraction from the matrix. This work was carried out on organoids from a healthy donor (LOs-WT) and on a patient with cystic fibrosis (LOs-CF). Results: High transduction efficiency was observed with rAdV5 (30% cells), rAAV6 (>80% cells), and rAAV9 (>40% cells). rAdV5 and rAAV9 transduced basal and secretory cells with >90% efficiency. For rAAV9, Protocol 1 (without extraction of organoids from the matrix) showed lower transduction efficiency (33% for LOs-WT, 9% for LOs-CF), significantly lower than that of Protocols 2 (60% for LOs-WT, 59% for LOs-CF) and 3 (46% for LOs-WT, 35% for LOs-CF) with organoid extraction from the matrix (p < 0.005). Conclusions: rAdV5 and rAAV9 are the most promising vectors for the delivery of transgenes to basal and secretory cells in a lung organoid model, providing a solid foundation for CF gene therapy development.

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.

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.