State Research Center of Virology and Biotechnology VECTOR

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m-TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m-TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.

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: The neonatal Fc receptor (FcRn) is a heterodimeric protein composed of a heavy α-chain with an MHC class I-like fold and β₂-microglobulin. It plays a crucial role in maintaining the homeostasis and pharmacokinetics of immuno-globulin G (IgG) and albumin through pH-dependent recycling. The production of soluble recombinant FcRn is technically challenging due to its heterodimeric structure and the presence of a transmembrane domain. This study aimed to develop a polycistronic construct enabling the co-expression of FcRn subunits from a single transcript and to evaluate the functional activity of the resulting protein in CHO-K1 cells. Methods: Integration vectors (pComV-FcRn-B2M) were designed to encode FcRn and β₂-microglobulin linked via self-cleaving 2A peptides (P2A, E2A, F2A, T2A). Stable producer cell lines were generated using the Sleeping Beauty transposon system. The purified proteins were characterized by SDS-PAGE, Western blotting, and size-exclusion chromatography (SEC). Functional activity was assessed by ELISA and bio-layer interferometry (BLI). Results: Electrophoretic and chromatographic analyses confirmed the expected subunit composition and demonstrated that over 95% of the recombinant protein was monomeric. Functional assays revealed pH-dependent IgG binding, with strong interaction at pH 6.0 and negligible binding at pH 7.5. BLI meas-urements showed high affinity consistent with native FcRn function (KD = 3.15 nM at pH 6.0). Conclusions: The developed polycistronic construct containing a P2A peptide with a GSG linker enabled efficient production of functional FcRn in CHO-K1 cells (yield up to 2.23 mg/mL). The P2A variant demonstrated the highest efficiency and can serve as a reference system for screening Fc-engineered antibodies with optimized pharmacokinetic properties.

The COVID-19 pandemic has focused the worldwide attention on the fight against this infection. Development of preventive vaccines based on different platforms, including DNA and RNA vaccines, genetic vectors and subunit vaccines A has been the key approach to combating COVID-19. The subunit vaccines were among these platforms, primarily, due to their unique safety profile. However, the safety of these vaccines is often associated with low efficiency. Hence, their application often requires usage of immune adjuvants, along with more complex immunization regimens. Meanwhile, an important advantage of subunit vaccines is their scalability and relative ease of production, since there is no need to work with live virus or viral vectors during the production process. The purpose of our work was to develop a candidate vaccine based on the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike S-protein from the Delta variant (B.1.617.2). The study used immunological techniques employing methods of genetic engineering and biotechnology. In the course of this work, we have developed a producer of recombinant RBD based on the CHO-K1 mammalian cells. To obtain a protein meeting the requirements of injectable drugs, a chromatographic purification scheme was developed, including affinity and ion exchange chromatography. We have proposed a variant of the subunit vaccine “Delta-Vac” developed on this recombinant protein. Immunogenicity of “Delta-Vac” vaccine was tested in the BALB/c mice. The animals were immunized twice at a dose of 50 μg of RBD combined with Al(OH)3, at a two-week interval. The ability of the candidate vaccine was assessed by induced production of specific IgG and neutralizing antibodies in BALB/c mice. Specific antibody titers of immunized animals ranged from 1/105 to 1/106 . Moreover, the blood sera showed neutralizing activity against SARS-CoV-2 (variant Delta, B.1.617.2) at a titer of up to 1/2000. The developed vaccine “Delta-Vac” is highly immunogenic and induces production of neutralizing antibodies against homologous Delta variant as well as heterologous Wuhan and Omicron SARS-CoV-2 variants. Hence, “Delta-Vac” may act as a vaccine candidate and serve as a prototype for the development of subunit vaccines against COVID-19.

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.

This study investigated the antiretroviral efficacy, toxicity profile, and cellular uptake of LNA-modified oligonucleotides within an in vitro HIV infection model. Phosphorothioate (PS) oligonucleotides, designed to bind conserved regions of the HIV-1 genome, were modified at the 3′ and/or 5′ ends with LNA nucleotides. The antiviral properties of oligonucleotides against HIV-1 subtype A6 were evaluated using human MT-4 cell cultures. The antiretroviral activity of LNA-oligonucleotides against HIV-1 has been established. Variations in the 50% inhibitory viral reproductive dose (IC50) values among the oligonucleotides were observed, depending upon both the target and the incorporated LNA modification. The optimal IC50 values (90 ± 10 nM) were achieved using a PS oligonucleotide lacking LNA modifications, which targeted the HIV-1 integrase-encoding genomic region. Optimal IC50 values (90 ± 10 nM) were achieved using a PS oligonucleotide lacking LNA modifications, which targeted the HIV-1 integrase-encoding genomic region. Optimal HIV inhibitory action among LNA constructs was observed in an oligonucleotide with a 5′-end LNA modification targeting the HIV integrase region (IC50 = 1.12 ± 0.03 μM). The introduction of LNA modifications to PS oligonucleotides failed to enhance antiviral activity, as demonstrated by IC50 values revealing significant in vitro HIV-1 inhibitory capacity. The internalization of oligonucleotides demonstrating optimal IC50 values was investigated via flow cytometry and imaging techniques. The PS modification has been demonstrated to exhibit the highest penetration efficiency. The characteristic features included low toxicity (maintaining >92% viable cells after 48 hours of culture), high cytoplasmic membrane sorption capacity (approximately 12% FAM+ cells after 48 hours), high penetration efficiency (approximately 98% FAM+ cells showing cytoplasmic signal), and elevated internalization and entropy ratios.

INTRODUCTION. Vaccination against smallpox was discontinued in 1980 following the success of the World Health Organisation’s Smallpox Eradication Programme. Consequently, more than half of the current world population lacks immunity to orthopoxviruses pathogenic to humans. Since 2022, several countries have been reporting outbreaks of mpox (previously known as monkeypox), which necessitates developing new small molecules active against orthopoxviruses.AIM. The aim was to evaluate the safety and tolerability of NIOCH-14, a Russian anti-smallpox medicinal product, after oral administration in a phase I clinical trial.MATERIALS AND METHODS. The clinical trial was conducted in 90 healthy volunteers at the Federal State Bud­getary Institution of Healthcare ‘Medical and Sanitary Unit No. 163 of the Federal Medical and Biological Agency’ in accordance with Protocol No. NIOCH-01/20 ‘An open, simple, randomised study of the safety, tolerability, pharmacokinetics of NIOCH-14 in volunteers aged 18–50 years in parallel groups’. Investigators used a standard set of clinical, laboratory, and instrument-based testing methods. The clinical trial included six experimental groups of 15 subjects each. Groups 1, 2, and 3 received NIOCH-14 as a single oral dose of 200, 600, and 1200 mg, respectively. Groups 4, 5, and 6 received NIOCH-14 at daily oral doses of 200, 600, and 1200 mg/day, respectively, for 6 days.RESULTS. After single and repeated administration of NIOCH-14 at doses of 200, 600, and 1200 mg to volunteers, the study did not demonstrate any changes in the blood test results (erythrocyte, leucocyte, platelet, and differential leucocyte counts; haemoglobin levels; erythrocyte sedimentation rate values; alanine transaminase, aspartate transaminase, and alkaline phosphatase activity levels; total protein, total bilirubin, glucose, creatinine, urea, and C-reactive protein levels; prothrombin time ratios; thymol turbidity). Similarly, the urinalysis results remained unaltered throughout the entire observation period. No adverse events were observed in volunteers receiving NIOCH-14 at all single doses or repeated doses of 200 and 600 mg. One volunteer receiving the investigational medicinal product at a repeated dose of 1200 mg/day experienced transient epigastric pain on days 2 and 5.CONCLUSIONS. The physical examination, haematology, blood chemistry, and urinalysis results did not reveal any pathological changes in healthy volunteers. The investigational medicinal product, formulated as hard gelatin capsules containing 200 mg of NIOCH-14 and excipients, demonstrated a favourable safety and tolerability profile.

Background: Since the emergence of SARS-CoV-2 in the human population, the virus genome has undergone numerous mutations, enabling it to enhance transmissibility and evade acquired immunity. As a result of these mutations, most monoclonal neutralizing antibodies have lost their efficacy, as they are unable to neutralize new variants. Antibodies that neutralize a broad range of SARS-CoV-2 variants are of significant value in combating both current and potential future variants, making the identification and development of such antibodies an ongoing critical goal. This study discusses the strategy of using heterologous antigens in biopanning rounds. Methods: After four rounds of biopanning, nanobody variants were selected from a phage display library. Immunochemical methods were used to evaluate their specificity to the S protein of various SARS-CoV-2 variants, as well as to determine their competitive ability against ACE2. Viral neutralization activity was analyzed. A three-dimensional model of nanobody interaction with RBD was constructed. Results: Four nanobodies were obtained that specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and exhibit neutralizing activity against various SARS-CoV-2 strains. Conclusions: The study demonstrates that performing several rounds of biopanning with heterologous antigens allows the selection of nanobodies with a broad reactivity spectrum. However, the fourth round of biopanning does not lead to the identification of nanobodies with improved characteristics.

The rapid rise in monkeypox virus infections among humans from 2022 to 2024 has captured the attention of the global healthcare community. In light of the lack of mandatory vaccination and limited data on next-generation vaccines for monkeypox prevention, the urgent development of therapeutic agents has become a priority. One promising approach involves the use of neutralizing monoclonal antibodies. This review highlights significant advancements in the search for antibodies against human pathogenic orthopoxviruses, particularly focusing on their potential application against the monkeypox virus. We also analyze viral proteins that serve as targets for identifying therapeutic antibodies capable of neutralizing a wide range of viruses. Finally, we deemed it essential to address the challenges associated with selecting an animal model that can adequately reflect the infectious process of each orthopoxvirus species in humans.