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Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, five virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and five intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certified for vaccine production in accordance with the “Guidelines for Clinical Trials of Medicinal Products”. PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.

Rheumatoid arthritis (RA) is a serious systemic disease of connective tissue, mainly affecting joints but also with different extra-articular manifestations. In the course of RA the degenerative changes occur in cartilage surfaces of affected joints and also in subchondral bone tissue, joints get deformed and lose their mobility. RA affects about 1 % of the global human population. Biological therapy with recombinant protein inhibitors of inflammatory cytokines is an effective and well-accepted treatment of RA. TNF inhibitors such as recombinant receptors or monoclonal antibodies are the most widely used biotherapeutics in clinical practice. However, this treatment has some serious side effects. The patients treated with TNF inhibitors are more susceptible to infection diseases, they are also at higher risk of developing neoplastic or autoimmune disorders. Biotherapeutics become less effective or even lose their efficiency with evoking specific antidrug antibodies. These drawbacks are in general associated with repeated systemic injections of large amounts of recombinant protein required to achieve the therapeutic efficacy. Genetic therapy might provide a good and effective solution. Viral genes coding for immunomodulatory factors could be used to create new gene therapy products to treat RA and other human disease. Poxviruses, as compared to other viral families, have an unprecedentedly rich set of such immunomodulatory genes. In particular, they have genes encoding TNF-binding proteins. Previously in a variety of laboratory models we have shown that recombinant TNF-binding protein CrmB can effectively block TNF. In this work we demonstrated that candidate antirheumatic genotherapeutic plasmid constructions encoding poxviral TNF-binding proteins have low immunogenicity.

In the world of today, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is selective destruction of cancer cells with minimizing the destructive effect on normal cells of the body. A promising basis for the creation of oncolytic drugs is orthopoxviruses, which have a number of advantages over other viral vectors, and one of these advantages is a large capacity of the genome, which allows genes encoding proteins with antitumor properties to be cloned into their genome. In this study, we compared the replicative properties of ten variants of vaccinia virus (the strain LIVP of VACV) using human glioblastoma cell culture; some of these viruses have additional genes, such as the gene encoding granulocyte-macrophage colony stimulating factor, gene encoding apoptosis-inducing protein TRAIL and gene encoding green fluorescent protein. Furthermore, the virus with five virulence genes deleted (genes encoding hemagglutinin, γ-interferonbinding protein, thymidine kinase, complementbinding protein and Bcl2-like inhibitor of apoptosis), which has significantly lower reactogenicity and neurovirulence compared to the original strain LIVP of VACV, was studied. These data suggest that variants of vaccinia virus with a defective gene encoding thymidine kinase most actively replicate in glioblastoma cell culture.