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:Intranasal vaccination using live vector vaccines based on non-pathogenic or slightly pathogenic viruses is the one of the most convenient, safe and effective ways to prevent respiratory infections, including COVID-19. Sendai virus is the best suited for this purpose, since it is respiratory virus and is capable of limited replication in human bronchial epithelial cells without causing disease. The aim of the work is to design and study the vaccine properties of recombinant Sendai virus, Moscow strain, expressing secreted receptor-binding domain of SARS-CoV-2 Delta strain S protein (RBDdelta) during a single intranasal immunization.MATERIALS AND METHODS:Recombinant Sendai virus carrying insertion of RBDdelta transgene between P and M genes was constructed using reverse genetics and synthetic biology methods. Expression of RBDdelta was analyzed by Western blot. Vaccine properties were studied in two models: Syrian hamsters and BALB/c mice. Immunogenicity was evaluated by ELISA and virus-neutralization assays. Protectiveness was assessed by quantitation of SARS-CoV-2 RNA in RT-PCR and histological analysis of the lungs.RESULTS:Based on Sendai virus Moscow strain, a recombinant Sen-RBDdelta(M) was constructed that expressed a secreted RBDdelta immunologically identical to natural SARS-CoV-2 protein. A single intranasal administration of Sen-RBDdelta(M) to hamsters and mice significantly, by 15 and 107 times, respectively, reduced replicative activity of SARS-CoV-2 in lungs of animals, preventing the development of pneumonia. An effective induction of virus-neutralizing antibodies has also been demonstrated in mice.CONCLUSION:Sen-RBDdelta(M) is a promising vaccine construct against SARS-CoV-2 infection and has a protective properties even after a single intranasal introduction.

The aim of the study was to create a highly immunogenic vaccine construct based on a recombinant variant of a replication-defective MVA strain of vaccinia virus, expressing virus-like particles that mimic natural infection with Marburg virus. Materials and methods. The recombinant virus was obtained through recombination between homologous viral DNA sequences and the insertion plasmid pDel2-GP-VP-Pat which carries transgenes of the structural proteins GP and VP40 of Marburg virus, flanked by fragments of MVA strain genome. Structure of the recombinant virus was confirmed in PCR and using sequencing, transgenes expression was analyzed by Western blotting, viruslike particles formation was recorded using electron microscopy. Evaluation of immunogenicity and protectivity was carried out using a guinea pig model. The antibody titer was determined in enzyme-linked immunosorbent assay. To assess T-cell response, the intracellular staining of cytokines was used, followed by analysis of samples on a flow cytometer. Results and discussion. On the basis of highly attenuated MVA strain of vaccinia virus a recombinant variant MVA-GP-VP40-MARV has been constructed, carrying a cassette of transgenes, GP and VP40, of Marburg virus in the region of deletion II of the genome. The expression of transgenes in MVA-permissive CER cells infected with recombinant MVA-GP-VP40-MARV strain and secretion of GP and VP40 proteins into culture medium have been demonstrated. Electron microscopy analysis has revealed the presence of Marburg virus-like particles in the culture medium of cells 12 hours after infection. Double vaccination of guinea pigs with MVA-GP-VP40-MARV strain at a dose of 108 PFU/animal induced the formation of antibodies to Marburg and vaccinia viruses, as well as 100 % protection against lethal Marburg virus infection (50 LD50). Using original TEpredict software, the structure of T-helper epitopes of GP protein has been predicted. Using the ICS method, the biological activity of these epitopes has been experimentally confirmed and it was shown that they provide the induction of a T-cell immune response as part of the MVA-GP-VP40-MARV vaccine construct.

Mucosal immunity is realized through a structural and functional system called mucose-associated lymphoid tissue (MALT). MALT is subdivided into parts (clusters) depending on their anatomical location, but they all have a similar structure: mucus layer, epithelial tissue, lamina propria and lymphoid follicles. Plasma cells of MALT produce a unique type of immunoglobulins, IgA, which have the ability to polymerize. In mucosal immunization, the predominant form of IgA is a secretory dimer, sIgA, which is concentrated in large quantities in the mucosa. Mucosal IgA acts as a first line of defense and neutralizes viruses efficiently at the portal of entry, preventing infection of epithelial cells and generalization of infection. To date, several mucosal antiviral vaccines have been licensed, which include attenuated strains of the corresponding viruses: poliomyelitis, influenza, and rotavirus. Despite the tremendous success of these vaccines, in particular, in the eradication of poliomyelitis, significant disadvantages of using attenuated viral strains in their composition are the risk of reactogenicity and the possibility of reversion to a virulent strain during vaccination. Nevertheless, it is mucosal vaccination, which mimics a natural infection, is able to induce a fast and effective immune response and thus help prevent and possibly stop outbreaks of many viral infections. Currently, a number of intranasal vaccines based on a new vector approach are successfully undergoing clinical trials. In these vaccines, the safe viral vectors are used to deliver protectively significant immunogens of pathogenic viruses. The most tested vector for intranasal vaccines is adenovirus, and the most significant immunogen is SARSCoV-2 S protein. Mucosal vector vaccines against human respiratory syncytial virus and human immunodeficiency virus type 1 based on Sendai virus, which is able to replicate asymptomatically in cells of bronchial epithelium, are also being investigated.

Accurate measurement of tumor size and margins is crucial for successful oncotherapy. In the last decade, non-invasive imaging modalities, including optical imaging using non-radioactive substrates, deep-tissue imaging with radioactive substrates, and magnetic resonance imaging have been developed. Reporter genes play the most important role among visualization tools; their expression in tumors and metastases makes it possible to track changes in the tumor growth and gauge therapy effectiveness. Oncolytic viruses are often chosen as a vector for delivering reporter genes into tumor cells, since oncolytic viruses are tumor-specific, meaning that they infect and lyse tumor cells without damaging normal cells. The choice of reporter transgenes for genetic modification of oncolytic viruses depends on the study objectives and imaging methods used. Optical imaging techniques are suitable for in vitro studies and small animal models, while deep-tissue imaging techniques are used to evaluate virotherapy in large animals and humans. For optical imaging, transgenes of fluorescent proteins, luciferases, and tyrosinases are used; for deep-tissue imaging, the most promising transgene is the sodium/iodide symporter (NIS), which ensures an accumulation of radioactive isotopes in virus-infected tumor cells. Currently, NIS is the only reporter transgene that has been shown to be effective in monitoring tumor virotherapy not only in preclinical but also in clinical studies.

Long-term efforts of the international community led to the development of highly efficient direct-acting antivirals (DAAs) that allow treatment of the vast majority of patients with chronic hepatitis C. The proteins encoded in the genome of the hepatitis C virus (HCV) that play a key role in its life cycle (NS3, NS5A, and NS5B) are the targets of this type of drug. There are three classes of DAAs each of which is directed to the inhibition of a specific target protein. Since the HCV has a sufficiently high rate of accumulation of mutations, the development of resistance to these drugs is a big problem. The current recommended treatment regimens with DAAs without the use of interferon and ribavirin are a combination of drugs of different classes providing an increase in the barrier of resistance. Due to the emergence of DAAs, a number of countries (WHO members, with the involvement of Russia) put forward a global strategy to eradicate the HCV. Taking into account the high cost of DAAs and a large number of HCV-infected individuals in Russia, achieving the goals declared by the WHO presents great financial difficulties for our country. However, federal funds allocated for hepatitis C therapy increased significantly over the past 3 years. In addition to increased funding, there is a great potential for reducing the cost of treatment, but its implementation is impossible without the organization of national production of quality generics, issuance of compulsory licenses (given that it is impossible to negotiate with patent holders on licensing), and/or negotiations on price reduction in exchange for volume (for example, the experience of Australia and Portugal). Anyways, Russia faces a very important task to provide therapy for several million patients with hepatitis C in the coming years to get closer to the goal of eradication of the HCV set by the international community.

Multiple lines of evidence indicate that CAR-T cell based therapy and oncolytic virotherapy display robust performance in both immunocompetent and immunodeficient mouse models. Rare, yet highly successful attempts to combine these therapeutic platforms have also been reported. Interestingly, both approaches have shown pronounced efficacy in human trials, albeit these were limited to just a handful of malignancies. Specifically, CD19-specific CAR-T cell products (Kymriah and Yescarta) have been highly effective against B cell lymphomas and leukemias, whereas administering oncolytic viruses resulted in pronounced responses in melanoma (Imlygic and Rigvir) and nasopharyngeal carcinoma (Oncorine) patients. It is well established that efficacy of virotherapy as a standalone approach is largely restricted by the pre-existing and mounting immune response against viral antigens, and requires a relatively functional immune system, which is not typical for cancer patients, with the current antitumor therapy schemes. On the other hand, the most important challenges faced by the current CAR-T cell therapy formats include the lack of targetable tumor-specific surface antigens, tumor cell heterogeneity, and immunosuppressive tumor microenvironment, not to mention the unacceptably high costs. Remarkably, combining the two approaches may help address their individual bottlenecks. Namely, local acute inflammatory reaction induced by the viral infection may reverse tumor-associated immunosuppression and lead to more efficient homing and penetration of CAR-expressing lymphocytes into the tumor stroma; combined viral and CAR-mediated cytotoxicity may ensure the production of immunogenic cell debris and efficient presentation of tumor neoantigens, and potently recruit the patient’s own bystander immune cells to attack cancer cells. Thus, testing the combinations of CAR-based and virolytic approaches in the clinical setting appears both logical and highly promising.

Both vaccinia virus and rat parvovirus H-1 are currently used to create drugs that have demonstrated their effectiveness against a number of human tumors. In preclinical studies it was demonstrated that the main factor responsible for the oncolytic activity of parvovirus H-1 is the NS1 protein and tumor cells of the brain were especially susceptible to its direct cytotoxic effect. We used NS1 gene insertion to arm two attenuated strains of vaccinia virus for virotherapy of glioblastoma: VV-NS1-dGF and MVA-NS1. Recombinant VV-NS1-dGF was engineered from replicative-competent L-IVP strain attenuated by deletion of the virus growth factor gene. MVA-NS1 was engineered from highly attenuated MVA strain, which is replicative-incompetent for mammalian cells. The NS1 transgene expression significantly enhanced the lytic activity of both recombinants against human U87MG glioblastoma cells regardless of the level of attenuation of the original virus strains in vitro. However, in in vivo experiments, the recombinant VV-NS1-dGF more effectively destroys the subcutaneous xenografts of U87MG cells in Nu/Nu mice compared with the strain MVA-NS1 after a single injection of viruses into the tumor region. Both recombinants selectively replicate in tumor cells, but reproductive capacity of the VV-NS1-dGF is higher, which ensures its rapid accumulation in xenografts and their subsequent destruction. Investigation of the antitumor properties of recombinants in the orthotopic model of U87MG human glioblastoma demonstrated an increase in the lifetime of mice and a significant decrease in tumor volume when treated with VV-NS1-dGF. The data obtained demonstrate that recombinant VV-NS1-dGF has good therapeutic potential against human glioblastoma.

This review summarizes the results of a comprehensive multi-year molecular-epidemiological research on HCV (2001–2016) in Altai krai and Novosibirsk oblast. The first eight cases of infection with the CRF01_1b2k recombinant form of HCV were revealed during this research. The recombination point in the NS2 gene was confirmed for all CRF01_1b2k Siberian isolates, which agrees with the data on the previously identified recombinants of the same form in other regions of Russia and the rest of the world. An analysis of the nucleotide sequences of the Core , E1 , NS2 , and NS5b gene fragments in the genomes of eight recombinant isolates of the 2k/1b type identified in Siberia showed a close phylogenetic relationship between them, as well as with 27 recombinants described in St. Petersburg, Azerbaijan, Armenia, Estonia, The Netherlands, Ireland, France, and the United States. The levels of homology for the Core , Е1 , NS2 , and NS5b genes were 97, 94, 92, and 96%, respectively. Phylogenetic analysis of the genomes of recombinants indicated the common origin of isolates of the 2k/1b type and their broad circulation in the territory of Russia. Analysis of the 5'‑region of the genome of recombinant isolates of subtype 2k showed that, apart from Russia, the closest isolates of the 2k subtype were previously identified in Moldova and Uzbekistan in 1996–2013. The incidence of recombinant isolates in the Siberian region is estimated at 1%. The molecular clock method has shown that the most probable time of the appearance of the CRF01_1b2k recombinant form is between 1957 and 1970.

Recombinant VV-GMCSF/lact-dGF was constructed using L-IVP strain of vaccinia virus (VACV). The recombinant strain possesses deletions of gene fragments of viral thymidine kinase and growth factor, and insertion of chimeric peptide GMCSF/lact into region of thymidine kinase gene deletion. The transgene includes gene adjuvant GM-CSF connected with oncotoxic peptide lactaptin by flexible linker GlyGlyGlySer. The transgene expresses under control of natural promoter P7.5k of VACV and produces a secreted form of chimera protein GMCSF/lact (46 kDa), which have biological activity of GM-CSF and lactaptin. Strain VV-GMCSF/lact-dGF demonstrated highly targeted oncolytic activity toward human tumor cells of various origination. Single injection of the VV-GMCSF/lact – dGF strain in a dose 10 7 PFU/mouse into xenografts of human breast cancer BT-549 cells in nude mice results in complete destruction of tumor tissue and its infiltration by effector cells of immune system. The strain VV-GMCSF/lact – dGF looks promising for elaboration of a preparation for malignancies treatment, and further studies of the mechanisms of its antitumor activity are reasonable.

Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity.

Human carcinoma A431 cells were subcutaneously injected into nude mice at points remote from each other. One of the two xenografts developed afterwards was used for the treatment with a recombinant vaccinia virus, while the other xenograft served as an artificial metastasis. We used the attenuated recombinant vaccinia virus (VACV) VVdGF-GFP2 of the L-IVP strain (GenBank accession number KP233807) with the deletion of two virulence genes, i.e., the genes of the virus growth factor and thymidine kinase, with the gene for the green fluorescent protein (GFP2) inserted in the place of the latter. The treatment was performed by a single intratumoral injection of recombinant VACV at a dose of 107 PFU/mouse. VACV was detected in cells of the artificial metastasis as early as two days after infection. After eight days, the virus concentration was comparable with that in the infected tumor (~109 PFU/mL). Electron microscopy revealed selective replication of the recombinant virus in the tumor cells. The targeted accumulation of GFP2 in both tumor and metastasis was shown in the UV images of the mice obtained using the In-vivo Multispectral Imaging System (Bruker, Germany). The complete destruction of the tumor was detected after 12 days, and that of metastasis, after 20 days after the injection of VVdGF-GFP2. The destruction process was accompanied by pronounced edema and leukocyte infiltration of the tumor tissue. The recombinant virus induced a significant reduction in the sizes of the tumor and metastasis. By the end of the experiment (35 days), the xenografts in the control mice were 10 times larger than those in the treated mice (5000 vs 500 mm3). Our study has shown that the attenuated VACV administered by the peripheral route can not only destroy the primary tumor but also has a distinct antimetastatic effect.

Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase-3 and -7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings.

The recombinant vaccinia virus strain VV-GMCSF-S1/3, which contains an insertion of full-length DNA copy of messenger RNA of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in the structural part of the viral thymidine kinase gene, was obtained. The expression of the GM-CSF gene as a part of the recombinant virus is under the control of the native vaccinia virus promoter р7.5K; this results in the production of a mature form of the secreted protein with a molecular mass of 32 kDa. The biological activity of GM-CSF was evaluated by stimulation of the proliferation of cytokine-dependent human TF-1 erythroleukemia cells. The secretion level of biologically active human GM-CSF in the system of recombinant vaccinia virus/mammalian cells was 1–40 μg/mL of culture medium. The recombinant strain VV-GMCSF-S1/3 can be used as a producer of the glycosylated mature form of human GM-CSF, as well as a vector for the construction of oncolytic viruses and multivalent vaccine preparations.

Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%.

Antigenic profiles of envelope glycoproteins of hepatitis C virus presented by three genotypes 1b, 2a/2c and 3a, which are most widespread in the territory of Russia and, in particular, in Novosibirsk, were studied using a panel of overlapping synthetic peptides. It was shown that highly immunogenic peptide epitopes of Е1 and Е2 proteins common for all HCV genotypes, are located in amino acid positions 250-260, 315-325 (Е1 protein), 390-400 (hypervariable region 1), 430-440, and 680-690 (Е2 protein). The greatest inter-genotypic differences were recorded in positions 280-290, 410-430 and 520-540. A novel antigenic determinant was detected in the region of aa 280-290 of the Е1 protein which was typical only for HCV 2a/2c genotype. A broad variation in the boundaries for the most epitopes suggests a high variability of the Е1 and Е2 viral proteins; however, a similar repertoire of antibodies induced by different HCV genotypes indicates to an opportunity of designing a new generation of cross-reactive HCV vaccines based on mapping of the E1 and E2 antigenic regions.