Search

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.

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.

Based on the highly attenuated vaccinia virus MVA strain, a recombinant variant MVA-GP-VP40-MARV was constructed, which expresses a cassette of the GP and VP-40 genes of the Marburg virus with the formation of immunogenic virus-like particles and protects Guinea pigs from a lethal infection by the Marburg virus.

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.

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.

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.

The chicken anemia virus gene that encodes apoptin, a selective killer of cancer cells, was synthe-sized and inserted into the vaccinia virus (strain L-IVP) genome. The insertion replaces a major part of the viral C11R gene that encodes viral growth factor, which is important for virulence. The recombinant virus VVdGF-ApoS24/2 was obtained by transient dominant selection using the gene of puromycin resistance as a selective marker. The expression of the apoptin gene from a synthetic early-late promoter of vaccinia virus ensured the efficient accumulation of the target protein in VVdGF-ApoS24/2-infected cells. Although recombinant apoptin carried the signal peptide of the virus growth factor at the N-end, the protein was not secreted into the culture medium. The recombinant virus VVdGF-ApoS24/2 exhibited significantly higher selective lytic activity in human cancer cell lines (A549, A431, U87MG, RD, and MCF7) than the parent strain L-IVP and its VVdGF2/6 variant with C11R deletion. These results suggest that the use of apoptin can be an efficient means of enhancing the natural anticancer activity of vaccinia virus.

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.

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%.

Modern data on selection and design of poxviruses, which are able to specifically lyse tumor cells of various origins and induce antitumor immunity and apoptosis of malignant cells, are presented in this review. Work is being carried out in several areas, including attenuation of viruses, insertion of immunomodulatory protein genes, and insertion of antitumor protein genes. Thymidine kinase and viral growth factor genes, inactivation of which results in an inability of the virus to replicate in nondividing cells and thus promotes an increase in selectivity for the tumor cells, make the greatest contribution in attenuation of the virus. Among the immunomodulatory proteins, interleukins 2 and 12 and granulocyte-macrophage colony-stimulating factor were the most promising in oncolytic viral therapy. An attempt to use the p53 protein gene, expressed by the Vaccinia virus, for targeted apoptosis of the tumor cells was described. Use of double and triple viral recombinants, which carry genes of differently directed effects, seems very promising. Encouraging results were obtained using the Vaccinia virus in cancer therapy by means of prodrugs and angiogenesis inhibitors. At present, two poxvirus strains are undergoing a third stage of clinical trials as antitumor drugs in the United States.

Identified are the occurrence, serotypic and genotypic variations of Hepatitis B virus isolates (HBV) among the Novosibirsk region inhabitants (n=2000), native population of the Alarsk District of the Irkutsk Region (n=487) and Shuryshkarsk Township of the Yamalo-Nenets Autonomous District (n=657). Occurrence rate of hepatitis В surface antigen (HBsAg) among different groups of the Novosibirsk Region population varied within the limits of 3,6-35,0 %. It was 8,2 % in Alarsk District, and 3,2 % in Shuryshkarsk Township. HBV isolates of D genotype (92-97 %) prevail among the population of Siberia; few are the cases of A (1,7 %) and C (1,2-8 %) genotypes. The identified varying occurrence of HBV sub-genotypes and HBsAg subtypes in two aboriginal groups of Siberia (D3 sub-genotype and ayw2 subtype - in the Alarsk District, D2 and ayw3 - in Shuryshkarsk Township) suggests the existence of, at least, two isolated HBV virus populations, circulating among different groups of Siberia native population .

Samples of blood serum obtained from patients with symptoms of acute hepatitis, admitted to infectious disease hospitals of Novosibirsk, Barnaul, and Irkutsk, were investigated. The serologic markers of hepatitis E virus (HEV): IgM and IgG antibodies, were determined by enzyme immunoassay (ELISA). Seropositive samples were investigated by the method of reverse transcription and the polymerase chain reaction (RTPCR). Two variants of HEV were detected, and the first human cases of HEV infection in this region were demonstrated. One of the variants was identified as genotype I, the other as genotype III of HEV. The HEV variants found were cultured in a continuous culture of green monkey kidney cells (4647). The ability of the HEV variant belonging to genotype I to persist in cell culture was shown.

The use of a scanning flow cytometer (SFC) to study the evolution of monomers, dimers and higher multimers of latex particles at the initial stage of the immunoagglutination is described. The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60 deg. A comparison of the experimentally measured and theoretically calculated indicatrices allows one to discriminate different types of latex particles (i.e. monomers, dimers, etc.) and, therefore, to study the evolution of immunoagglutination process. Validity of the approach was verified by simultaneous measurements of light-scattering patterns and fluorescence from individual polymer particles. Immunoagglutination was initiated by mixing bovine serum albumin (BSA)-covered latex particles (of 1.8 um in diameter) with anti-BSA IgG. The analysis of experimental data was performed on the basis of a mathematical model of diffusion-limited immunoagglutination aggregation with a steric factor. The steric factor was determined by the size and the number of binding sites on the surface of a latex particle. The obtained data are in good agreement with the proposed mathematical modeling., 12 pages, 6 figures

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264–318) and E2 (aa448–483 and aa496–515) of the HCV glycoproteins, the third of which (aa496–515) is highly conserved (85–95%) amongst genotypes. Antibodies to aa496–515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496–515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.

Western Siberia is the region with little information on the prevalence of hepatitis C virus (HCV) infection, genotypic diversity of HCV isolates and risk factors. A molecular epidemiological survey was conducted to clarify these issues. Four groups of volunteers were included in a cross-sectional study (n = 500 in each group): health care workers; daycare patients from a hospital for drug users, daycare patients from an AIDS prevention and control center; and persons admitted to a local general practice clinic for any reason (outpatients). The anti-HCV IgG prevalence was 4.6% in health care workers, 48.0% in a narcological center, 35.8% in AIDS center, and 5.6% in outpatients. HCV RNA was found in 79.3%-86.3% of seropositives. A total of 388 HCV isolates were genotyped by direct sequencing and phylogenetic analysis of the 5'-UTR and NS5B regions of HCV genome. The genotypes distribution was: 1b--50.3%, 2a--4.4%, 2c--0.3%, 3a--44.8%. One isolate (0.3%) could not be typed unambiguously. This genotypic diversity is intermediate between that of European Russia and China. Genotype 1 prevailed in an older age group (75% among 51-60 years old), and genotype 3 was most prevalent in young people (51.4% in 16-20 years old). A statistically significant (P0.05) increase in risk was found in intravenous drug users (odds ratio (OR) = 77.5), unemployed persons (OR = 16.3), persons having4 sexual partners during lifetime (OR = 4.3), and male homosexuals (OR = 6.6).

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.

The mousepox (ectromelia) virus genome contains four genes encoding kelch-like proteins EVM018, EVM027, EVM150, and EVM167. A complete set of insertion plasmids was constructed to produce recombinant ectromelia viruses with targeted deletions of one to four genes of the kelch family, both individual (single mutants) and combined (double, triple, and quadruple mutants). It was shown that deletions EVM018, EVM027, or EVM167 resulted in a reduction of the 50% lethal dose (LD50) in outbred white mice infected intraperitoneally by five and more orders. Deletion of the mousepox kelch-gene EVM150 did not affect the virus virulence. Deletions of two or more kelch-genes also resulted in high attenuation levels, evidently due to the lack of EVM167, EVM018, and/or EVM027 products identified as virulence factors. The local inflammatory process assessed on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more pronounced for the wild type virus and virulent mutant ΔEVM150 compared to avirulent mutant ΔEVM167.