[Intranasal vaccine against COVID-19 based on a recombinant variant of the Sendai virus (Paramyxoviridae: Respirovirus) strain Moscow].

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.

Reporter Transgenes for Monitoring the Antitumor Efficacy of Recombinant Oncolytic Viruses.

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.

[Mucosal immunity and vaccines against viral infections].

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.

[Combination of Oncolytic Virotherapy and CAR T/NK Cell Therapy for the Treatment of Cancer].

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.

[Apoptin enhances the oncolytic activity of vaccinia virus].

Chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain L-IVP) genome. The insertion has replaced major part of the viral C11R gene encoding viral growth factor (VGF), which is important for the virulence. The recombinant virus VVdGF-ApoS24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. The expression apoptin gene from a synthetic early-late promoter of vaccinia virus effectively provides accumulation of the protein in the cells infected with the VVdGF-ApoS24/2 virus. Despite the presence of virus growth factor signal peptide at apoptin N-terminal secretion of the recombinant protein into culture medium did not occur. The recombinant virus VVdGF-ApoS24/2 was found to have a significantly greater selective lyticactivity on human cancer cell lines (A549, A431, U87MG, RD and MCF7) as compared with the parent strain L-IVP and its variant VVdGF2/6 with the deletion of the C11R gene. The results suggest that the use of apoptin represents a promising approach for improving the natural anticancer activities of vaccinia virus.

[Oncolytic poxviruses].

The latest data on selection and construction of poxviruses capable of specifically lysing tumor cells of different genesis, inducing antitumor immunity and apoptosis of malignant cells are discussed. The review concerns several directions: virus attenuation, insertion of immunomodulatory protein genes, and anti-tumor protein genes. Thymidine kinase and viral growth factor genes make the greatest contribution to the virus attenuation as their inactivation results in the virus inability to replicate in non-dividing cells, thereby contributing to increased selectivity with respect to tumor cells. Among the immunomodulatory proteins, interleukins 2, 12, and granulocyte-macrophage colony-stimulating factor proved to be most promising for oncolytic virotherapy. An attempt to use p53 protein gene expressed by vaccinia virus for addressed apoptosis of tumor cells was reported. The use of the double and triple viral recombinants carrying genes of multidirectional action seems to be most promising. Encouraging results were obtained using vaccinia virus in the oncotherapy with prodrugs and angiogenesis inhibitors. At present, two poxviral strains are undergoing Phase III clinical trials as anti-tumor preparations in the USA.

[Orthopoxvirus genes for kelch-like proteins. III. Construction of mousepox (ectromelia) virus variants with targeted gene deletions].

Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.

[Detection of the virus of hepatitis E isolates and elucidation of the possibility of their replication in vitro].

Blood serum samples collected from patients with acute hepatitis symptoms admitted to Infectious Disease Hospitals of Novosibirsk, Barnaul, and Irkutsk were studied. The serum samples were tested for the IgM and IgG antibodies to HEV using ELISA. Seropositive samples were tested using RT-PCR for HEV RNA. Two HEV strains were isolated, and thus HEV infection was identified for West Siberia. One of this strains is classified as HEV genotype I; the other, as genotype III. Cell culturing of these strains in green monkey kidney (4647) cells showed an ability of HEV genotype I strain to cause persistent infection.

[Genetic diversity of hepatitis A virus in Siberia].

The nucleotide sequences of a region of VP1/2A genes of a large group of hepatitis A virus (HAV) isolates circulating in Siberia (the Altai Territory, the Irkutsk and Novosibirsk Regions) were determined. Comparison of these sequences with those of prototype HAV of genotypes IA, IB, and IIA revealed their high similarity to prototype genotype IA strains. The above domains were shown to contain the types of viruses, which were close to both the European subtypes of HAV genotypes IA (78.3%) and the Far Eastern subtypes of this genotype (21.7%). The similar comparison of the derived amino acid sequences suggests that VP1 and 2A contains the amino acid substitutions that are typical of this geographical region.

Molecular epidemiology of the hepatitis C virus in Western Siberia.

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 (P < 0.05) increase in risk was found in intravenous drug users (odds ratio (OR) = 77.5), unemployed persons (OR = 16.3), persons having >4 sexual partners during lifetime (OR = 4.3), and male homosexuals (OR = 6.6).

[The occurrence rate of HGV/GBV-C RNA and risk factors in patients of narcological dispensary in Novosibirsk].

The occurrence rate of HGV/GBV-C RNA, genotypic variety of isolates and various risk factors of infection with HGV/GBV-C were evaluated in 500 patients of the narcological dispensary of Novosibirsk. The occurrence rate of HGV/GBV-C RNA among all examined blood sera was 33.6%. At the same time in blood sera with HCV markers the occurrence rate of HGV/ GBV-C was 42.9% and in sera with negative results for markers HCV--25%. For gene typing of obtained isolates the direct sequencing of the amplification products of fragment NS3B and the phylogenetic analysis of the sequences thus obtained were used. Almost all isolates subjected to gene typing belonged to genotype 2, widespread in Europe, and only 1 isolate was classified with genotype 4. Statistically significant (p<0.05) risk of HGV/GBV-C infection among the examined subjects was linked with the intravenous use of drugs (OR 2.15), risky sexual behavior (OR 1.8) and the presence of virus hepatitis C (OR 2.26).

[Occurrence of markers, distribution of genotypes and risk factors for viral hepatitis C among some groups of the population in the Novosibirsk region]. / Vstrechaemost' markerov, raspredelenie genotipov i faktory riska virusnogo gepatita C sredi naseleniia Novosibirskoi oblasti.

The occurrence of markers, genotypic variability of isolates and risk factors for viral hepatitis C (HCV) were studied in 4 groups of residents of the Novosibirsk region (altogether 2,000 persons). Anti-HCV IgG were detected within the range from 4.6% among medical personnel to 48% among the patients of the drug-abuse clinic. The detection rate of HCV RNA in seropositive samples varied from 79.3% to 86.3%. The determination of genotype was carried out for 388 isolates: 1b--50.3%, 2a--4.4%, 2c--0.3%, 3a--44.8%. The highest risk indices with respect to HCV among the residents of the region were linked with the drug use (OR=77.5; p<0.05) as well as with risky behavior and low social status. The elevated numbers of seropositive persons were detected among unemployed (OR=16.3), alcohol abusers (OR=3.9), persons having more than 4 sex partners in their lifetime (OR=4.3) and persons having homosexual contacts (OR=6.6). In some groups blood transfusions also played a definite role in the transmission of HCV. In the analysis, carried out separately for two different genotypes the intravenous use of drugs was perceptibly stronger linked with VHC of genotype 3 (OR=85.5) in comparison with HCV of genotype 1 (OR=49.3) and genotype 2 (OR=41.1). Genotype 1 prevailed in the older age group and genotype 3, among young people.

[Frequency of occurrence of Hepatitis C virus markers and risk factors among hospital personnel in the Novosibirsk region]. / Chastota vstrechaemosti markerov gepatita C i faktory riska u personala bol'nits Novosibirskoi oblasti.

The occurrence of markers, the genotypic variety of isolates and the profile of risk factors with respect to viral hepatitis C among 629 employees of the Regional Clinical Hospital (RCH) in Novosibirsk and 1,020 employees of the Central District Hospital (CDH) in Iskitim were studied in a cross-sectional investigation. The occurrence of hepatitis C virus (HCV) markers was 5.1% in RCH and 2.2% in CDH. Among the risk factors in the population under study were: the medical history of blood transfusions (TF) with 0 TF, anti-HCV = 2.3%; 1 TF, = 5.7% > 1 TF, = 13.5% (p < 0.001); general anesthesia (GA) with < or = 2 GA, anti-HCV = 2.8%; > 2 GA, = 7.8% (p = 0.002); surgical interventions (SU) with 0 SU, = 1.9%; > 0 SU, = 4.3% (p = 0.012); the intravenous use of drugs (OR = 31.8); age (< or = 25 years, anti-HCV IgG = 8.6% > 25 years, = 4.5%); the number of partners of the opposite sex < or = 4 partners, = 2.4%; > 4 partners, = 6.9%; p < 0.001). The probable risk factors at a working place (pricks and cuts, contamination of mucous membranes with blood and other biological fluids, etc.) proved to be faintly related with the status of HBV infection. HBV isolates detected in the examined persons (35 examinees) were distributed by genotypes as follows: 60% of subtype 1b, 28.6% of subtype 2a/2c, 11.4% of subtype 3a. HBV of genotype 1a was not detected in the examined specimens, while the detection rate of genotype 2a/2c was considerably greater than in specimens obtained in the European and Asian parts of Russia (according to the data reported earlier).

Individual Escherichia coli cells studied from light scattering with the scanning flow cytometer.

BACKGROUND: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. METHODS: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. RESULTS: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. CONCLUSIONS: The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.

Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis.

Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.

[Directed reconstruction of the influenza virus hemagglutinin gene]. / Napravlennaia rekonstruktsiia gena gemaggliutinina virusa grippa.

A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed. By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained. These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule. The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.

[Construction and expression in Escherichia coli of a gene of hybrid hemagglutinin H1-H3 of influenza virus]. / Konstruirovanie i ékspressiia v Escherichia coli gena gibridnogo gemaggliutinina podtipa H1-H3 virusa grippa.

The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed. The product of expression of this gene in E. coli was obtained as a fusion protein with beta-galactosidase. The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.

[Antiviral activity of mutant interferons. A new approach to the study of structural-functional organization of interferons]. / Antivirusnoe deistvie mutantnykh interferonov. Novyi podkhod k issledovaniiu strukturno-funktsional'noi organizatsii interferonov.

The developed approach to investing the structure-functional organization of interferon has been developed consisting in: 1) fusing genes of interferon and alpha-peptide of beta-galactosidase, the resultant protein having the interferon properties and being determined by the beta-galactosidase alpha-complementation test; 2) constructing mutant genes of interferon by the localized mutagenesis; 3) determining the mutant interferon activity; 4) deducing the amino acid sequence of mutant interferon by sequencing mutant genes; 5) analyzing structure-functional organization of interferon. In accordance with this approach, ten mutant interferons with up to 15 changes of amino acid substitutions are obtained and their antiviral activity is determined. The role of some amino acid residues in antiviral activity of interferon alpha 2 is revealed.