[Detection rates and high concentration of herpesvirus (Orthoherpesviridae) DNA in autopsy materials from patients with COVID-19 fatal outcome].

INTRODUCTION: SARS-CoV-2 infection causes immune disorders that create conditions for the reactivation of human herpesviruses (HHVs). However, the estimates of the HHVs effect on the course and outcome of COVID-19 are ambiguous. Аim - to study the possible relationship between the HHV reactivation and the adverse outcome of COVID-19. MATERIALS AND METHODS: Postmortem samples from the brain, liver, spleen, lymph nodes and lungs were obtained from 59 patients treated at the Moscow Infectious Diseases Hospital No.1 in 2021-2023. The group 1 comprised 39 patients with fatal COVID-19; group 2 (comparison group) included 20 patients not infected with SARS-CoV-2 who died from various somatic diseases. HHV DNA and SARS-CoV-2 RNA were determined by PCR. RESULTS: HHV DNA was found in autopsy samples from all patients. In group 1, EBV was most often detected in lymph nodes (94%), HHV-6 in liver (68%), CMV in lymph nodes (18%), HSV in brain (16%), VZV in lung and spleen (3% each). The detection rates of HHVs in both groups was similar. Important differences were found in viral load. In patients with COVID-19, the number of samples containing more than 1,000 copies of HHV DNA per 100,000 cells was 52.4%, in the comparison group - 16.6% (p < 0.002). An association has been established between the reactivation of HSV and HHV-6 and the severity of lung damage. Reactivation of EBV correlated with increased levels of liver enzymes. CONCLUSION: Reactivation of HHVs in patients with fatal COVID-19 was associated with severe lung and liver damages, which indicates a link between HHV reactivation and COVID-19 deaths.

The adjuvant effect of polymuramil, a NOD1 and NOD2 agonist, differs when immunizing mice of different inbred lines with nonstructural hepatitis C virus (Flaviviridae: Hepacivirus)proteins and is synergistically enhanced in combination with pyrogenalum, a TLR4 agonist.

INTRODUCTION: Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines. The aim of the work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum. MATERIALS AND METHODS: BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response - by antigen-specific lymphocyte proliferation and by production of IFN-γ in vitro. RESULTS: Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN-γ in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins. CONCLUSION: Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.

[Exosomes in the life cycle of viruses and the pathogenesis of viral infections].

Exosomes are extracellular vesicles of endosomal origin, with a bilayer membrane, 30160 nm in diameter. Exosomes are released from cells of different origins and are detected in various body fluids. They contain nucleic acids, proteins, lipids, metabolites and can transfer the contents to recipient cells. Exosome biogenesis involves cellular proteins of the Rab GTPase family and the ESCRT system, which regulate budding, vesicle transport, molecule sorting, membrane fusion, formation of multivesicular bodies and exosome secretion. Exosomes are released from cells infected with viruses and may contain viral DNA and RNA, as well as mRNA, microRNA, other types of RNA, proteins and virions. Exosomes are capable of transferring viral components into uninfected cells of various organs and tissues. This review analyzes the impact of exosomes on the life cycle of widespread viruses that cause serious human diseases: human immunodeficiency virus (HIV-1), hepatitis B virus, hepatitis C virus, SARS-CoV-2. Viruses are able to enter cells by endocytosis, use molecular and cellular pathways involving Rab and ESCRT proteins to release exosomes and spread viral infections. It has been shown that exosomes can have multidirectional effects on the pathogenesis of viral infections, suppressing or enhancing the course of diseases. Exosomes can potentially be used in noninvasive diagnostics as biomarkers of the stage of infection, and exosomes loaded with biomolecules and drugs - as therapeutic agents. Genetically modified exosomes are promising candidates for new antiviral vaccines.

[Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein].

INTRODUCTION: A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid. MATERIALS AND METHODS: An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated. RESULTS: Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60. CONCLUSIONS: Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

Mechanisms of Survival of Cytomegalovirus-Infected Tumor Cells.

Human cytomegalovirus (HCMV) DNA and proteins are often detected in malignant tumors, warranting studies of the role that HCMV plays in carcinogenesis and tumor progression. HCMV proteins were shown to regulate the key processes involved in tumorigenesis. While HCMV as an oncogenic factor just came into focus, its ability to promote tumor progression is generally recognized. The review discusses the viral factors and cell molecular pathways that affect the resistance of cancer cells to therapy. CMV inhibits apoptosis of tumor cells, that not only promotes tumor progression, but also reduces the sensitivity of cells to antitumor therapy. Autophagy was found to facilitate either cell survival or cell death in different tumor cells. In leukemia cells, HCMV induces a "protective" autophagy that suppresses apoptosis. Viral factors that mediate drug resistance and their interactions with key cell death pathways are necessary to further investigate in order to develop agents that can restore the tumor sensitivity to anticancer drugs.

[Mechanisms of Survival of Cytomegalovirus-Infected Tumor Cells].

Human cytomegalovirus (HCMV) DNA and proteins are often detected in malignant tumors, warranting studies of the role that HCMV plays in carcinogenesis and tumor progression. HCMV proteins were shown to regulate the key processes involved in tumorigenesis. While HCMV as an oncogenic factor just came into focus, its ability to promote tumor progression is generally recognized. The review discusses the viral factors and cell molecular pathways that affect the resistance of cancer cells to therapy. CMV inhibits apoptosis of tumor cells, that not only promotes tumor progression, but also reduces the sensitivity of cells to antitumor therapy. Autophagy was found to facilitate either cell survival or cell death in different tumor cells. In leukemia cells, HCMV induces a "protective" autophagy that suppresses apoptosis. Viral factors that mediate drug resistance and their interactions with key cell death pathways are necessary to further investigate in order to develop agents that can restore the tumor sensitivity to anticancer drugs.

Daidzein-directed methionine γ-lyase in enzyme prodrug therapy against breast cancer.

Thiosulfinates in situ formed by "pharmacological pair" C115H methionine γ-lyase/S-(allyl/alkyl)-l-cysteine sulfoxides possess cytotoxic activity against various malignant cell lines. To investigate in vivo antitumor activity of thiosulfinates generated directly at the surface of tumor cells, a chemical conjugate between Clostridium novyi C115H methionine γ-lyase (C115H MGL) and isoflavone daidzein was prepared. The binding of conjugate (C115H-Dz) to various breast cancer cell lines was demonstrated, as well as its cytotoxicity in the presence of S-(allyl/alkyl)-l-cysteine sulfoxides. The most promising among thiosulfinates was dipropyl thiosulfinate (IC50 < 0.53 µM). The pharmacokinetic parameters of C115H MGL and C115H-Dz were obtained. Plasma half-lives of the enzyme and conjugated enzyme were 4.4 and 7.2 h, respectively. In vivo antitumor effect of pharmacological pairs on SKBR-3 xenografts was demonstrated. Treatment of tumor-bearing mice with a pair of C115H-Dz/propiin inhibited tumor growth by 85%.

Preventive Vaccination with Mesenchymal Stem Cells Protects Mice from Lethal Infection Caused by Herpes Simplex Virus 1.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect almost all organs and tissues, cause genital herpes-the most common sexually transmitted disease-disorders of the central nervous system (CNS), and lead to severe complications in children. Despite the available drugs, the incidence of HSV-1/2 continues to rise. None of the prophylactic vaccine candidates have shown a protective effect in trials nor approval for use in clinical practice. We have investigated the protective properties of mesenchymal stem cells (MSC) isolated from the bone marrow of mice. A comparative analysis of the protective response to the introduction of primary and modified MSCs (mMSC) was carried out using the plasmid containing gene of the HSV and an inactivated virus in a model of lethal HSV-1 infection in mice. mMSCs were obtained by transfection of the Us6 gene encoding glycoprotein D (gD) of the HSV, the plasmid contained the same gene. After twofold immunization with primary MSCs, the formation of antibodies interacting with the viral antigen (according to enzyme immunoassay data) and neutralizing the infectious activity of HSV-1 in the reaction of biological neutralization was observed in the peripheral blood of mice. In addition, the introduction of primary MSCs induced the production of interferon gamma (INF-γ) which is detected in the peripheral blood of mice. After infection with HSV-1, the immunized mice showed significantly increased titers of virus-specific antibodies, an increased level of IFNγ, and were completely protected from lethal HSV-1 infection. The protective effect of the other three immunogens was lower and did not exceed 50-65%. Considering the wide availability of MSCs, the proven safety of intravenous administration, and the results obtained in this work on the ability to induce innate, adaptive and protective immunity to HSV-1, MSCs can be considered a promising basis for the development of new cellular vaccines for the prevention of herpesvirus and other viral infections.

[Preventive Vaccination with Mesenchymal Stem Cells Protects Mice from Lethal Infection Caused by Herpes Simplex Virus 1].

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect almost all organs and tissues, cause genital herpes-the most common sexually transmitted disease-disorders of the central nervous system (CNS), and lead to severe complications in children. Despite the available drugs, the incidence of HSV-1/2 continues to rise. None of the prophylactic vaccine candidates have shown a protective effect in trials nor approval for use in clinical practice. We have investigated the protective properties of mesenchymal stem cells (MSC) isolated from the bone marrow of mice. A comparative analysis of the protective response to the introduction of primary and modified MSCs (mMSC) was carried out using the plasmid containing gene of the HSV and an inactivated virus in a model of lethal HSV-1 infection in mice. mMSCs were obtained by transfection of the Us6 gene encoding glycoprotein D (gD) of the HSV, the plasmid contained the same gene. After twofold immunization with primary MSCs, the formation of antibodies interacting with the viral antigen (according to enzyme immunoassay data) and neutralizing the infectious activity of HSV-1 in the reaction of biological neutralization was observed in the peripheral blood of mice. In addition, the introduction of primary MSCs induced the production of interferon gamma (INF-γ) which is detected in the peripheral blood of mice. After infection with HSV-1, the immunized mice showed significantly increased titers of virus-specific antibodies, an increased level of IFNγ, and were completely protected from lethal HSV-1 infection. The protective effect of the other three immunogens was lower and did not exceed 50-65%. Considering the wide availability of MSCs, the proven safety of intravenous administration, and the results obtained in this work on the ability to induce innate, adaptive and protective immunity to HSV-1, MSCs can be considered a promising basis for the development of new cellular vaccines for the prevention of herpesvirus and other viral infections.

Conjugates of methionine γ-lyase with polysialic acid: Two approaches to antitumor therapy.

The methionine dependence is a well known phenomenon in metabolism of cancer cells. Methionine γ-lyase (EC 4.4.1.11, MGL) catalyzes the γ-elimination reaction of L-methionine and thus could effectively inhibit the growth of malignant cells. Recently we have demonstrated that the mutant form of the enzyme C115H MGL can be used as a component of the pharmacological pair enzyme/S-(allyl/alkyl)-L-cysteine sulfoxides to yield thiosulfinates in situ. Thiosulfinates were shown to be toxic to various cancer cell lines. Therefore the application of the enzyme in enzyme pro-drug therapy may be promising. The conjugates of MGL and C115H MGL with polysialic acid were obtained and their kinetic and pharmacokinetic parameters were determined. The formation of polysialic shell around the enzyme was confirmed by atomic force microscopy. The half-life of conjugated enzymes increased 3-6 times compared to the native enzyme. The cytotoxic effect of conjugated MGL against methionine dependent cancer cell lines was increased two times compared to the values for the native enzymes. The anticancer efficiency of thiosulfinates produced by pharmacological pair C115H MGL/S-(allyl/alkyl)-L-cysteine sulfoxides was demonstrated in vitro. The results indicate that the conjugates of MGL with polysialic acid could be new antitumor drugs.

[The role of herpesviruses in development of diseases of the urogenital tract and infertility in women].

This review presents the data on the spreading of all known human herpesviruses (НHVs) in female urogenital tract. According to the WHO almost 500 million people worldwide suffer from genital infection caused by НHVs. НHVs were detected in various inflammatory diseases of female upper and lower genital tract (vaginitis and cervicitis), in extrauterine pregnancy (in fallopian tubes), in infertility (cervical channel, endometrium and ovaries). Herpes simplex virus 1 (HSV­1) was identified for the first time in oocytes after failed in vitro fertilization (IVF). НHVs produce negative effect on the entire reproductive process from conception to childbirth. It was established that HSV, cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) markedly increase the risk of spontaneous abortion, preterm birth and stillbirth. Intrauterine НHV infection is a major cause of congenital malformations. Data on humoral and cell immunity in genital herpesvirus infections (НHVI) are also reviewed. Intravaginal HSV­2 infection changes cell composition of vaginal mucosa, i.e., together with cells mobilized from the blood, protective role is performed by resident memory T­cells (TRM), natural killer cells (NK­cells) and regulatory T­cells (Treg) whose function consists in maintaining the balance of the activities of lymphocytes. Constant НHVI spreading is largely explained by transition of primary infection to potentially reactivating latent form, since latent virus is unavailable to immune recognition and medicines. The genome editing system CRISPR/Cas9 can recognize and modify not only active but also latent viruses. The promising pilot results with the use of this system offer the possibility of developing innovative technologies for НHV elimination and НHVI eradication.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

[Change of phenotypic properties of escape mutants and readaptants of influenza virus A (H1N1)pdm09 under the influence of selected mutations in the molecule of hemagglutinin.]

INTRODUCTION: After the emergence and spread of pandemic H1N1 viruses in 2009, antigenic epitopes recognized by neutralizing antibodies against the hemagglutinin of influenza A/Moscow/01/09(H1N1)pdm09 viruses were studied. PURPOSE: The purpose of the study was to obtain readapted variants of the virus from a low-virulent escapemutant that has an increased affinity of the avian and the human types cellular receptors compared to the wild type and the comparative study of their antigenic and receptor specificity. MATERIAL AND METHODS: Viruses were accumulated in 10-day-old chicken embryos. The MAB panel against HA of influenza virus strain A/IIV-Moscow/01/09(H1N1)sw1 was used in the form of ascites fluids from mice. Immunization of mice, HI testing, elution of viruses from chicken erythrocytes, PCR and sequencing of readapted variants were performed by standard methods. RESULTS: The amino acid substitution A198E acquired in the process of readaptation leads to changes in the antigenic specificity. A correlation was found between a decrease in virulence of a low-virulent escape mutant associated with the substitution D190N in the hemagglutinin molecule and an increase in the hemagglutinating titer to inhibitors in normal mouse serum. Viruses with low affinity of cellular receptor analogs and carrying amino acid substitutions have an increased ability to elute from chicken erythrocytes. DISCUSSION: The results discuss the effect of mutations in the HA molecule of the influenza A(H1N1) pdm09 virus to the change in antigen specificity; virulence for mice, adsorption-elution at cellular receptors. CONCLUSION: A comparative study of the antigenic specificity and receptor-binding activity of the escape mutants was conducted for the hemagglutinin of the influenza virus A/Moscow/01/2009 (H1N1)swl, and the readapted variants obtained for one of the escape mutants with reduced virulence for mouse. Monitoring the pleiotropic effect of mutations in the hemagglutinin H1 molecule is necessary to predict variants of the virus with pandemic potential.

[Development and properties of neutralizing monoclonal antibodies for fusion protein of respiratory syncytial virus.]

INTRODUCTION: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and the elderly. The absence of a wide range of therapeutic drugs and vaccines indicates to the high relevance of the development of new effective drugs for the prevention and treatment of RSV infections. PURPOSE: to obtain highly active and specific monoclonal antibodies (MAbs) capable of detecting RSV in infected cells and neutralizing the infectious activity of the virus in vitro. MATERIAL AND METHODS: RSV reference strains of group A 2 subgroups (A2 and Long) were propagated in HEp-2 and MA-104 cell lines, respectively. Mice were immunized with purified RSV A2 virus. MAbs were obtained using hybridoma technology. RESULTS: A panel of 6 MAbs reacting with RSV strains А2 and Long has been obtained. Four MAbs were IgG (IgG2a or IgG2b subtype), two MAbs were IgM. All MAbs reacted with RSV F-protein in immunochemical tests. The MAbs actively reacted with RSV in ELISA, in immufluorescence and peroxidase staining of infected cells, and in immunodot test. Five out of 6 MAbs neutralized of RSV in cell culture. Different properties of MAbs suggest that they target different antigenic sites of F-protein. DISCUSSION: Comparative analysis suggests that the obtained MAbs can be used for the development of diagnostic preparations, for RSV detection in clinical materials and confirmation of infection etiology by rapid culture method. CONCLUSION: High activity and specificity of MAbs indicate that they can serve as a basis for development vaccines and preventive medicines.

[Diagnosis and treatment of abacterial category III CP/CPPS, associated with herpes viruses].

INTRODUCTION: The etiology of abacterial CP/CPPS (category III) has not been studied enough. Currently, there is no gold standard of diagnostic study and optimal treatment algorithm. AIM: The aim of our study was to study three human herpes viruses (HHV) in clinical samples from patients with inflammatory diseases of urogenital tract and to evaluate the efficiency of proposed treatment algorithm for abacterial CP/CPPS. MATERIALS AND METHODS: The biological samples from the urogenital tract (urethral swab, ejaculate and expressed prostatic secretions) from 101 patients with category III CP/CPPS were studied. Quantitative analysis of HHV DNA (CMV, EBV and HHV-6) was performed by PCR. RESULTS: HHV DNA was detected in 38/101 patients (37.6%) in Group 1. Among the detected viral types, HHV-6 was the most common (52%). Analysis of biological samples form the three sources revealed that viral DNA was determined in urethral swab in concentration of 3,703,900 copies/ml. In Group 2, viral DNA was not detected in 63 patients. Evaluation of results of the standard treatment in HHV-negative patients (n=63) and antibiotic-free scheme, including the immunoregulatory drug Viferon, in HHV-positive patients (n=38) showed that the number of HHV-positive samples after treatment decreased by 54.3%. In addition, severity of all symptoms according to NIH-CPSI scale significantly decreased in both groups (p<0.0001). There was an improvement in all clinical symptoms in Group 1 by 47.9%, especially for pain + urination (52%). It should be noted that a positive response to treatment, which was confirmed by the changes in total score of NIH-CPSI scale, was noted in all patients in Group 1. CONCLUSION: Detection of herpes viruses in the urogenital tract of patients with abacterial CP/CPPS suggests possible role of viral infections in its etiology. The comparative analysis of the results of standard treatment including antiviral, immunomodulatory and antioxidant drugs showed that the use of complex therapy without antibiotics allowed to eliminate or significantly reduce the concentration of viruses in urogenital tract, as well as significantly reduce the clinical manifestations of abacterial CP/CPPS.

[A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture].

Herpesviruses are widespread in the human population. Herpes simplex virus type 1 (HSV1) alone infects more than 3.7 billion people. In most of these, the virus establishes a latent form resistant to the action of all antiviral drugs. Moreover, completely drug-resistant strains of herpesviruses are known, which has prompted the search for alternative approaches to the treatment of herpesviruses, including genome editing with prokaryotic CRISPR/Cas. The CRISPR/Cas9 system of Streptococcus pyogens effectively suppresses HSV1 infection when expressed from genome-integrated lentiviral vectors. However, there are concerns about the safety of this approach. Here we describe the system built upon the plasmid-encoded CRISPR/Cas9 targeted against UL52 and UL29 genes of the HSV1 primase-helicase complex. The construct was transfected into Vero cells with no significant cytotoxic effects detected. Complete suppression of HSV1 infection within two days was observed, raising the possibility that the proposed plasmid-expressed CRISPR/Cas9 system may be used for the screening of genes important for the HSV1 life cycle and for development of novel strategies for targeted therapy of herpesvirus infections.

Resistance of THP-1 Leukemia Cells Infected with Cytomegalovirus to Anti-tumor Antibiotic Doxorubicin and Restoration of the Sensitivity by Inhibitors of the PI3K/AKT/mTOR Molecular Pathway.

Results obtained showed that infection with HCMV prevented the death of THP-1 cells treated with DOX in both active and latent forms of infection. In the presence of mTOR inhibitors (rapamycin and Torin2), the sensitivity of the infected cells to DOX was restored. Rapamycin inhibited the expression of the HCMV protein IE1-p72 and increased sensitivity to DOX. Molecular targets for the creation of new drugs for the treatment of leukemia in patients infected with HCMV were determined.

Polyprenyl Phosphates Induce a High Humoral and Cellular Response to Immunization with Recombinant Proteins of the Replicative Complex of the Hepatitis C Virus.

The search for new adjuvants remains the critical task for the creation of hepatitis C vaccines due to the weak immunogenicity of biotechnological products. When immunizing mice with the recombinant proteins NS3 and NS5B of the hepatitis C virus (HCV), the adjuvant activity of three immunomodulators was compared. Phosprenyl® on the basis of polyprenyl phosphate (PPP), chemically synthesized analogue of the bacterial cell wall glucosaminyl muramyl dipeptide (GMDP), and IFN-α recombinant protein were tested. GMDP increased the activity of IgG1 antibodies 4-6 times but did not stimulate the production of IFN-γ; IFN-α has not shown any adjuvant properties. The introduction of recombinant HCV proteins together with PPP in low doses increased the activity of IgG2a isotype antibodies 4-7 times and increased IFN-γ secretion 3 times. Thus, it was first shown that PPP polarizes the immune response to Th1-type and is a promising adjuvant for the development of a vaccine against hepatitis C.

[Mesenchymal stem cells enhance immune response and protect mice against lethal herpes viral infection.]

The objective of this study was to evaluate immunoregulatory and protective potential of mesenchymal stem cells (MSC) in a mouse model of lethal HSV1 infection. MSC were isolated from bone marrow of DBA mice and cultured in flasks with DMEM containing 10% FBS, insulin, transferrin, selenite, fbroblast growth factor, glutaminе and gentamicin. Antiviral activity was tested on HSV1-infected Vero cells. In vivo experiments were performed on DBA mice divided into 5 groups (10 animals each): group 1, intact (naïve) mice; group 2, intravenous (iv) MSC injection; group 3, ntraperitoneal infection with 20 LD50 HSV1 followed by MSC injection; group 4, HSV1 infection followed by acyclovir (ACV) injection; group 5, HSV1 infection and iv injection of saline. Isolated cells were consistent with MSC morphologically, by adhesive ability and surface receptors. Conditioned media from MSC collected after 4-5 passages inhibited HSV1 infection in vitro by 64-70% and contained IL-6 and TNF-α, whose concentrations were 5- and 20-fold higher, respectively, than in the control. MSC and ACV injections protected 70% and 60% of DBA mice, respectively, compared with the control (group 5, 10% survival). High activity of virus neutralizing anti-HSV1 antibodies and activation of T cell proliferation were observed in survived mice from group 3. Serum levels of IL-6 and TNF-α in these mice were lower and that of INF-γ much higher than in agonizing animals of this group (Р<0.05). These fndings indicate that MSC therapy is a prospective approach to the development of new effective management of generalized HSV1 infection.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1ß, TNF-α, and TGF-ß) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-ß in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1ß in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.