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Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

Цель настоящей работы состояла в оценке иммунорегуляторных и протективных свойств мезенхимальных стволовых клеток (МСК) на экспериментальной модели летальной ВПГ-1-инфекции (вирус простого герпеса 1-го типа) мышей. МСК получали из костного мозга мышей линии DBA и высаживали в культуральные флаконы в среде DMEM, содержащей 10% эмбриональной телячьей сыворотки, инсулин, трансферрин, селенит, фактор роста фибробластов, глутамин и гентамицин. Прикрепившиеся клетки пассировали, на каждом пассаже отбирали культуральную жидкость (к/ж) и изучали противовирусную активность на клетках Vero, заражённых ВПГ-1. В опытах in vivo использовали 5 групп по 10 мышей DBA: 1-я группа-интактные (naïve); 2-я-внутривенное (в/в) введение МСК; 3-я-заражение 20 полулетальными дозами (ЛД 50) ВПГ-1 внутрибрюшинно с последующим в/в введением МСК; 4-я-заражение ВПГ-1 с последующим введением ацикловира (АЦВ); 5-я-заражение ВПГ-1 и в/в введение физиологического раствора. Показано, что выделенные клетки по морфологии, адгезивной способности и поверхностным рецепторам соответствовали общепринятым характеристикам МСК. Установлено, что к/ж от МСК на 4-5-м пассажах подавляла ВПГ-1-инфекцию in vitro на 64-70%, и в то же время в к/ж были обнаружены иЛ-6 и ФнО-α, концентрация которых на 3-4-м пассажах в 5 и 20 раз соответственно превышала контрольный уровень. При введении заражённым ВПГ-1 мышам 3-й группы МСК выживаемость составила 70%, ацикловира-60%, что значимо превышало число выживших животных (10%) в контрольной 5-й группе. У выживших животных 3-й группы обнаружена высокая активность вируснейтрализующих антител к ВПГ-1, а также активация пролиферации Т-клеток. У выживших мышей 3-й группы уровни иЛ-6 и ФнО-α оказались ниже, а иФн-γ-значительно выше по сравнению с таковыми у агонизирующих животных этой группы (р < 0,05). Таким образом впервые показано, что клеточная терапия МСК является перспективным подходом к разработке новых эффективных методов лечения генерализованной ВПГ-1-инфекции. К л ю ч е в ы е с л о в а : мезенхимальные стволовые клетки (МСК); летальная ВПГ-1-инфекция; клеточная терапия; активация иммунного ответа; защитный эффект МСК.

Antiviral activity of new AТ-specific fluorescent symmetric dimeric bisbenzimidazoles of DBА(n) series was assessed in the cell models of infections caused by type 1 herpes simplex virus (HSV1) and human cytomegalovirus (CMV). In DBA(n) molecules bisbenzimidazole fragments are bound to an oligomethylene liner with varied number of methylene groups in the linker (n = 1, 3, 5, 7, 9, 11). In contrast to DB(n) dimeric bisbenzimidazoles, in DBA(n) series terminal fragments of macromolecules contain N-dimethylaminopropylcarboxamide groups instead of N-methylpiperazine groups. DBА(n) compounds better dissolve in water, pass across plasma and nuclear membrane, and stain DNA in living cells. DBA(1) and DBA(7) produced therapeutic effects in HSV1 infection; DBA(7) completely suppressed the infection. DBA(11) displayed in vitro therapeutic activity in HSV1 and CMV infections. In addition, DBA(7) and DBA(1) showed microbicidal activity. Thus, DBA(11), which is active against two viruses causing severe diseases with serious health consequences for immunodeficient individuals, should be further investigated. High antiviral activity of DBA(7) in all test models indicates that this compound is a promising active agent for innovative antiviral drugs.

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

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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.