[Evaluation of the dynamics of detection of viable SARS-CoV-2 (Coronaviridae: Betacoronavirus: Sarbecovirus) in biological samples obtained from patients with COVID-19 in a health care setting, as one of the indicators of the infectivity of the virus].

INTRODUCTION: The study of the mechanisms of transmission of the SARS-CoV-2 virus is the basis for building a strategy for anti-epidemic measures in the context of the COVID-19 pandemic. Understanding in what time frame a patient can spread SARS-CoV-2 is just as important as knowing the transmission mechanisms themselves. This information is necessary to develop effective measures to prevent infection by breaking the chains of transmission of the virus. The aim of the work is to identify the infectious SARS-CoV-2 virus in patient samples in the course of the disease and to determine the duration of virus shedding in patients with varying severity of COVID-19. MATERIALS AND METHODS: In patients included in the study, biomaterial (nasopharyngeal swabs) was subjected to analysis by quantitative RT-PCR and virological determination of infectivity of the virus. RESULTS: We have determined the timeframe of maintaining the infectivity of the virus in patients hospitalized with severe and moderate COVID-19. Based on the results of the study, we made an analysis of the relationship between the amount of detected SARS-CoV-2 RNA and the infectivity of the virus in vitro in patients with COVID-19. The median time of the infectious virus shedding was 8 days. In addition, a comparative analysis of different protocols for the detection of the viral RNA in relation to the identification of the infectious virus was carried out. CONCLUSION: The obtained data make it possible to assess the dynamics of SARS-CoV-2 detection and viral load in patients with COVID-19 and indicate the significance of these parameters for the subsequent spread of the virus and the organization of preventive measures.

Development and Characterization of a Vector System Based on the Simian Adenovirus Type 25

Technological versatility and the humoral and cellular immune response induction capacity have conditioned wide spread of adenoviral vectors as vaccine and gene therapy drugs. However, vaccination with Sputnik V made a significant portion of the population immune to the types 5 and 26 (Ad5 and Ad26) recombinant human adenovirus vectors, which are some of the most frequently used bases for candidate vaccines. Today, vaccine designers tend to select alternative adenovirus serotypes as platforms to develop vaccines against new pathogens on. A good example is simian adenovirus type 25 (SAd25), which belongs to subgroup E. It is genetically distant from Ad5 and exhibits extremely low seroprevalence in human beings, which makes it an appealing alternative vaccine vector. The purpose of this work was to design and study a new vaccine platform based on simian adenovirus type 25. We relied on the advanced methods of molecular biology and virology to construct and make recombinant adenoviruses;the phylogenetic analysis in the context of this study was enabled with bioinformatic methods. The resulting recombinant adenoviral vector can effectively replicate itself in the HEK293 cell line (human embryonic kidney cells). This work substantiates the expediency of further investigation into the SAd25 vector as a platform for development of the prevention vaccines against various infectious diseases.Copyright © 2023 Pirogov Russian National Research Medical University. All rights reserved.

Immunogenicity evaluation of Gam-COVID-Vac (Sputnik V).

In November 2020, the Armed Forces of the Russian Federation began mass immunisation of the personnel with Gam-COVID-Vac (Sputnik V), the first Russia vaccine against the new coronavirus infection (COVID-19). Thus, it became necessary to assess post-vaccination antibody levels and the duration and intensity of humoral immunity to COVID-19. The aim of the study was to investigate the immunogenicity and efficacy of Gam-COVID-Vac in military medical staff after vaccination. Material(s) and Method(s): the authors determined the presence of specific antibodies in the serum of individuals immunised with Gam-COVID-Vac (477 volunteers) and COVID-19 convalescents (73 patients), using virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) with reagent kits by several manufacturers, and immunoblotting. The results of the study were evaluated using analysis of variance. Result(s): VN detected virus neutralising antibodies in 90.7% of vaccinated subjects;ELISA, in 95.4%. Both VN and ELISA showed lower antibody levels in the vaccinated over 50 years of age. ELISA demonstrated a significantly higher concentration of anti-SARS-CoV-2 spike IgG in the Gam-COVID-Vac group than in the COVID-19 convalescent group. The correlation between antibody detection results by VN and ELISA was the strongest when the authors used their experimental reagent kit for quantitative detection of virus neutralising antibodies by competitive ELISA with the recombinant human ACE2 receptor. Having analysed the time course of neutralising antibody titres, the authors noted a significant, more than two-fold decrease in geometric means of the titres three months after administration of the second vaccine component. Conclusion(s): the subjects vaccinated with Gam-COVID-Vac gain effective humoral immunity to COVID-19. The decrease in titres indicates the need for revaccination in 6 months.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.

Efficacy of Favipiravir and Molnupiravir against Novel Sars-Cov-2 Variants in Vitro and in Vivo

The COVID-19 disease pandemic remains a significant global problem, resulting in hundreds of millions of cases and millions of deaths. The search for specific inhibitors of SARS-CoV-2 for the treatment of this infection remains relevant. Drugs such as Favipiravir and Molnupiravir, which exhibit specific antiviral activity against SARS-CoV-2, are already being used to treat patients. However, there is limited evidence of their effectiveness, especially against novel genetic variants of the COVID-19 pathogen. The aim of this study was to investigate the antiviral effect of these drugs using an in vitro experimental model of SARS-CoV-2 infection in Vero E6 cell culture and an animal model of infection using Syrian hamsters. It has been established that Molnupiravir has an inhibitory effect against variants of the SARS-CoV-2 with IC50 values from 16.51 to 7.88 microM in vitro, and reduces the infectious titer of the virus in the lungs of animals by ~1.5 Log10 in vivo, in while Favipiravir shows lower activity and severe toxicity. Dose selection and frequency of use remain unexplored.Copyright © 2022 Pirogov Russian National Research Medical University. All rights reserved.

Immunogenicity evaluation of Gam-COVID-Vac (Sputnik V)

In November 2020, the Armed Forces of the Russian Federation began mass immunisation of the personnel with Gam-COVID-Vac (Sputnik V), the first Russia vaccine against the new coronavirus infection (COVID-19). Thus, it became necessary to assess post-vaccination antibody levels and the duration and intensity of humoral immunity to COVID-19. The aim of the study was to investigate the immunogenicity and efficacy of Gam-COVID-Vac in military medical staff after vaccination. Materials and methods: the authors determined the presence of specific antibodies in the serum of individuals immunised with Gam-COVID-Vac (477 volunteers) and COVID-19 convalescents (73 patients), using virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) with reagent kits by several manufacturers, and immunoblotting. The results of the study were evaluated using analysis of variance. Results: VN detected virus neutralising antibodies in 90.7% of vaccinated subjects;ELISA, in 95.4%. Both VN and ELISA showed lower antibody levels in the vaccinated over 50 years of age. ELISA demonstrated a significantly higher concentration of anti-SARS-CoV-2 spike IgG in the Gam-COVID-Vac group than in the COVID-19 convalescent group. The correlation between antibody detection results by VN and ELISA was the strongest when the authors used their experimental reagent kit for quantitative detection of virus neutralising antibodies by competitive ELISA with the recombinant human ACE2 receptor. Having analysed the time course of neutralising antibody titres, the authors noted a significant, more than two-fold decrease in geometric means of the titres three months after administration of the second vaccine component. Conclusions: the subjects vaccinated with Gam-COVID-Vac gain effective humoral immunity to COVID-19. The decrease in titres indicates the need for revaccination in 6 months.

Modification of the Spike Protein for Vaccines against Enveloped RNA Viruses.

Most vaccines work by inducing neutralizing antibodies that target the viral envelope. Enveloped RNA viruses have evolved mechanisms for surface glycoproteins to evade host immune responses, which exhibit substantial variability, even among different strains. Natural infection and vaccines using native forms of surface proteins may induce broadly neutralizing antibodies, yet with low and ineffective levels. Class I membrane-fusion proteins of enveloped RNA viruses, HIV-1, influenza A virus, SARS-CoV-2, yield a stable conformation (so-called "pre-fusion") in providing fusion between viral and host cell membranes. Modified viral surface proteins that are based on these features induce neutralizing antibodies with activity available against a broad spectrum of circulating strains and make it possible to overcome the difficulties associated with escape/variability of viral antigen.

[Modification of the Spike Protein for Vaccines against Enveloped RNA Viruses].

Most vaccines work by inducing neutralizing antibodies that target the viral envelope. Enveloped RNA viruses have evolved mechanisms for surface glycoproteins to evade host immune responses, which exhibit substantial variability, even among different strains. Natural infection and vaccines using native forms of surface proteins may induce broadly neutralizing antibodies, yet with low and ineffective levels. Class I membrane-fusion proteins of enveloped RNA viruses, HIV-1, influenza A virus, SARS-CoV-2, yield a stable conformation (so-called "pre-fusion") in providing fusion between viral and host cell membranes. Modified viral surface proteins that are based on these features induce neutralizing antibodies with activity available against a broad spectrum of circulating strains and make it possible to overcome the difficulties associated with escape/variability of viral antigen.

Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome.

The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.

A heterologous virus-vectored vaccine for prevention of Middle East respiratory syndrome induces long protective immune response against MERS-CoV

Introduction. Middle East respiratory syndrome (MERS) is acute inflammatory disease of respiratory system with a high mortality, caused Ьу coronavirus MERS-CoV At present moment, we still lack specific therapeutic preparations and vaccines against MERS. Vaccine administration can help to limit the spread of the disease and lower the mortality. Duration of vaccine-induced immune response is one of the key characteristics of a vaccine, which is connected with duration of its protective effectiveness. Unfortunately, the data on duration of vaccine-induced immune response against MERS is scarce. The aim of the study was to determine duration of humoral immune response in mice and primates and duration of protective immune response in mice after immunization with the heterologous virus-vectored vaccine against MERS (BVRS-GamVac-Combi), developed earlier Ьу our research group. Material and methods. To study duration of humoral immune response, we used mice of C57BL/6 strain and common marmosets. Animals were immunized with the vaccine BVRS-GamVac-Combi, based on recomЬinant adenoviral vectors rAd26 and rAd5. Antigen-specific-antibody titers were determined with ELISA, virus-neutralizing antibody titers were measured with virus neutralization assay using MERS-CoV (EMC/2012). To study duration of protective immune response, we used a model of lethal infection on transgenic mice, carrying human DPP4 gene of viral receptor. Results. In present research, we showed that vaccination of animals with BVRS-GamVac-Combi induced robust humoral immune response, which persisted at least 18 months after immunization. In addition, our vaccine protected 100 % of animals from lethal infection for at least 7 months after immunization. Concluslon. Strength of vaccine-induced immune response is generally connected with a protective effectiveness of a vaccine. One of the key problems of vaccine design is to find a way to provide as long and robust immune response as possible. Duration of vaccine-induced immune response is one of the key characteristics of a vaccine, which demands quality control during multiple steps of a vaccine development. Введение. Ближневосточный респираторный синдром (БВРС) - это острое воспалительное заболевание дыхательной системы с высокой летальностью, возбудителем которого является коронавирус БВРС-КоВ. В настоящее время в мире не существует специфических профилактических и терапевтических средств против БВРС. Вакцино-профилактика позволит ограничить распространение данного заболевания и снизить летальность. Одной из ключевых характеристик вакцин является длительность индуцируемого иммунного ответа, от которой зависит продолжительность протективного эффекта вакцины. К сожалению, данных по длительности поствакцинального иммунного ответа для вакцин против БВРС сейчас недостаточно. Цель исследования - определение длительности гуморального иммунного ответа у грызунов и приматов и протективного иммунного ответа после иммунизации комбинированной векторной вакциной против БВРС (БВРС-ГамВак-Комби), разработанной нами ранее. Материал и методы. Длительность гуморального иммунитета исследовали на мышах линии C57BL/6 и обыкновенных игрунках. ЖивРтных иммунизировали вакциной БВРС-ГамВак-Комби на основе рекомбинантных векторов rAd26 и rAd5. Титр антиген-специфических антител определяли методом иммуноферментного анализа (ИФА). Титр вирус-нейтрализующих антител определяли с помощью реакции вирус-нейтрализации против вируса БВРС-КоВ (MERS-CoV EMC/2012). Длительность протективного иммунитета исследовали на модели летальной инфекции у трансгенных мышей, несущих ген человека DPP4, кодирующий рецептор к БВРС-КоВ. Результаты. Исследование длительности поствакцинального гуморального иммунного ответа у грызунов и приматов показало, что вакцинация животных БВРС-ГамВак-Комби индуцирует формирование напряженного гуморального иммунного ответа к гликопротеину S БВРС-КоВ, который сохраняется на протяжении не менее 18 мес. Также было показано, что вакцинация позволяет защитить 100 % животных от летальной инфекции, вызванной БВРС-КоВ (MERS-CoV EMC/2012, 100 ЛД50/мышь), через 7 мес после иммунизации. Заключение. Напряженность поствакцинального гуморального иммунного ответа, как правило, связана с протективностью вакцины. Одной из ключевых задачпри дизайне вакцин является обеспечение наиболее длительного напряженного иммунного ответа. Длительность поствакцинального иммунного ответа - одна из ключевых характеристик вакцин, которая требует контроля на различных этапах их разработки.