Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

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.

Formation of a cohort of anticovid plasma donors with high-titer antibodies neutralizing SARS-CoV-2

Background. The application of convalescent plasma (CP) is currently seen as a feasible therapeutic approach in the treatment of COVID-19.Aim. To analyze the experience of recruiting a donor cohort from COVID-19 convalescents for banking of CP as part of a pilot project at the Moscow Healthcare Department.Materials and methods. A retrospective research included 493 COVID-19 convalescents as potential CP donors, all examined at the Sklifosovsky Research Institute for Emergency Medicine. CP was banked using the plasmapheresis method. Only those donors with a documented medical history of COVID-19, which was confi rmed by polymerase chain reaction of SARS-CoV-2 RNA in pharyngeal swabs, and no sooner than 14 days after complete recovery were eligible for donation. Viral neutralizing activity (VNA) was chosen as the key characteristic of the immunological viability of CP. All the donors having VNA titers were characterized in terms of gender, age, time interval since the disease onset, regression of clinical symptoms and clinical features of the COVID 19 course.Results. Effective (1:160 or more) and acceptable (1:80) VNA titers were found in 21.1 % and 24.75 % of donors, respectively. Signifi cant predictors for a donor having a high VNA titer included: male sex, age over 36 years and verifi ed viral pneumonia. The absence of a signifi cant body temperature response (38.5 °С) can be considered as a negative marker of a potential donor. Введение. Трансфузии плазмы антиковидной патогенредуцированной (ПАП) рассматривается как один из методов лечения COVID-19. Цель: проанализировать опыт формирования донорского резерва из реконвалесцентов COVID-19 для заготовки ПАП.Материалы и методы. В ретроспективное исследование включено 493 реконвалесцента COVID-19, проходивших обследование в ГБУЗ «НИИ скорой помощи им. Н. В. Склифосовского ДЗМ» в качестве потенциальных доноров ПАП. Заготовку ПАП осуществляли методом плазмафереза. К процедуре плазмодачи допускали доноров, у которых в анамнезе была перенесенная инфекция COVID-19, подтвержденная выявлением методом полимеразной цепной реакции РНК SARS-CoV-2 в мазках из глотки, наличие медицинской документации, срок с момента разрешения заболевания не менее 14 суток. В качестве характеристики иммунологической состоятельности ПАП был выбран титр вируснейтрализующих антител (ВНА). Сопоставлены характеристики доноров (пол, возраст, давность с момента начала заболевания и регресса клинической симптоматики, особенности течения COVID-19) с титром ВНА.Результаты. Эффективный титр ВНА (1:160 и более) выявлен у 21,10 % доноров, допустимый (1:80) - у 24,75 %. Значимыми предикторами высокого титра ВНА оказались: мужской пол донора, возраст старше 36 лет, наличие верифицированной вирусной пневмонии. Отсутствие значимой температурной реакции (38,5 °C) может рассматриваться как отрицательный маркер для привлечения потенциального донора. Заключение. Для получения ПАП с высокими титрами ВНА оптимально привлечение в качестве доноров-реконвалесцентов мужчин, переболевших COVID-19 с клинической картиной вирусной пневмонии и значимой температурной реакцией.

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.