Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product

Production and characterisation of a SARS-CoV-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 SARS-CoV-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.Materials and methods: 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 mono clonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones.Results: 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.Conclusions: 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

Получение и характеристика гомодимерной формы RBD S-белка SARS-CoV-2, обладающей повышенной авидностью к специфическим антителам

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 w as t o 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. Materials and methods: 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. Results: 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. Conclusions: 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.Важным параметром, оцениваемым при мониторинге иммунной прослойки у населения и эффективности вакцинации населения, является уровень вируснейтрализующих антител. Разработка подхода к выявлению вируснейтрализующих антител к вирусу SARS-CoV-2 с помощью безопасного, простого и быстрого метода, не требующего использования живых вирусов, имеет большое значение для борьбы с пандемией COVID-19. Для разработки тест-систем для проведения иммуноферментного анализа (ИФА), детектирующих потенциально вируснейтрализующие антитела, необходимо получение высокоочищенного рекомбинантного рецептор-связывающего домена (RBD) S-белка, обладающего высокой авидностью к специфическим антителам. Цель работы: получение и характеристика гомодимерной формы RBD S-белка вируса SARS-CoV-2, а также клеточной линии, продуцирующей рекомбинантный RBD, для создания ИФА тест-системы для выявления потенциально вируснейтрализующих антител. Материалы и методы: дизайн генетической конструкции проводили in silico. Стабильную клеточную линию получали при помощи трансфекции клеток CHO-S, селекции на антибиотике и отбора оптимального клона. Рекомбинантный RBD очищали с использованием хроматографических методов, получали мономерную и гомодимерную формы RBD. Активность полученных форм анализировали с использованием методов Вестерн-блот, биослойной интерферометрии и непрямго ИФА. Для анализа использовали моноклональные антитела GamXRH19, GamP2C5 и h6g3, а также образцы сывороток крови добровольцев, вакцинированных препаратом Гам-КОВИД-Вак, и невакцинированных добровольцев. Результаты: получена клеточная линия CHO-S, стабильно продуцирующая рекомбинантный RBD S-белка вируса SARS-CoV-2. Показано, что при культивировании данной клеточной линии в режиме fed-batch более 7 суток рекомбинантный RBD способен образовывать гомодимеры за счет наличия неспаренных цистеинов. Количественный выход очищенного рекомбинантного RBD из культуральной жидкости составил 30–50 мг/л. Мономерная и гомодимерная формы RBD были разделены при помощи гель-фильтрации и охарактеризованы по способности взаимодействовать со специфическими моноклональными антителами, а также сыворотками крови от вакцинированных добровольцев. Продемонстрировано, что именно гомодимерная форма рекомбинантного RBD обладает повышенной авидностью к моноклональным антителам и антителам в сыворотке крови вакцинированных. Выводы: гомодимерная форма рекомбинантного RBD может являться более предпочтительной для анализа уровня антител к рецептор-связывающему домену S-белка вируса SARS-CoV-2

Сравнение различных технологий получения рекомбинантного аденоассоциированного вируса в лабораторном масштабе

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.Векторы на основе аденоассоциированного вируса являются одними из наиболее перспективных для доставки трансгенов в различные органы и ткани. Рекомбинантный аденоассоциированный вирус (rAAV) способен трансдуцировать как делящиеся, так и неделящиеся клетки, обладает низкой иммуногенностью и способен обеспечивать долгосрочную экспрессию трансгенов. На сегодняшний день существуют технологии, позволяющие получать rAAV для применения in vivo, однако они не лишены недостатков, связанных с трудоемкостью, сложностями масштабирования и высокой стоимостью, поэтому вопрос об усовершенствовании технологических схем получения rAAV является актуальным. Цель работы: сравнение технологических подходов к получению rAAV, основанных на различных условиях культивирования трансфицированной клеточной линии HEK293 в лабораторном масштабе. Материалы и методы: в исследовании использовали культуру клеток HEK293, плазмидную систему AAV-DJ Packaging System, систему PlasmidSelect Xtra Starter Kit. В качестве модели для сравнения технологий использовали вектор rAAV с трансгеном однодоменного антитела, слитого с Fc-фрагментом IgG1, специфичного к ботулотоксину. Применяли метод трансфекции клеток HEK293 суперскрученной плазмидной ДНК, выделенной при помощи трехступенчатой хроматографической очистки. Определение подлинности препарата rAAV проводили методами электрофореза, иммуноблоттинга и полимеразной цепной реакции в режиме реального времени. Результаты: продемонстрирована эффективность получения суперскрученной формы плазмидной ДНК, применимой для эффективной трансфекции с целью получения rAAV. Проведено сравнение процесса транзиентной трансфекции и культивирования трансфицированных клеток HEK293 в условиях суспензии в колбах, адгезии в культуральных флаконах и адгезии в биореакторе BioBLU 5p на матрице из дисков Fibra-Cel с целью продукции rAAV. Выводы: показана возможность применения описанных подходов к очистке плазмидной ДНК, трансфекции и культивированию трансфицированных клеток в различных условиях для получения препарата rAAV, эспрессирующего ген антитела. Реактор BioBLU 5p с дисками Fibra-Cel был впервые использован для получения препаративных количеств rAAV в лабораторном масштабе, что позволило увеличить площадь поверхности адгезии при культивировании и трансфекции клеток и, как следствие, увеличить выход целевого продукта

rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19

IntroductionNumerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis.MethodsIn current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing.Results and discussionsA P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants

Safety and immunogenicity of rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine against SARS-CoV-2 in healthy adolescents: an open-label, non-randomized, multicenter, phase 1/2, dose-escalation study

To protect young individuals against SARS-CoV-2 infection, we conducted an open-label, prospective, non-randomised dose-escalation Phase 1/2 clinical trial to evaluate the immunogenicity and safety of the prime-boost “Sputnik V” vaccine administered at 1/10 and 1/5 doses to adolescents aged 12–17 years. The study began with the vaccination of the older cohort (15-to-17-year-old participants) with the lower (1/10) dose of vaccine and then expanded to the whole group (12-to-17-year-old participants). Next, 1/5 dose was used according to the same scheme. Both doses were well tolerated by all age groups. No serious or severe adverse events were detected. Most of the solicited adverse reactions were mild. No significant differences in total frequencies of adverse events were registered between low and high doses in age-pooled groups (69.6% versus 66.7%). In contrast, the 1/5 dose induced significantly higher humoral and T cell-mediated immune responses than the 1/10 dose. The 1/5 vaccine dose elicited higher antigen-binding (both S and RBD-specific) as well as virus-neutralising antibody titres at the maximum of response (day 42), also resulting in a statistically significant difference at a distanced timepoint (day 180) compared to the 1/10 vaccine dose. Higher dose resulted in increased cross-neutralization of Delta and Omicron variants.;Clinical Trial RegistrationClinicalTrials.gov, NCT04954092, LP-007632

Some aspects of the legal protection of Lake Baikal at the present stage

Lake Baikal is a unique natural complex, an ecosystem, each element of which is inimitable and invaluable. The importance of the natural monument is recognized at the national and international levels. Legal standards have been developed and are in force, establishing a regime for the protection of the ecology of the reservoir and adjacent territories. They regulate the rules of the special regime of economic activity around the lake, the boundaries of the fish protection zone and the features of animal protection, the prohibitions on the chemical and biological pollution of the lake, as well as the prohibition of any type of activity that could damage the “holy see”. The debatable issues of the core provisions of” Baikal Law” have been considered in the article. The contradictions in the zoning of the lands of the Baikal National Park have been identified. The amendments to the standards of maximum permissible impacts on the ecological system of Lake Baikal proposed by the Ministry of Natural Resources and Ecology of the Russian Federation have been analyzed

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice

The bacterium Clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A

Combined influence of teichoic acids from Staphylococcus aureus and heterometallik Cu/Cd ethylenediamine complex on peritoneal macrophages and tumor cells

We investigated the effects of teichoic acid (TA) from Staphylococcus aureus Wood 46 on tumor growth and metastasis of the experimental Lewis lung carcinoma (LLC) in mice. Intranasal administration of TA alone aggravated both tumor growth and metastasis, whereas combined administration of TA with a synthetic bimetallic (copper: cadmium) ethylene diamine complex PO244 resulted in pronounced antitumor and antimetastatic effects. The group of animals subjected to the combined treatment with TA and PO244 manifested the highest degree of lymphocyte infiltration into the tumor tissue, compared to the control group and those exposed to TA or PO244 alone. Moreover, the combined treatment negatively affected the adhesive properties of peritoneal macrophages in the LLC bearing mice. Co-cultivation of the isolated macrophages with primary LLC cultures revealed significant (p < 0.05) cytotoxic and cytostatic effects, detected as an increased level of apoptosis and a reduced fraction of replicating cells.Исследовали модифицирующее влияние тейхоевой кислоты (ТК) на рост и метастазирование перевиваемой карциномы легких Льюис у мышей. Выявлена гиперактивация роста и метастазирования первичной опухоли при интраназальном введении животным ТК, в то время как при комбинированном влиянии с РО244 наблюдали усиление противо-опухолевого эффекта. Самый высокий уровень инфильтрации опухолевой ткани лимфоцитами зафиксировали в группе животных, которым проводили комбинированную терапию ТК с РО 244. Показано снижение адгезивных свойств перитонеальных макрофагов под влиянием биметаллического комплекса. После сокультивирования макрофагов от животных, которым проводили комбинированную терапию с первичной культурой LLC, выявлено значительное (p < 0.05) цитотоксическое/цитостатическое влияние, что проявлялось в увеличении уровня апоптоза и уменьшении популяции клеток пролиферативного пула

DataSheet_1_Estimation of anti-orthopoxvirus immunity in Moscow residents and potential risks of spreading Monkeypox virus.docx

WHO has declared the outbreak of monkeypox as a public health emergency of international concern. In less than three months, monkeypox was detected in more than 30 000 people and spread to more than 80 countries around the world. It is believed that the immunity formed to smallpox vaccine can protect from monkeypox infection with high efficiency. The widespread use of Vaccinia virus has not been carried out since the 1980s, which raises the question of the level of residual immunity among the population and the identification of groups requiring priority vaccination. We conducted a cross-sectional serological study of remaining immunity among Moscow residents. To do this, a collection of blood serum samples of age group over 30 years old was formed, an in-house ELISA test system was developed, and a virus neutralization protocol was set up. Serum samples were examined for the presence of IgG antibodies against Vaccinia virus (n=2908), as well as for the ability to neutralize plaque formation with a Vaccinia virus MNIIVP-10 strain (n=299). The results indicate the presence of neutralizing antibody titer of 1/20 or more in 33.3 to 53.2% of people older than 45 years. Among people 30-45 years old who probably have not been vaccinated, the proportion with virus neutralizing antibodies ranged from 3.2 to 6.7%. Despite the higher level of antibodies in age group older than 66 years, the proportion of positive samples in this group was slightly lower than in people aged 46-65 years. The results indicate the priority of vaccination in groups younger than 45, and possibly older than 66 years to ensure the protection of the population in case of spread of monkeypox among Moscow residents. The herd immunity level needed to stop the circulation of the virus should be at least 50.25 – 65.28%.</p