The use of quantitative enzyme-linked immunosorbent assay for the determination of S-antigen concentration in whole-virion inactivated adsorbed coronavirus vaccines

The severe consequences and high mortality of COVID-19 prompted the development of a wide range of preventive vaccines. The first vaccines to be tested were developed in China and formulated as inactivated SARS-CoV-2 adsorbed on aluminium hydroxide. One of the quality indicators for inactivated adsorbed vaccines is the degree of adsorption, which can be used to control the content not only of non-adsorbed antigen, but also of specific antigen in one dose of a vaccine.The aim of the study was to investigate the possibility of desorbing SARS-CoV-2 antigen from formulated adsorbed vaccines and the possibility of measuring its concentration using the BioScan-SARS-CoV-2 (S) ELISA kit for SARS-CoV-2 S-protein content determination.Materials and methods: the study used four batches of BBIBP-CorV by CNBG, Sinopharm (China) and three batches of CoronaVac by Sinovac Biotech (China). The authors desorbed SARS-CoV-2 S antigen in accordance with monograph FS.3.3.1.0029.15 of the State Pharmacopoeia of the Russian Federation (Ph. Rus.), edition XIV, and quantified it using the BioScan-SARS-CoV-2 (S) ELISA kit by Bioservice Biotechnology Co. Ltd. (Russia).Results: mean S-antigen concentrations in the desorbed samples ranged from 61 to 129 ng/mL for BBIBP-CorV and from 461 to 533 ng/mL for CoronaVac.Conclusions: the study demonstrated the possibility of specific SARS-CoV-2 antigen desorption from the surface of aluminium hydroxide using the Ph. Rus. method, as well as the possibility of S-antigen quantification in desorbed medicinal products and supernatants using the BioScan-SARS-CoV-2 (S) ELISA kit. The authors observed 3.6- to 8.7-fold difference between the S-antigen concentrations of the desorbed preparations by the two manufacturers

Эффективность применения индуктора интерферонов против вируса Чикунгунья in vitro

Scientific relevance. To date, no specific antivirals have been approved to treat and prevent Chikungunya fever, its complications, and sequelae. Therefore, the development of therapeutic and preventive medicinal products against Chikungunya virus (CHIKV), including interferon inducers, is gaining relevance.Aim. The authors aimed to study the effectiveness of prophylactic administration of an interferon inducer against CHIKV in an in vitro model.Materials and methods. The study used two cell lines (Vero and А549), a CHIKV strain (Nika2021), and an interferon-inducing medicinal product (double-stranded RNA sodium salt) at two doses (250 μg/mL and 500 μg/mL) administered at two schedules: Prevention (4 h prior to the virus challenge) and Emergency Prevention (at the time of the virus challenge). The authors determined the CHIKV titre by its cytopathogenic effect, the CHIKV RNA content by the cycle threshold value in real-time reverse-transcription polymerase chain reaction, and the concentration of cytokines using the enzyme immunoassay method. The study monitored the changes in CHIKV biological activity, CHIKV RNA levels, and the production of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) in cells over time. The statistical analysis of the resulting data used Microsoft Office Excel 2016 and StatTech.Results. The medicinal product at doses of 250 μg/mL and 500 μg/mL stimulated the production of both IFN-α and IFN-γ (IFN-α to a greater extent than IFN-γ) in both cell lines (in A549 to a greater extent than in Vero). The changes in CHIKV RNA levels with time corresponded to those of the virus titre. In general, CHIKV RNA levels in Vero cells were significantly higher than those in A549 cells (р<0.002 at 250 μg/mL and р<0.0005 at 500 μg/mL). The CHIKV RNA content after preventive interferon inducer administration was significantly lower than that in the control experiment (challenge without administration of the medicinal product) for both doses and both cell lines (р<0.002 for Vero cells; р<0.0003 for А549 cells). The CHIKV RNA content after interferon inducer administration as emergency prevention was significantly lower than that in the control experiment (р<0.05 for Vero cells; р<0.003 for А549 cells). The study demonstrated the efficacy of the interferon inducer against CHIKV and a higher applicability of the A549 cell line to studying antiviral activity in vitro. The authors observed the production of IL-6 and TNF-α by intact cells of both lines.Conclusions. According to the results, the studied interferon inducer has a positive antiviral effect against CHIKV in vitro, with the antiviral effect degree depending on the cell line used. This experimental study demonstrated the need to carefully select the cell line for a study in accordance with its objectives and to evaluate the production of cytokines by a monolayer of cells before stimulation with viruses and/or medicinal products.Актуальность. Препараты для специфического лечения и профилактики лихорадки Чикунгунья, ее осложнений и последствий в настоящее время отсутствуют, в связи с этим особую актуальность приобретают вопросы разработки терапевтических и профилактических препаратов против вируса Чикунгунья (ЧИКВ), в том числе препаратов индукторов интерферонов.Цель. Изучение эффективности профилактического применения препарата индуктора интерферонов против вируса Чикунгунья в модели in vitro.Материалы и методы. Использовали штамм ЧИКВ Nika2021, две линии клеток — Vero и А549, а также препарат индуктора интерферонов (РНК двуспиральной натриевая соль) в двух дозировках (250 и 500 мкг/мл) и двух режимах применения (за 4 ч до заражения и одновременно с ним). Определяли титр вируса по его цитопатическому действию; содержание РНК ЧИКВ — методом ОТ-ПЦР-РВ и оценивали по показателю порогового числа циклов ПЦР; концентрацию продуцируемых клетками цитокинов — методом ИФА. В динамике анализировали активность ЧИКВ и содержание РНК ЧИКВ, а также продукцию цитокинов клетками (ИФН-α, -γ, ИЛ-6 и ФНО-α). Полученные данные обрабатывали при помощи пакетов программ Microsoft Office Excel 2016 и StatTech.Результаты. Препарат в дозах 250 и 500 мкг/мл стимулировал продукцию в большей степени ИФН-α и в меньшей степени ИФН-γ; в большей степени — в линии клеток А549, в меньшей — в линии клеток Vero. Динамика содержания РНК ЧИКВ соответствовала динамике его титра в клетке. В целом содержание РНК ЧИКВ в линии клеток Vero было значительно выше, чем в линии клеток А549 (р<0,002 при дозировке препарата 250 мкг/мл, р<0,0005 при дозировке 500 мкг/мл). Препарат применяли в двух режимах: профилактическом — за 4 ч до заражения клеток вирусом; экстренно профилактическом — одновременно с инфицированием. Содержание РНК ЧИКВ в режиме профилактического применения препарата относительно контрольного опыта (инфицирование без препарата) было значительно ниже в обеих дозах в обеих линиях клеток (р<0,002 для Vero, р<0,0003 для А549), в режиме экстренной профилактики — р<0,05 и р<0,003 соответственно. Выявлена и статистически подтверждена эффективность применения препарата индуктора интерферонов против ЧИКВ, показано преимущество линии клеток А549 для изучения противовирусной активности in vitro. В обеих линиях интактных клеток отмечена продукция ИЛ-6 и ФНО-α.Выводы. Обнаружен положительный противовирусный эффект от применения препарата индуктора интерферонов против ЧИКВ in vitro, при этом степень эффективности зависела от линии клеток. Показана необходимость тщательного отбора клеточной линии в зависимости от целей исследования, а также изучения продукции цитокинов клетками в монослое до стимуляции вирусами и (или) препаратами

Молекулярно-генетическое исследование стабильности и подтверждение подлинности штамма Внуково-32, применяемого для производства вакцины антирабической культуральной концентрированной очищенной инактивированной сухой

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form. Бешенство – острая вирусная инфекция, вызываемая вирусом семейства Rhabdoviridae рода Lyssavirus и характеризующаяся симптомами поражения центральной нервной системы и абсолютной летальностью. Единственной возможностью предотвратить возникновение данного заболевания у людей является вакцинопрофилактика. Одним из препаратов, используемых в этих целях, является вакцина антирабическая культуральная концентрированная очищенная инактивированная сухая, выпускаемая ФГБНУ «ФНЦИРИП им. М. П. Чумакова РАН».Цель работы: исследование структуры производственного, рабочего посевного вируса бешенства штамма Внуково-32, используемого ФГБНУ «ФНЦИРИП им. М. П. Чумакова РАН» для производства антирабической вакцины, его генетической стабильности на этапах производства, изучение возможности применения молекулярно-генетических методов для подтверждения подлинности производственного штамма в готовой форме вакцины и изучение нуклеотидной последовательности штамма CVS.Материалы и методы: производственный штамм вируса бешенства Внуково-32, рабочие посевные вирусы, готовые серии вакцины антирабической, штамм CVS фиксированного вируса бешенства, используемый для оценки специфического иммунитета. Молекулярно-генетическое исследование проведено с использованием ОТ-ПЦР с последующей рестрикцией и секвенированием.Результаты: представлены результаты анализа нуклеотидных последовательностей фрагмента гена G, полученного из производственного штамма Внуково-32, серий рабочего посевного вируса и готовых серий вакцины антирабической, изготовленных в 2012, 2018, 2019 г., штамма фиксированного вируса бешенства CVS, используемого для оценки специфической активности вакцины. Показана возможность применения рестрикционного анализа для подтверждения подлинности штамма Внуково-32 на всех этапах производства, включая готовую форму вакцины.Заключение: штаммы Внуково-32 и CVS, используемые в ФГБНУ «ФНЦИРИП им. М. П. Чумакова РАН», являются вирусами бешенства. Анализ нуклеотидной последовательности фрагмента гена G показал, что штамм Внуково-32 стабилен на разных этапах производства. Полученная нуклеотидная последовательность гена G штамма Внуково-32 депонирована в GenBank (номер MN116503). Показана возможность применения рестрикционного анализа для подтверждения подлинности штамма Внуково-32 вируса бешенства на всех этапах производства, включая готовую форму вакцины

Molecular Genetic Testing of Stability and Identification of Vnukovo-32 Strain Used for Production of the Cultural Concentrated Purified Inactivated Dry Rabies Vaccine

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form

Indication and Identification of Dengue and Chikungunya Viruses in Aedes spp. Mosquitoes Captured in Central America

The purpose of study was to isolate arboviruses from mosquitoes of different species in the cell culture and to identify them by using molecular and immunochemical techniques.Materials and methods. Viruses were isolated in C6/36 cell cultures. The pathogens were identified by using enzyme-linked immunosorbent assay (ELISA) kits for detection of antigens of dengue, Chikungunya, West Nile and Sindbis viruses as well as the reverse transcription polymerase chain reaction (RT-PCR) with specific primers and Sanger sequencing.Results. A total of 102 mosquitoes belonging to three genera, Culex spp, Culiseta spp., Aedes spp., were studied. Mosquitoes of each species or genus were divided into pools, each containing 4–5 mosquitoes. The study of suspensions of only 2 mosquito pools obtained from Aedes aegypti and Aedes albopictus, starting from the 3rd passage, showed changes in the C6/36 cell monolayer. Starting from the 4th passage, an antigen of Chikungunya virus was detected using ELISA test in the suspension obtained from the Aedes albopictus pool. Dengue virus was detected in the 5th passage from the materials obtained from the Aedes aegypti pool. Thus, antigens of the Chikungunya and dengue viruses were detected only in 2 of 23 examined pools of mosquitoes of different genera. Materials of the 5th passage were analyzed by RT-PCR with specific primers for dengue and Chikungunya viruses. It was confirmed that the isolate obtained from Aedes albopictus mosquitoes contained RNA of the Chikungunya virus and corresponded to the East/Central/South African genotype, while the isolate obtained from Aedes aegypti mosquitoes contained RNA of the dengue type 2 virus.Conclusion. The obtained nucleotide sequences of the Chikungunya virus were deposited in the GenBank international database under accession numbers MN271691 and MN271692