The tissue distribution of SARS-CoV-2 in transgenic mice with inducible ubiquitous expression of hACE2

The novel coronavirus disease COVID-19 has become one of the most socially significant infections. One of the main models for COVID-19 pathogenesis study and anti-COVID-19 drug development is laboratory animals sensitive to the virus. Herein, we report SARSCoV- 2 infection in novel transgenic mice conditionally expressing human ACE2 (hACE2), with a focus on viral distribution after intranasal inoculation. Transgenic mice carrying hACE2 under the floxed STOP cassette [(hACE2-LoxP(STOP)] were mated with two types of Cre-ERT2 strains (UBC-Cre and Rosa-Cre

Development and laboratory production of virus-like immune-stimulating complexes based on saponins and evaluation of their adjuvant potential using mice immunisation with influenza antigens

The COVID-19 pandemic has exacerbated the public’s need for effective vaccines. Consequently, significant financial support has been provided to developers of a number of innovative vaccines, including the vaccines with saponin-based adjuvants. In 2021, the World Health Organisation recommended Mosquirix, the first malaria vaccine, which contains a saponin adjuvant. An anti-covid vaccine by Novavax is in the approval phase. A promising approach to vaccine development is presented by the use of virus-like immune-stimulating complexes (ISCOMs) containing saponins and by the creation of combinations of ISCOMs with antigens. The aim of the study was to develop, produce and characterise virus-like immune-stimulating complexes based on saponins of Quillaja saponaria, as well as similar saponins of Russian-sourced Polemonium caeruleum. Materials and methods: The ISCOM adjuvants, Matrix-BQ and Matrix-BP, were produced using liquid chromatography and examined using electron microscopy. Balb/c mice were immunised intraperitoneally and intramuscularly with ISCOM-antigen preparations. Afterwards, the immunised animals were challenged with the influenza virus strain, A/California/4/2009(H1N1)pdm09, adapted and lethal to mice. The serum samples were examined using haemagglutination inhibition (HI) tests. Results: The authors produced the ISCOMs containing saponins of Quillaja saponaria and Polemonium caeruleum. After one intramuscular injection of either of the ISCOM-antigen preparations with 1 µg of each of A/Brisbane/02/2018 (H1N1) pdm09, A/Kansas/14/2017 (H3N2), and B/Phuket/3073/2013 haemagglutinin antigens (HAs), HI tests detected serum antibody titres to the corresponding antigens of ≥1:40. Two intramuscular injections of the ISCOM-antigen preparation containing 50 ng of each of the HAs and Matrix-BQ resulted in a protective response. In some animals, two intraperitoneal injections of ISCOM-antigen preparations resulted in the maximum antibody titre to the A/Kansas/14/2017 (H3N2) vaccine strain of 1:20,480. Two intramuscular injections of a test preparation containing 5 µg, 1 µg, 200 ng, or 50 ng of each of the HAs and Matrix-BQ or a control preparation containing 5 µg, 1 µg, or 200 ng of each of the HAs (commercially available vaccines) to the mice that were afterwards infected with the lethal influenza strain protected the experimental animals from death. Conclusions: The ISCOM-based preparations had high immunostimulatory activity in the mouse-model study. The presented results indicate the potential of further studies of ISCOM-based preparations in terms of both vaccine and immunotherapeutic development

Разработка и лабораторное получение вирусоподобных иммуностимулирующих комплексов на основе сапонинов, оценка их адъювантных свойств при иммунизации мышей гриппозными антигенами

The COVID-19 pandemic has exacerbated the public’s need for effective vaccines. Consequently, significant financial support has been provided to developers of a number of innovative vaccines, including the vaccines with saponin-based adjuvants. In 2021, the World Health Organisation recommended Mosquirix, the first malaria vaccine, which contains a saponin adjuvant. An anti-covid vaccine by Novavax is in the approval phase. A promising approach to vaccine development is presented by the use of virus-like immune-stimulating complexes (ISCOMs) containing saponins and by the creation of combinations of ISCOMs with antigens. The aim of the study was to develop, produce and characterise virus-like immune-stimulating complexes based on saponins of Quillaja saponaria, as well as similar saponins of Russian-sourced Polemonium caeruleum. Materials and methods: The ISCOM adjuvants, Matrix-BQ and Matrix-BP, were produced using liquid chromatography and examined using electron microscopy. Balb/c mice were immunised intraperitoneally and intramuscularly with ISCOM-antigen preparations. Afterwards, the immunised animals were challenged with the influenza virus strain, A/California/4/2009(H1N1)pdm09, adapted and lethal to mice. The serum samples were examined using haemagglutination inhibition (HI) tests. Results: The authors produced the ISCOMs containing saponins of Quillaja saponaria and Polemonium caeruleum. After one intramuscular injection of either of the ISCOM-antigen preparations with 1 µg of each of A/Brisbane/02/2018 (H1N1) pdm09, A/Kansas/14/2017 (H3N2), and B/Phuket/3073/2013 haemagglutinin antigens (HAs), HI tests detected serum antibody titres to the corresponding antigens of ≥1:40. Two intramuscular injections of the ISCOM-antigen preparation containing 50 ng of each of the HAs and Matrix-BQ resulted in a protective response. In some animals, two intraperitoneal injections of ISCOM-antigen preparations resulted in the maximum antibody titre to the A/Kansas/14/2017 (H3N2) vaccine strain of 1:20,480. Two intramuscular injections of a test preparation containing 5 µg, 1 µg, 200 ng, or 50 ng of each of the HAs and Matrix-BQ or a control preparation containing 5 µg, 1 µg, or 200 ng of each of the HAs (commercially available vaccines) to the mice that were afterwards infected with the lethal influenza strain protected the experimental animals from death. Conclusions: The ISCOM-based preparations had high immunostimulatory activity in the mouse-model study. The presented results indicate the potential of further studies of ISCOM-based preparations in terms of both vaccine and immunotherapeutic development.Пандемия COVID-19 обострила потребность общества в эффективных вакцинных препаратах. В этих условиях существенную финансовую поддержку получили разработчики ряда инновационных вакцин, в том числе вакцин, в состав которых входят адъюванты на основе сапонинов. В 2021 г. ВОЗ была одобрена первая противомалярийная вакцина Mosquirix, содержащая сапонины. На стадии одобрения находится вакцина Novavax против COVID-19. Перспективным подходом к созданию вакцин является использование вирусоподобных иммуностимулирующих комплексов (ИСКОМ) на основе сапонинов и создание на их основе комплексов с антигеном (ИСКОМ-антиген). Цель работы: получение и изучение вирусоподобных иммуностимулирующих комплексов на основе сапонинов Квиллайи мыльной (Quillaja saponaria), а также аналогов на основе сапонинов Синюхи голубой (Polemonium caeruleum), полученных из отечественного сырья. Материалы и методы: с применением метода жидкостной хроматографии получали препараты ИСКОМ адъювантов — Матрикс-BQ и Матрикс-BP. Проведено электронно-микроскопическое исследование препаратов. Иммунизацию мышей Balb/c препаратами ИСКОМ-антиген проводили интраперитонеально и внутримышечно. Иммунизированных животных заражали адаптированным летальным для мышей штаммом вируса гриппа A/California/4/2009 (H1N1) pdm09. Образцы сыворотки крови иммунизированных животных исследовали в реакции торможения гемагглютинации (РТГА). Результаты: получены ИСКОМ, содержащие сапонины Синюхи голубой и Квиллайи мыльной. В образцах сыворотки крови животных, однократно внутримышечно иммунизированных препаратом ИСКОМ-антиген, содержащим по 1 мкг гемагглютинина каждого из штаммов вирусов гриппа A/Brisbane/02/2018 (H1N1) pdm09, A/Kansas/14/2017 (H3N2), B/ Phuket/3073/2013, значения титров антител в РТГА составили более 1:40 к соответствующим антигенам. При двукратном внутримышечном введении препарата ИСКОМ-антиген, содержащего 50 нг каждого антигена, был выявлен протективный ответ. Максимальные значения титров антител в РТГА выявлены при двукратном интраперитонеальном введении препарата ИСКОМ-антиген и составили 1:20480 к гемагглютинину вакцинного штамма A/Kansas/14/2017 (H3N2). Показано, что двукратное внутримышечное введение 5 мкг, 1 мкг, 200 нг, 50 нг препарата ИСКОМ-антиген и 5 мкг, 1 мкг, 200 нг контрольного антигена коммерчески доступной вакцины мышам, впоследствии зараженным летальным штаммом вируса гриппа A/California/4/2009 (H1N1)pdm09, защищает экспериментальных животных от гибели. Выводы: полученные препараты на основе ИСКОМ обладали высокой иммуностимулирующей активностью в исследовании на мышиной модели. Представленные результаты свидетельствуют о перспективности дальнейшего изучения препаратов на основе ИСКОМ при разработке как противовирусных, так и иммунокорректирующих препаратов

A single blind, placebo-controlled randomized study of the safety, reactogenicity and immunogenicity of the “EpiVacCorona” Vaccine for the prevention of COVID-19, in volunteers aged 18–60 years (phase I–II)

Vaccination of the population is one of the most effective countermeasures in responding to the pandemic caused by novel coronavirus infection. Therefore, scientists all over the world have been working to develop effective and safe vaccines. We have developed a synthetic peptide vaccine, EpiVacCorona, against novel SARS-CoV-2 coronavirus, which is a suspension for intramuscular administration containing a composition of chemically synthesized peptide immunogens of the S protein of SARS-CoV-2 coronavirus conjugated to a carrier protein and adsorbed on aluminum hydroxide. Phase I–II clinical trials of the vaccine have started that consist of two stages: Stage 1 is an open study of the safety, reactogenicity, and immunological activity of the vaccine with the involvement of 14 volunteers aged 18–30 years; Stage 2 is a single blind, comparative, randomized placebo-controlled study with the involvement of 86 volunteers. The study involved volunteers aged 18–60 years; the vaccine was injected intramuscularly twice, spaced 21 days apart between injections. All local reactions in response to vaccine administration were mild, such as a short-term pain at the injection site. There were no signs of development of local or systemic adverse reactions. The two-dose vaccination scheme induced the production of antibodies, specific to the antigens that make up the vaccine, in 100% of the volunteers. Seroconversion with a neutralizing antibody titer ≥ 1:20 was reported in 100% of the volunteers 21 days following the second immunization dose. No seroconversion was reported in the groups of volunteers vaccinated with a placebo. The peptide-based EpiVacCorona Vaccine has low reactogenicity and is a safe, immunogenic product. Clinical Trials Identifier: NCT04527575

Оценка гуморального иммунного ответа экспериментальных животных на введение рекомбинантного эктодомена поверхностного S-гликопротеина вируса SARS-CoV-2 с ИСКОМ-адъювантом

Scientific relevance. The use of recombinant antigens in vaccine production is limited because vaccines based on such antigens tend to have low immunogenicity. However, a COVID-19 vaccine that combines recombinant SARS-CoV-2 spike glycoprotein as its antigen and virus-like immune-stimulating complexes (ISCOMs) as its adjuvant (Nuvaxovid) induces a protective virus-neutralising response. The State Research Center of Virology and Biotechnology “Vector” (hereinafter, Vector) has developed the ISCOM adjuvant Matrix-V, which plays a key role in inducing virus-neutralising antibodies. Studying Matrix-V will provide for the wide use of recombinant antigens combined with this adjuvant in the development and production of novel Russian vaccines.Aim. This study aimed to evaluate the humoral immune responses of experimental animals to intramuscular injections of a complex combining the recombinant Wuhan-type SARS-CoV-2 spike RBD antigen and the virus-like ISCOM adjuvant containing Quillaja saponaria saponins.Materials and methods. The Matrix-V ISCOM adjuvant was produced using Vector’s proprietary technology, which involves cross-flow filtration through Sartorius VivaFlow cassettes. To determine the saponin and residual detergent concentrations in Matrix-V, the authors conducted high-performance liquid chromatography. Having produced the recombinant SARS-CoV-2 RBD antigen, the authors used electron microscopy to analyse the ultrastructure of the ISCOM–antigen complex. In the study of the ISCOM–antigen complex, 25 female Balb/c mice (5 groups) and 15 male and female outbred guinea pigs (3 groups) received two intramuscular injections with a 14-day interval. Serum tests relied on virus neutralisation (VN) and enzyme-linked immunosorbent assay (ELISA) methods and used antigens of 8 SARS-CoV-2 variants (State Collection of Viruses and Rickettsia, Vector). The authors used Statistica 10 to analyse the results.Results. Two injections of the SARS-CoV-2 RBD antigen (mice: 7 μg, guinea pigs: 1 μg) alone did not induce statistically significant virus-neutralising antibody responses, as shown by the VN results. Two injections of the SARS-CoV-2 RBD antigen (mice: 7 μg, guinea pigs: 1 μg) adjuvanted with Matrix-V (25 μg) resulted in geometric mean antibody titres of 1:83–1:178 (mice) and 1:174–1:587 (guinea pigs) in the VN tests with the Wuhan variant. One injection of the antigen (1 μg or 7 μg) with Matrix-V (25 μg) induced antibodies only in individual cases, as demonstrated by the VN and/or ELISA results. The most intensive immune response was observed in ELISA tests with the Delta variant after two injections of the Ecto-S-Wuhan (1 μg) and Matrix-V (25 μg) complex. Immune responses did not differ between the group that received two injections of the Ecto-S-Wuhan antigen (1 μg) without the ISCOM adjuvant and the negative control group (titres below 1:100; p=0.95). Two injections of the SARS-CoV-2 RBD antigen (7 μg) without the ISCOM adjuvant induced antibodies in mice (titres between 1:248 and 1:1477).Conclusions. Two intramuscular injections of the complex containing the recombinant SARS-CoV-2 RBD antigen and the Matrix-V ISCOM adjuvant induce virus-neutralising antibodies. The approach proposed by the authors has the potential for use in the development of immunobiological medicinal products to prevent and treat a wide range of infectious diseases.Актуальность. Использование рекомбинантных антигенов в вакцинах ограничено низкой иммуногенностью таких препаратов. Однако вакцина от COVID-19 (Nuvaxovid), содержащая не только рекомбинантный антиген гликопротеина Spike вируса SARS-CoV-2, но и вирусоподобный иммуностимулирующий комплекс ИСКОМ-адъювант, индуцирует выработку протективного вируснейтрализующего ответа. Исследование разработанного в ФБУН ГНЦ ВБ «Вектор» Роспотребнадзора ИСКОМ-адъюванта «Матрикс-В», играющего ключевую роль в формировании вируснейтрализующего иммунного ответа, позволит широко использовать рекомбинантные антигены в комплексе с адъювантом для разработки и производства новых отечественных вакцин.Цель. Оценка гуморального иммунного ответа на внутримышечное введение животным комплекса рекомбинантного антигена RBD поверхностного Spike-гликопротеина SARS-CoV-2 (штамм «Ухань») и полученных вирусоподобных ИСКОМ с сапонинами Quillaja saponaria.Материалы и методы. ИСКОМ-адъювант «Матрикс-В» получали по разработанной в ФБУН ГНЦ ВБ «Вектор» Роспотребнадзора технологии с применением метода тангенциальной фильтрации в системе Sartorious VivaFlow. Оценивали концентрацию сапонинов и детергента в препарате методом высокоэффективной жидкостной хроматографии. Получен рекомбинантный антиген RBD SARS-CoV-2, проведено электронно-микроскопическое исследование ультраструктуры комплекса «ИСКОМ — антиген». Комплексами «ИСКОМ — антиген» внутримышечно двукратно (через 14 сут) иммунизировали 25 (5 групп) мышей линии Balb/c и 15 (3 группы) разнополых аутбредных морских свинок. Образцы сыворотки крови исследовали в реакции нейтрализации и методом ИФА с антигенами 8 штаммов вируса SARS-CoV-2 (Государственная коллекция возбудителей вирусных инфекций и риккетсиозов ФБУН ГНЦ ВБ «Вектор» Роспотребнадзора). Результаты обрабатывались статистически в программе Statistica 10.Результаты. Двукратное введение антигена RBD SARS-CoV-2 морским свинкам (по 1 мкг) и мышам (по 7 мкг) не приводило в реакции нейтрализации к достоверному формированию вируснейтрализующего ответа. В реакции нейтрализации со штаммом «Ухань» средние геометрические значения титров антител сыворотки крови животных, двукратно иммунизированных комплексом RBD SARS-CoV-2 (мыши — по 7 мкг, морские свинки — по 1 мкг) + Матрикс-В (по 25 мкг), были в пределах 1:83–1:178 и 1:174–1:587 соответственно. В группах животных, однократно иммунизированных комплексом (1 мкг и 7 мкг антигена + Матрикс-В) антитела определялись в реакции нейтрализации (и/или ИФА) только в единичных случаях. В опытах ИФА наиболее интенсивный ответ (на антиген штамма Delta) получен после двукратной иммунизации комплексом Ecto-S-Wuhan (1 мкг) + Матрикс-В (25 мкг). Ответ на двукратное введение антигена Ecto-S-Wuhan (1 мкг) без ИСКОМ-адъюванта (p=0,95) не отличался от отрицательного контроля (значение титра менее 1:100). Двукратное введение мышам антигена RBD SARS-CoV-2 (по 7 мкг) приводило к формированию антител (значения титров 1:248–1:1477).Выводы. Комплекс рекомбинантного антигена RBD SARS-CoV-2 и адъюванта «Матрикс-В» при двукратном внутримышечном введении индуцирует выработку вируснейтрализующих антител. Предложенный подход перспективен для разработки иммунобиологических препаратов профилактики и терапии широкого спектра инфекционных заболеваний

Cyclamen europaeum (<i>Cyclamen purpurascens</i>) extract as adjuvant for nasal immunization of mice with influenza antigens

The article describes the first attempt to use the juice and extract of Cyclamen europaeum (Cyclamen purpurascens) tubers as an adjuvant for intranasal immunization of mice with influenza antigens. The concentration of antigens used for immunization was 300 μg/ml for each subtype. The adjuvant was added at the concentration of 10 and 20 mg/ml. Blood serum was studied using the hemagglutination inhibition reaction (HI) and enzyme immunoassay (ELISA). After two immunizations with a dose of 7.5 μg, the maximum inverse titers to the H1/H3/B components in the HI were 320/80/80, respectively. The administration of an intranasal comparator without an adjuvant did not result in seroconversion which can be detected by the HI. The analysis of the blood sera of mice, immunized intranasally by the antigen only, showed no increase in the antibody levels between the first and second injections. For mice immunized intranasally by a preparation containing 10 mg/ml (0.5 mg per 50 μl dose) of adjuvant the ELISA detected a significant growth of antibody levels for all components, and GMT antibody levels were comparable to GMT antibody levels after a single intramuscular injection of 5 μg of each antigen. Despite a significant serum titer dispersion (which the authors explain by the impossibility of ensuring absolute uniformity in administration of 50 μl of substance via the nasal route) the use of the extract as an adjuvant for intranasal immunization of mice with highly concentrated influenza antigens showed a significant humoral response. The level of this response after two immunizations in some animals was comparable to that after intramuscular administration. The obtained data open the possibility of using Cyclamen europaeum tuber extract or its chemical analogues in further studies in guinea pigs, ferrets or other animal models in order to develop an efficacious adjuvant for intranasal immunization

Экстракт цикламена европейского (Cyclamen purpurascens) в качестве адъюванта при интраназальной иммунизации мышей гриппозными антигенами

The article describes the first attempt to use the juice and extract of Cyclamen europaeum (Cyclamen purpurascens) tubers as an adjuvant for intranasal immunization of mice with influenza antigens. The concentration of antigens used for immunization was 300 μg/ml for each subtype. The adjuvant was added at the concentration of 10 and 20 mg/ml. Blood serum was studied using the hemagglutination inhibition reaction (HI) and enzyme immunoassay (ELISA). After two immunizations with a dose of 7.5 μg, the maximum inverse titers to the H1/H3/B components in the HI were 320/80/80, respectively. The administration of an intranasal comparator without an adjuvant did not result in seroconversion which can be detected by the HI. The analysis of the blood sera of mice, immunized intranasally by the antigen only, showed no increase in the antibody levels between the first and second injections. For mice immunized intranasally by a preparation containing 10 mg/ml (0.5 mg per 50 μl dose) of adjuvant the ELISA detected a significant growth of antibody levels for all components, and GMT antibody levels were comparable to GMT antibody levels after a single intramuscular injection of 5 μg of each antigen. Despite a significant serum titer dispersion (which the authors explain by the impossibility of ensuring absolute uniformity in administration of 50 μl of substance via the nasal route) the use of the extract as an adjuvant for intranasal immunization of mice with highly concentrated influenza antigens showed a significant humoral response. The level of this response after two immunizations in some animals was comparable to that after intramuscular administration. The obtained data open the possibility of using Cyclamen europaeum tuber extract or its chemical analogues in further studies in guinea pigs, ferrets or other animal models in order to develop an efficacious adjuvant for intranasal immunization.В статье описано первое использование сока и экстракта клубней цикламена европейского (Cyclamen purpurascens) в качестве адъюванта при интраназальной иммунизации мышей гриппозными антигенами. Для иммунизации использовали концентрацию антигенов 300 мкг/мл каждого субтипа. Адъювант добавлялся в концентрации 10 и 20 мг/мл. Сыворотки крови изучали в реакции торможения гемагглютинации (РТГА) и иммуноферментном анализе (ИФА). После двух иммунизаций дозой 7,5 мкг максимальные обратные титры к H1/H3/B-компонентам в РТГА составили 320/80/80 соответственно. Введение интраназального препарата сравнения без адъюванта не привело к сероконверсии, детектируемой в РТГА. В сыворотках крови мышей, иммунизированных интраназально препаратом сравнения без адъюванта, не отмечено достоверного нарастания уровня антител между первым и вторым введением. В группах, иммунизированных интраназально препаратом, содержащим 10 мг/мл (0,5 мг в 1 дозе, объемом 50 мкл) адъюванта, отмечено значимое нарастание уровня антител между однократной и двукратной иммунизацией для всех компонентов, детектированных в ИФА, а средние уровни антител сопоставимы с ответом, полученным на однократное внутримышечное введение дозы препарата, содержащей 5 мкг каждого антигена. Несмотря на разброс значений титров сывороток крови между животными, который мы связываем с невозможностью стандартного внесения 50 мкл препарата в носоглотку мыши, применение экстракта в качестве адъюванта при интраназальной иммунизации мышей высококонцентрированными гриппозными антигенами показало выраженный гуморальный ответ. Уровень этого ответа после двукратной иммунизации у некоторых животных сравним с ответом на гриппозные вакцины при внутримышечном введении. Полученные данные позволяют рассматривать сок и экстракт клубней цикламена европейского или его синтетические аналоги для дальнейших исследований на морских свинках, хорьках или других животных-моделях с целью разработки эффективного адъюванта для интраназальной вакцинации

Human Adenovirus and Influenza A Virus Exacerbate SARS-CoV-2 Infection in Animal Models

In this study, we investigated the features of the infectious process by simulating co-infection with SARS-CoV-2 and human adenovirus type 5 (HAdV-5) or influenza A virus (IAV) in vitro and in vivo. The determination of infectious activity of viruses and digital PCR demonstrated that during simultaneous and sequential HAdV-5 followed by SARS-CoV-2 infection in vitro and in vivo, the HAdV-5 infection does not interfere with replication of SARS-CoV-2. The hamsters co-infected and mono-infected with SARS-CoV-2 exhibited nearly identical viral titers and viral loads of SARS-CoV-2 in the lungs. The hamsters and ferrets co-infected by SARS-CoV-2- and IAV demonstrated more pronounced clinical manifestations than mono-infected animals. Additionally, the lung histological data illustrate that HAdV-5 or IAV and SARS-CoV-2 co-infection induces more severe pathological changes in the lungs than mono-infection. The expression of several genes specific to interferon and cytokine signaling pathways in the lungs of co-infected hamsters was more upregulated compared to single infected with SARS-CoV-2 animals. Thus, co-infection with HAdV-5 or IAV and SARS-CoV-2 leads to more severe pulmonary disease in animals

Severe cases of seasonal influenza in Russia in 2017-2018.

The 2017-2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017-2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017-2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017-2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33-47% and 24-30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017-2018 influenza epidemic season in Russia

Correction: Severe cases of seasonal influenza in Russia in 2017-2018.

[This corrects the article DOI: 10.1371/journal.pone.0220401.]