Research Institute of Influenza

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Intranasal immunization with a recombinant protein based on the M2e peptide and second subunit of influenza A viral hemagglutinin fragment induces a cross-protective humoral and Tcell response in mice

Development of vaccines with a broad-spectrum of protection is one of the priorities in the programs of influenza prevention. Recently, the conserved fragments of influenza virus proteins (M1, M2, NP, the second subunit of the hemagglutinin HA2) provoke interest of investigators as the object of the development a broad-spectrum vaccines. Low immunogenicity present a problem when developing vaccines based on such conserved fragments. However, fusion of low immunogenic antigens into the high immunogenic carrier protein may significantly enhance their immunogenicity. The candidate vaccine protein Flg-HA2-2-4M2e was developed which containins two highly conserved viral antigens (the ectodomain of the M2 protein (M2e), 76130 region of the second subunit of HA2), fused with flagellin as a carrier protein. Flagellin (bacterial flagella protein) is a natural ligand of TLR-5, and has a strong adjuvant activity at different ways of its administration. The purpose of this study was to assess development of humoral and T cell immune response, along with broad-spectrum protection after mice immunization with the candidate Flg-HA2-2-4M2e vaccine protein. Mice were immunized intranasally three times with two-week intervals. Two weeks after the final immunization, the mice were challenged at the 5 LD50 dose with influenza viruses A/California/07/09 (H1N1) pdm09 (phylogenetic group I), or A/Shanghai/2/2013 (H7N9) (phylogenetic group II). The results obtained in this study showed induction of strong M2e-specific humoral response (serum IgG and A) in the immunized mice. Immunization with recombinant protein stimulated formation of M2e-specific and virus-specific CD4+ and CD8+T cells in lung which produced TNFα or IFNγ. Production of antigen-specific effector and central memory T cells was also detected in lungs of immunized mice. The formation of cross-protective immunity in immunized mice was demonstrated in a model of lethal influenza infection. The experimental animals were almost completely protected from the high dose of the pandemic virus A/H1N1pdm09, and highly pathogenic avian influenza A/H7N9 (90-100% survival). We also evaluated the changes of antigen-specific immune response in immunized mice after sublethal infection with A/H3N2 influenza virus. Mice of control and experimental groups were infected with MID100 of influenza virus A/Aichi/2/68 (H3N2). It was shown that the M2e-specific response (IgG, IgA) was significantly increased in immunized mice after sublethal infection with influenza virus A/H3N2, and we detected the changes in profile of M2e-specific IgG subclasses. Following sublethal infection in immunized mice, the proportion of M2e-specific IgG2a was increased 10-fold. The results showed that the recombinant protein Flg-HA2-2-4M2e is a promising candidate for development of universal vaccines, which induces a protective humoral and T-cell response to conserved viral epitopes and protects against influenza A viruses of both phylogenetic groups

Генетические маркеры крови у больных гриппом и ОРЗ, осложненных пневмониями

Distribution rates of blood genetic markers (antigens of ABO- and HLA- systems) have been evaluated. The study results obtained among 119 patients with severe influenza and ARD forms are presented. It was found that in patients hospitalized during influenza A epidemia HLA—B8 and A(II) blood group antigens predominated. A(II) blood group antigens were registered more frequently in patients with confluent pneumonia than in ones with local pneumonia (51.3% and 28.2%, respectively); HLA—B8 antigens were registered near 3 times less frequently in confluent pneumonia patients than in local pneumonia ones (5.6% and 18%, respectively).Представлены данные изучения частоты распределения генетических маркеров—антигенов систем ABO и HLA среди 119 госпитализированных больных с тяжелыми формами гриппа и ОРЗ. Выявлено, что среди больных, госпитализированных в период эпидемий гриппа А преобладали носители антигенов А(II) и HLA-B8. Среди больных сливными пневмониями достоверно чаще, чем среди больных очаговыми пневмониями, встречалась вторая группа крови (51,3% против 28,2%) и в 3 раза реже антиген HLA-B8 (5,6% против 18,0%)

ЭФФЕКТИВНОСТЬ КРОСС-ПРОТЕКТИВНОЙ РЕКОМБИНАНТНОЙ ПРОТИВОГРИППОЗНОЙ ВАКЦИНЫ, ВКЛЮЧАЮЩЕЙ КОНСЕРВАТИВНЫЕ ЭПИТОПЫ ВИРУСНЫХ БЕЛКОВ М2 И ГЕМАГГЛЮТИНИНА

The influenza virus is the most unique in the level of variability of antigenic and biological properties. Because of constant mutations into genes coding surface viral proteins, in modern vaccines it is necessary to replace 1–2 virus components annually. Traditional influenza vaccines are the strain – specific and have limited efficiency in prevention of new strains of influenza viruses. In this regard, creation of influenza vaccines based on conserved determinants of viral proteins with broad spectrum protection and the short period of production is one of priority tasks which decision will lead to real control of an influenza infection. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based the combination of conserved viral proteins or peptides. The goals of this study: to develop the candidate recombinant flu vaccine based on the two conserved influenza proteins (М2 and НА); to investigate immune response; and to measure the protection activity in an animal model. Results: In this study we investigated the humoral and T-cell response in mice after intranasal immunization with recombinant proteins (Flg-4M2ehs and Flg-HA2-2-4M2ehs). Both proteins induce a robust M2e-specific humoral and CD4+ T-cell response in mice lung. The recombinant protein with two target antigens (M2e and HA2) induces virusspecific CD4+ and CD8+ T-cell response and full protection (100% survival) of mice from lethal challenge human and avian influenza viruses A (A/H3N2, A/H2N2, A/H5N1). In mice immunized with Flg-4M2ehs, the survival after lethal challenge was 60–75%. Conclusion: Our results show an essential role of a conserved fragment of the HA2 in the formation of protective T-cell response and protection of mice from lethal challenge with influenza viruses A of various subtypes. The prospects of the development of vaccine formulation based on two conserved antigenic determinants of influenza virus A are shown.Вирус гриппа является наиболее уникальным по уровню изменчивости антигенных и биологических свойств. Из-за постоянных мутаций в генах, кодирующих поверхностные белки вируса, в существующих «сезонных» вакцинах приходится ежегодно заменять 1–2 вирусных компонента. Кроме того, традиционные вакцины являются штамм-специфическими и обладают ограниченной эффективностью в предотвращении заболеваний, вызванных новыми штаммами вирусов гриппа. В связи с этим создание противогриппозных вакцин на основе консервативных детерминант вирусных белков с широким спектром защиты и коротким периодом производства является одной из приоритетных задач, решение которой приведет к реальному контролю гриппозной инфекции. Перспективной тенденцией в создании универсальных гриппозных вакцин является конструирование рекомбинантных белков на основе комбинации консервативных вирусных белков или пептидов. Цель: разработка кандидатной рекомбинантной гриппозной вакцины на основе двух консервативных белков вируса гриппа А (М2 и НА) и оценка ее иммуногенности и защитного эффекта на животной модели. Результаты: исследовали гуморальный и Т-клеточный ответ у мышей (Balb/c) после интраназальной иммунизации рекомбинантными белками (Flg4M2ehs и Flg-HA2-2-4M2ehs). Оба белка стимулировали формирование выраженного М2е-специфического гуморального и CD4+ Т-клеточного ответа в легких мышей. Рекомбинантный белок с двумя таргетными антигенами (М2е и полипептид 76–130 второй субъединицы гемагглютинина) стимулирует формирование вирус-специфического Т-клеточного ответа и полную защиту (100% выживаемость) мышей от летального заражения вирусами гриппа А человека и птиц (A/H3N2, A/H2N2, A/H5N1). У мышей, иммунизированных рекомбинантным белком с одним таргетным антигеном (Flg4M2ehs), выживаемость после летального заражения составила 60–75%. Заключение: полученные результаты демонстрируют существенную роль консервативного фрагмента второй субъединицы гемагглютинина в формировании протективного Т-клеточного иммунитета и защите мышей от летального заражения вирусами гриппа А различных субтипов. Показана перспективность разработки вакцинного препарата на основе двух консервативных антигенных детерминант вирусов гриппа А.

EFFECTIVENESS OF CROSS-PROTECTIVE RECOMBINANT INFLUENZA VACCINE BASED ON CONSERVED EPITOPES OF VIRAL PROTEINS М2 AND HEMAGGLUTININ

The influenza virus is the most unique in the level of variability of antigenic and biological properties. Because of constant mutations into genes coding surface viral proteins, in modern vaccines it is necessary to replace 1–2 virus components annually. Traditional influenza vaccines are the strain – specific and have limited efficiency in prevention of new strains of influenza viruses. In this regard, creation of influenza vaccines based on conserved determinants of viral proteins with broad spectrum protection and the short period of production is one of priority tasks which decision will lead to real control of an influenza infection. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based the combination of conserved viral proteins or peptides. The goals of this study: to develop the candidate recombinant flu vaccine based on the two conserved influenza proteins (М2 and НА); to investigate immune response; and to measure the protection activity in an animal model. Results: In this study we investigated the humoral and T-cell response in mice after intranasal immunization with recombinant proteins (Flg-4M2ehs and Flg-HA2-2-4M2ehs). Both proteins induce a robust M2e-specific humoral and CD4+ T-cell response in mice lung. The recombinant protein with two target antigens (M2e and HA2) induces virusspecific CD4+ and CD8+ T-cell response and full protection (100% survival) of mice from lethal challenge human and avian influenza viruses A (A/H3N2, A/H2N2, A/H5N1). In mice immunized with Flg-4M2ehs, the survival after lethal challenge was 60–75%. Conclusion: Our results show an essential role of a conserved fragment of the HA2 in the formation of protective T-cell response and protection of mice from lethal challenge with influenza viruses A of various subtypes. The prospects of the development of vaccine formulation based on two conserved antigenic determinants of influenza virus A are shown

Strengthening the Effectiveness of the Candidate Influenza Vaccine by Combining Conserved Sequences of Hemagglutinin and M2 protein

The development of universal influenza vaccine - a vaccine directed to all subtypes of human influenza A viruses - is the really actual problem task. This paper presents the comparative characteristic of the specific activity of various recombinant proteins consisting of antigenic determinants of influenza A virus - the ectodomain of the M2 protein (M2e) and a fragment of the second subunit of the hemagglutinin (the amino acid sequence 76 - 130). Flagellin - Salmonella typhimurium protein was used as carrier protein and as adjuvant. We use two forms of flagellin: full size and with deleted hypervariable region. The proteins showed high immunogenicity, and the ability to prevent lethal infection of influenza virus in mice. Full-length flagellin with HA2 (76 - 130) and M2e on the C-terminus (protein Flg-HA2-4M2e) demonstrated the most protective properties. It provides 100% survival immunized mice that were challenge with a high dose of influenza A (H3N2) - 10 LD50. Proteins containing only full sized flagellin with M2e or flagellin truncated form with M2e at the C-terminus and HA2 within the hypervariable region, protected 75% of animals from lethal infection. Protein Flg-HA2-4M2e is promising for further study as a vaccine