mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-IIW-1584mimmun-1584Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESИнтраназальная иммунизация рекомбинантным белком на основе М2е-пептида и фрагмента второй субъединицы гемагглютинина вирусов гриппа А индуцирует кросс-протективный гуморальный и Т-клеточный ответ у мышейIntranasal 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 miceШуклинаМ. А.ShuklinaM. A

Шуклина Марина Александровна — младший научный сотрудник.

195030, Санкт-Петербург, пр. Энтузиастов, 46, корп. 1, кв. 313, Тел.: 8(812) 526-72-25

Shuklina Marina A. - Junior Research Associate.

195030, St.Petersburg, Enthusiasts ave., 46, bldg 1, apt 313, Phone: 7(812) 526-72-25

ma.shuklina@yandex.ruСтепановаЛ. А.StepanovaL. A

Степанова Людмила Алексеевна — кандидат биологических наук, ведущий научный сотрудник.

Санкт-Петербург

Stepanova Liudmila A., PhD (Biology), Leading Research Associate.

St. Petersburg

stepanoval60@mail.ruКовалеваА. А.KovalevaA. A.

Ковалева Анна Александровна — научный сотрудник.

Korotkov Anna V. - Research Associate.

anna.kovaleva@influenza.spb.ruКоротковА. В.KorotkovA. V.

Коротков Александр Викторович — научный сотрудник.

Korotkov Anna V. - Research Associate.

St. Petersburg

lex9268@mail.ruШалджянА. А.ShaldzhyanA. A.

Шалджян Арам Арутюнович — лаборант-исследователь.

Shaldzhyan Aram A. - Research Assistant.

St. Petersburg

aram.shaldzhyan@influenza.spb.ruЗайцеваМ. В.ZaitcevaM. V.

Зайцева Марина Вячеславовна — младший научный сотрудник.

Санкт-Петербург

Zaitseva Marina V. - Junior Research Associate.

St. Petersburg

marina.zaitceva@influenza.spb.ruЕлецкаяЕ. И.EletskayaE. I.

Елецкая Елизавета Игоревна — лаборант-исследователь.

Eletskaya Elizaveta I. - Research Technical Assistant.

St. Petersburg

eletckaya_l95@mail.ruЦыбаловаЛ. М.TsybalovaL. M.

Цыбалова Людмила Марковна — докторр медицинских наук, профессор, заместитель директора по научной работе, заведующий отделом вакцинологии, заведующий лабораторией гриппозных вакцин.

Tsybalova Liudmila M. - PhD, MD (Medicine), Professor, Deputy Director for Research, Head, Vaccinology Department, Chief, Influenza Vaccinal Laboratory.

sovet@influenza.spb.ruФедеральное государственное бюджетное учреждение «Научно-исследовательский институт гриппа имени А.А. Смородинцева» Министерства здравоохранения Российской ФедерацииРоссияSmorodintsev Research Institute of Influenza, Ministry of Health of the Russian FederationRussian Federation202014042020222357370Copyright © Шуклина М.А., Степанова Л.А., Ковалева А.А., Коротков А.В., Шалджян А.А., Зайцева М.В., Елецкая Е.И., Цыбалова Л.М., 20202020Шуклина М.А., Степанова Л.А., Ковалева А.А., Коротков А.В., Шалджян А.А., Зайцева М.В., Елецкая Е.И., Цыбалова Л.М.Shuklina M.A., Stepanova L.A., Kovaleva A.A., Korotkov A.V., Shaldzhyan A.A., Zaitceva M.V., Eletskaya E.I., Tsybalova L.M.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/1584

Создание вакцин с широким спектром защиты является одной из приоритетных задач в области профилактики гриппа. В последнее время интерес исследователей вызывают консервативные фрагменты белков вируса гриппа (M1, M2, NP, вторая субъединица гемагглютинина HA2) как объект разработки рекомбинантных вакцин широкого спектра действия. Сложность в разработке вакцин на основе таких фрагментов заключается в их низкой иммуногенности. Однако присоединение слабоиммуногенных антигенов к белку-носителю позволяет значительно усилить их иммуноген-ность. Был разработан кандидатный вакцинный белок Flg-HA2-2-4M2e, содержащий два высококонсервативных вирусных антигена (эктодомен белка M2 — М2е и участок 76-130 второй субъединицы НА2), слитых с белком-носителем флагеллином. Флагеллин — белок жгутиков бактерий, является естественным лигандом Toll-подобного рецептора 5 (TLR-5) и обладает сильной адъювантной активностью при различных способах введения. Цель настоящего исследования состояла в оценке формирования гуморального и Т-клеточного иммунного ответа и широты протективного действия кандидат-ного вакцинного белка Flg-HA2-2-4M2e при введении мышам. Мышей иммунизировали трехкратно интраназально с двухнедельными интервалами. Через две недели после последней иммунизации животных заражали летальной дозой (5 LD50) вирусов, относящихся к обеим филогенетическим группам (A/California/07/09 (H1N1)pdm09 или A/Shanghai/2/2013 (H7N9)). Полученные результаты показали формирование выраженного М2е-специфического гуморального ответа (сывороточных IgG и IgA) у иммунизированных мышей. Иммунизация приводила также к формированию M2e- и вирус-специ-фических CD4+ и CDS+T-клеток в легких, которые продуцировали TNFα и IFNγ. Выявлено формирование антиген-специфических эффекторных и центральных Т-клеток памяти в легких мышей опытной группы. Формирование кросс-протективного защитного иммунитета у иммунизированных мышей было продемонстрировано на модели летальной гриппозной инфекции. Опытные животные были практически полностью защищены (90-100% выживаемость) от высокой дозы пандемического вируса A/H1N1pdm09 и высокопатогенного вируса гриппа птиц A/H7N9. Второй задачей была оценка иммунного ответа на сублетальную инфекцию вирусом гриппа у иммунизированных животных. Мыши контрольной и опытной групп были инфицированы дозой MID100 вируса гриппа А/Aichi/2/68 (H3N2). Было показано, что у иммунизированных мышей, по сравнению с контрольными, в ответ на сублетальное инфицирование изменился профиль иммунного ответа иммуноглобулинами — в 10 раз увеличилась доля IgG2a, более оптимальных в защите от инфекции. Полученные результаты показали, что рекомбинантный белок Flg-HA2-2-4M2e является перспективным кандидатом для разработки универсальной вакцины нового поколения, формирующий протективный гуморальный и Т-клеточный ответ на консервативные вирусные эпитопы и защищающий от вирусов гриппа А обеих филогенетических групп.

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.

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The authors declare that there are no conflicts of interest present.