vetpressАграрная наукаAgrarian science0869-81552686-701XРедакция журнала "Аграрная наука"10.32634/0869-8155-2024-383-6-39-43vetpress-3105Research ArticleВЕТЕРИНАРИЯVETERINARY MEDICINEФункциональная оценка in vivo рекомбинантных аденоассоциированных вирусов, несущих гены протективно значимых антигенов вируса африканской чумы свинейIn vivo functional assessment of recombinant adeno-associated viruses carrying genes of protectively significant antigens of the African swine fever virushttps://orcid.org/0000-0003-2650-6459ГалееваА. Г.GaleevaA. G.

Антонина Глебовна Галеева, кандидат ветеринарных наук, старший научный сотрудник; кандидат ветеринарных наук, старший научный сотрудник

Сибирский тракт, 35, Казань, 420029;

Научный городок, 2, Казань, 420075

 

Antonina Glebovna Galeeva, Candidate of Veterinary Sciences, Senior Researcher; Candidate of Veterinary Sciences, Senior Researcher

35 Sibirsky trakt, Kazan, 420029;

2 Nauchny gorodok, Kazan, 420075

antonina-95@yandex.ruhttps://orcid.org/0000-0001-8786-1310ЕфимоваМ. А.EfimovaM. A.

Марина Анатольевна Ефимова, доктор биологических наук, профессор; доктор биологических наук, ведущий научный сотрудник

Сибирский тракт, 35, Казань, 420029;

Научный городок, 2, Казань, 420075

Marina Anatolyevna Efimova, Doctor of Biological Sciences, Professor; Doctor of Biological Sciences, Leading Researcher

35 Sibirsky trakt, Kazan, 420029;

2 Nauchny gorodok, Kazan, 420075

marina-2004r@mail.ruФроловГ. С.FrolovG. S.

Сибирский тракт, 35, Казань, 420029

35 Sibirsky trakt, Kazan, 420029

https://orcid.org/0009-0007-2186-1300ЗубринкинД. А.ZubrinkinD. A.

Данил Александрович Зубринкин, аспирант

Сибирский тракт, 35, Казань, 420029

Danil Aleksandrovich Zubrinkin, Graduate Student

35 Sibirsky trakt, Kazan, 420029

zubrinkin-yande2013@yandex.ruХисамутдиновА. Г.HisamutdinovA. G.

Алмаз Габтраупович Хисамутдинов, кандидат ветеринарных наук

ул. Федосеевская, 36, Казань, 420111

Almaz Gabtraupovich Hisamutdinov, Candidate of Veterinary Sciences

36 Fedoseevskaya Str., Kazan, 420111

guv@tatar.ruГариповЛ. Н.GaripovL. N.

Ленар Наилевич Гарипов, заместитель министра

ул. Федосеевская, 36, Казань, 420111

Lenar Nailevich Garipov, Deputy Minister

36 Fedoseevskaya Str., Kazan, 420111

Garipov.Lenar@tatar.ruhttps://orcid.org/0000-0001-7217-4083МингалеевД. Н.MingaleevD. N.

Данил Наильевич Мингалеев, доктор ветеринарных наук, доцент; доктор ветеринарных наук, врио директора

Сибирский тракт, 35, Казань, 420029;

Научный городок, 2, Казань, 420075

Danil Nailevich Mingaleev, Doctor of Veterinary Sciences, Associate Professor, Doctor of Veterinary Sciences, Acting Director 

35 Sibirsky trakt, Kazan, 420029;

2 Nauchny gorodok, Kazan, 420075

damin80@mail.ruhttps://orcid.org/0000-0001-7210-7470РавиловР. Х.RaviloR. Kh.

Рустам Хаметович Равилов, доктор ветеринарных наук, профессор, член-корреспондент Aкадемии наук Республики Татарстан

Сибирский тракт, 35, Казань, 420029

Rustam Khametovich Ravilov, Doctor of Veterinary Sciences; Professor, Corresponding Member of the Academy of Sciences of the Republic of Tatarstan

35 Sibirsky trakt, Kazan, 420029

rustam.ravilov@mail.ruКазанская государственная академия ветеринарной медицины им. Н.Э. Баумана; Федеральный центр токсикологической, радиационной и биологической безопасностиРоссияKazan state academy of veterinary medicine named after N.E. Bauman; Federal Center for Toxicological, Radiation and Biological SafetyRussian FederationКазанская государственная академия ветеринарной медицины им. Н.Э. БауманаРоссияKazan state academy of veterinary medicine named after N.E. BaumanRussian FederationГлавное управление ветеринарии Кабинета министров Республики ТатарстанРоссияMain Directorate of Veterinary, Cabinet of Ministers of Republic of TatarstanRussian FederationМинистерство сельского хозяйства и продовольствия Республики ТатарстанРоссияMinistry of Agriculture and Food of Republic of TatarstanRussian Federation202413062024063943Copyright © Галеева А.Г., Ефимова М.А., Фролов Г.С., Зубринкин Д.А., Хисамутдинов А.Г., Гарипов Л.Н., Мингалеев Д.Н., Равилов Р.Х., 20242024Галеева А.Г., Ефимова М.А., Фролов Г.С., Зубринкин Д.А., Хисамутдинов А.Г., Гарипов Л.Н., Мингалеев Д.Н., Равилов Р.Х.Galeeva A.G., Efimova M.A., Frolov G.S., Zubrinkin D.A., Hisamutdinov A.G., Garipov L.N., Mingaleev D.N., Ravilo R.K.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.vetpress.ru/jour/article/view/3105Актуальность

Актуальность. Африканская чума свиней (АЧС) – вирусное геморрагическое заболевание с исключительно высокой летальностью представителей семейства Suidae, влекущее за собой серьезные экономические последствия, связанные с производственными потерями, торговыми ограничениями и реализацией программ эрадикации. По сей день эффективная коммерческая вакцина против АЧС не разработана. Особый интерес в конструировании кандидатных вакцин представляют вирусные векторы, в частности аденоассоциированный вирус 2-го серотипа (AAV2), успешно зарекомендовавший себя в качестве генотерапевтического средства. Ранее авторами сообщалось о способности rAAV2 эффективно доставлять гены вируса АЧС B646L, E183L, CP530R, CP204L в клетки свиней in vitro.

Цель исследования – оценка функциональности in vivo аденоассоциированных вирусов 2-го серотипа, несущих гены протективно значимых антигенов вируса африканской чумы свиней.

Методы

Методы. Путем клонирования попарно объединенных генов B646L-CP530R, E183L-CP204L в вектор pAAV-MCS были созданы бицистронные конструкции с самощепящимся пептидом Р2А. Сборка rAAV2 осуществлялась путем кальций-фосфатной трансфекции клеток AAV293. После очистки в градиенте плотности йодиксанола rAAV2 вводили свиньям в дозе 3 × 1011 вирусных частиц и оценивали показатели гуморального и клеточного иммунитета в течение 180 дней. Динамику антителогенеза оценивали в непрямом ИФА, иммунофенотипирование Т-лимфоцитов периферической крови – методом проточной цитометрии.

Результаты

Результаты. Установлено, что разработанные бицистронные конструкции на основе rAAV2 безопасны и легко переносимы животными и вызывают индукцию как гуморального, так и клеточного иммунного ответа: наблюдалось образование вирусспецифических антител, сохранявшихся до конца эксперимента, а также повышенная экспрессия CD8+ и СD4+ лимфоцитов. Предлагаемая AAV-платформа является перспективным инструментом для создания вакцины, однако комплексная характеристика rAAV2 может быть составлена только после оценки их протективного эффекта.

Relevance

Relevance. African swine fever (ASF) is a viral hemorrhagic disease with exceptionally high mortality in members of the family Suidae, with serious economic consequences associated with production losses, trade restrictions and eradication programs. To date, no effective commercial vaccine against ASF has been developed. Of particular interest in the design of candidate vaccines are viral vectors, in particular the adenoassociated virus of the 2nd serotype (AAV2), which has successfully proven itself as a gene therapy agent. We previously reported the ability of rAAV2 to effectively deliver ASF virus genes B646L, E183L, CP530R, CP204L into porcine cells in vitro.

The aim of the study was to evaluate the in vivo functionality of adenoassociated viruses of the 2nd serotype carrying genes of protectively significant antigens of the African swine fever virus.

Methods

Methods. By cloning pairwise combined genes B646L-CP530R, E183L-CP204L into the pAAV-MCS vector, bicistronic constructs with the self-cleaving P2A peptide were created. Assembly of rAAV2 was accomplished by calcium phosphate transfection of AAV293 cells. After iodixanol density gradient purification, rAAV2 was administered to pigs at a dose of 3 × 1011 viral particles and humoral and cellular immunity was assessed for 180 days. The dynamics of antibody genesis were assessed by indirect ELISA, and immunophenotyping of peripheral blood T-lymphocytes was assessed by flow cytometry.

Results

Results. It was found that the developed bicistronic constructs based on rAAV2 are safe and easily tolerated by animals and cause the induction of both humoral and cellular immune responses: the formation of virus-specific antibodies was observed, which persisted until the end of the experiment, as well as increased expression of CD8+ and CD4+ lymphocytes. The AAV platform we propose is a promising tool for creating a vaccine, however, a comprehensive characterization of rAAV2 can only be compiled after assessing its protective effect.

африканская чума свинейаденоассоциированные вирусывирусный векторгуморальный иммунитетклеточный иммунитетafrican swine feveradeno-associated virusviral vectorhumoral immunitycellular immunityИсследование выполнено за счет гранта Российского научного фонда № 22-76-00013 «Оценка эффективности векторной системы на основе аденоассоциированного вируса для доставки генов, кодирующих иммунодоминантные белки вируса африканской чумы свиней, в клетки млекопитающих».The materials were prepared as part of the grant of Russian Science Foundation № 22-76-00013 «Evaluation of the effectiveness of a vector system based on adeno-associated virus for the delivery of genes encoding immunodominant proteins of the African swine fever virus into mammalian cells».ReferencesМакаров В.В. Африканская чума свиней. Российский ветеринарный журнал. 2018; 6: 15–19. https://doi.org/10.32416/article_5c050abbcf8d70.94861250Makarov V.V. African swine fever. 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The authors declare that there are no conflicts of interest present.