sibmedСибирский научный медицинский журналСибирский научный медицинский журнал2410-25122410-2520ИЦиГ СО РАН10.15372/SSMJ20190407sibmed-211Research ArticleМЕДИКО-БИОЛОГИЧЕСКИЕ НАУКИBIOMEDICINEПОЛУЧЕНИЕ ХИМЕРНЫХ ВАРИАНТОВ HBсAg, ЭКСПОНИРУЮЩИХ ФРАГМЕНТЫ MPER ВИЧ-1OBTAINING CHIMERIC VARIANTS HBcAg EXPOSING HIV-1 MPER FRAGMENTSРудометовА. П.RudometovA. P.

630559, р. п. Кольцово Новосибирской области

630559, Koltsovo, Novosibirsk region

rudometov_ap@vector.nsc.ruРудометоваН. Б.RudometovaN. B.

630559, р. п. Кольцово Новосибирской области

630559, Koltsovo, Novosibirsk region

andreeva_nb@vector.nsc.ruЗайцевБ. Н.ZaytsevB. N.

к.ф.-м.н.,

 630559, р. п. Кольцово Новосибирской области

candidate of physical mathematical sciences

630559, Koltsovo, Novosibirsk region

zaitsev@vector.nsc.ruЛебедевЛ. Р.LebedevL. R.

д.м.н., 

630559, р. п. Кольцово Новосибирской области 

doctor of medical sciences

630559, Koltsovo, Novosibirsk region

lebedev_lr@vector.nsc.ruИльичевА. А.IlyichevA. A.

д.б.н., 

 630559, р. п. Кольцово Новосибирской области

doctor of biological sciences

630559, Koltsovo, Novosibirsk region

ilyichev@vector.nsc.ruКарпенкоЛ. И.KarpenkoL. I.

д.б.н., 

630559, р. п. Кольцово Новосибирской области

doctor of biological sciences

630559, Koltsovo, Novosibirsk region

karpenko@vector.nsc.ruГосударственный научный центр вирусологии и биотехнологии «Вектор» РоспотребнадзораРоссияState Research Center of Virology and Biotechnology «Vector» of RospotrebnadzorRussian Federation2019050920193945561Copyright © Рудометов А.П., Рудометова Н.Б., Зайцев Б.Н., Лебедев Л.Р., Ильичев А.А., Карпенко Л.И., 20192019Рудометов А.П., Рудометова Н.Б., Зайцев Б.Н., Лебедев Л.Р., Ильичев А.А., Карпенко Л.И.Rudometov A.P., Rudometova N.B., Zaytsev B.N., Lebedev L.R., Ilyichev A.A., Karpenko L.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://sibmed.elpub.ru/jour/article/view/211

Эпидемия ВИЧ-1 является одной из самых острых проблем мирового здравоохранения. По ряду причин эффек-тивной вакцины против этой инфекции на данный момент не создано. В настоящее время важным направлениемв разработке вакцины против ВИЧ/СПИДа является конструирование иммуногенов, которые были бы способныиндуцировать антитела, нейтрализующие широкий спектр штаммов ВИЧ-1 (bNAbs). Одним из подходов созда-ния таких иммуногенов является конструирование химерных вирусоподобных частиц (VLPs), экспонирующихэпитопы, узнаваемые bNAbs. Целью исследования являлись получение и характеризация химерных VLPs наоснове HBcAg, экспонирующих эпитопы, узнаваемые bNAbs 2F5 и 4E10. Материал и методы. Штаммы-проду-центы химерных вариантов HBcAg получали путем трансформации клеток Escherichia coli BL21 рекомбинант-ными плазмидами, несущими гены HBcAg и содержащими встройки, кодирующие эпитопы bNAbs 2F5 и 4E10.Очистку рекомбинантных белков проводили с помощью гель-фильтрации на колонке с сефарозой CL-6B. Спо-собность рекомбинантного HBcAg образовывать вирусоподобные частицы оценивали с помощью электронноймикроскопии. Антигенные свойства эпитопов в составе химерных вариантов HBcAg анализировали с помощьюиммуноблотинга. Результаты. Получена модифицированная нуклеотидная последовательность гена HBcAg, всостав которой были введены уникальные сайты рестрикции, фланкирующие район главной антигенной детер-минанты кора. На основе данной генетической конструкции получены три рекомбинантные плазмиды, кодиру-ющие химерные варианты НВсAg, включающие эпитопы bNAbs 2F5 и 4E10. С помощью иммуноблотинга уста-новлено, что эпитопы, узнаваемые bNAbs, сохраняют свои антигенные свойства в составе химерных НВсAg.

The HIV-1 epidemic is one of the most acute global health problems. For several reasons, an effective vaccine against this infection has not yet been created. Currently, an important direction in the development of a vaccine against HIV / AID S is the design of immunogens that would be able to induce antibodies that neutralize a high diversity of HIV-1 strains (bNAbs). One approach to creating such immunogens is the construction of chimeric virus-like particles (VLPs) exposing epitopes recognized by bNAbs. The aim of the study was to obtain and characterize chimeric VLPs based on HBcAg, exposing epitopes recognized by bNAbs 2F5 and 4E10. Material and methods. The producing strains of  chimeric HBcAg variants were obtained by transforming E. coli BL21 cells with recombinant plasmids carrying the HBcAg genes and containing insertions encoding bNAbs epitopes 2F5 and 4E10. Purification of recombinant proteins was performed using gel filtration on a sepharose CL-6B column. The ability of recombinant HBcAg to form virus-like particles was assessed using electron microscopy. Analysis of the antigenic properties of epitopes in the composition of chimeric variants of HBcAg was performed using immunoblotting. Results. A modified nucleotide sequence of the HBcAg gene was obtained, which included the introduction of unique restriction sites flanking the region of the main antigenic determinant of the core. Based on this genetic construct, three recombinant plasmids encoding chimeric HBcAg variants, including epitopes of bNAbs 2F5 and 4E10, were obtained. Using immunoblotting, it was found that epitopes recognized by bNAbs retain their antigenic properties after insertion into the HBcAg.

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Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008; 38 (5): 1404–1413.Grgacic E.V.L., Anderson D .A. Virus-like particles: passport to immune recognition. Methods. 2006; 40 (1): 60–65.McCoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017; 275 (1): 11–20.Grgacic E.V.L., Anderson D .A. Virus-like particles: passport to immune recognition. Methods. 2006; 40 (1): 60–65.Medina-Ramírez M., Garces F., Escolano A., Skog P., de Taeye S.W., Del Moral-Sanchez I ., McGuire A.T., Yasmeen A., Behrens A.J., Ozorowski G. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017; 214 (9): 2573–2590.Haynes B.F., Burton D.R. Developing an HIV vaccine. Science. 2017; 355 (6330): 1129–1130.Montero M., van Houten N.E., Wang X., Scott J.K. 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Polyvalent vaccine approaches to combat HIV-1 diversity. Immunol. Rev. 2017; 275 (1): 230–244.Manolova V., Flace A., Bauer M., Schwarz K., Saudan P., Bachmann M.F. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008; 38 (5): 1404–1413.Manolova V., Flace A., Bauer M., Schwarz K., Saudan P., Bachmann M.F. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008; 38 (5): 1404–1413.Manolova V., Flace A., Bauer M., Schwarz K., Saudan P., Bachmann M.F. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008; 38 (5): 1404–1413.Manolova V., Flace A., Bauer M., Schwarz K., Saudan P., Bachmann M.F. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008; 38 (5): 1404–1413.McCoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017; 275 (1): 11–20.McCoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017; 275 (1): 11–20.McCoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017; 275 (1): 11–20.McCoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017; 275 (1): 11–20.Medina-Ramírez M., Garces F., Escolano A., Skog P., de Taeye S.W., Del Moral-Sanchez I ., McGuire A.T., Yasmeen A., Behrens A.J., Ozorowski G. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017; 214 (9): 2573–2590.Medina-Ramírez M., Garces F., Escolano A., Skog P., de Taeye S.W., Del Moral-Sanchez I ., McGuire A.T., Yasmeen A., Behrens A.J., Ozorowski G. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017; 214 (9): 2573–2590.Medina-Ramírez M., Garces F., Escolano A., Skog P., de Taeye S.W., Del Moral-Sanchez I ., McGuire A.T., Yasmeen A., Behrens A.J., Ozorowski G. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017; 214 (9): 2573–2590.Medina-Ramírez M., Garces F., Escolano A., Skog P., de Taeye S.W., Del Moral-Sanchez I ., McGuire A.T., Yasmeen A., Behrens A.J., Ozorowski G. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017; 214 (9): 2573–2590.Montero M., van Houten N.E., Wang X., Scott J.K. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design. Microbiol. Mol. Biol. Rev. 2008; 72: 54–84.Montero M., van Houten N.E., Wang X., Scott J.K. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design. Microbiol. Mol. Biol. Rev. 2008; 72: 54–84.Montero M., van Houten N.E., Wang X., Scott J.K. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design. Microbiol. Mol. Biol. Rev. 2008; 72: 54–84.Montero M., van Houten N.E., Wang X., Scott J.K. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design. Microbiol. Mol. Biol. Rev. 2008; 72: 54–84.Muñoz-Barroso I., Salzwedel K., Hunter E., Blumenthal R. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J. Virol. 1999; 73 (7): 6089–6092.Muñoz-Barroso I., Salzwedel K., Hunter E., Blumenthal R. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J. Virol. 1999; 73 (7): 6089–6092.Muñoz-Barroso I., Salzwedel K., Hunter E., Blumenthal R. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J. Virol. 1999; 73 (7): 6089–6092.Muñoz-Barroso I., Salzwedel K., Hunter E., Blumenthal R. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J. Virol. 1999; 73 (7): 6089–6092.Pumpens P., Grens E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology. 2001; 44. 98–114.Pumpens P., Grens E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology. 2001; 44. 98–114.Pumpens P., Grens E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology. 2001; 44. 98–114.Pumpens P., Grens E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology. 2001; 44. 98–114.Pumpens P., Grens E. The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016. Mol. Biol. 2016; 50: 489–509.Pumpens P., Grens E. The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016. Mol. Biol. 2016; 50: 489–509.Pumpens P., Grens E. The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016. Mol. Biol. 2016; 50: 489–509.Pumpens P., Grens E. The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016. Mol. 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The authors declare that there are no conflicts of interest present.