Журнал микробиологии, эпидемиологии и иммунобиологии. 2020; 97: 7-14
Получение и характеристика моноклональных антител к G-белку респираторно-синцитиального вируса
https://doi.org/10.36233/0372-9311-2020-97-1-7-14Аннотация
Цель работы состояла в получении гибридом, продуцирующих моноклональные антитела (МКА) к G-белку респираторно-синцитиального вируса (РСВ), и в изучении их иммунологических характеристик и вируснейтрализующей активности.
Материал и методы. Мышиные МКА получали с помощью гибридомной технологии. Свойства МКА изучали методами иммуноферментного анализа (ИФА), иммунофлюоресцентного окрашивания (ИФл) зараженных клеток культуры, а также в реакции биологической нейтрализации in vitro (РБН). Взаимное расположение эпитопов, выявляемых МКА на G-белке, определяли с помощью теста аддитивности в ИФА.
Результаты. Гибридизация спленоцитов с клетками миеломы Sp2/0 и первичный скрининг показали, что 75 гибридом продуцируют МКА, взаимодействующие с очищенным вирусом, 17 из которых реагируют также и с рекомбинантным G-белком в ИФА. В РБН 4 гибридомы подавляли РСВ-инфекцию in vitro более чем на 50%. Клонирование этих гибридом позволило выявить 4 моноклона, продуцирующих наиболее активные МКА. МКА 1С11 принадлежали к IgG2a, 3 других (5D4, 5G11 и 6H4) — к IgM. 3 МКА IgM активно реагировали как с РСВ А2 и Long, так и с G-белком, МКА 1С11 были менее активны со всеми антигенами. Все МКА подавляли РСВ-инфекцию, причем МКА 5D4 — практически полностью (98%). ИФл-анализ показал, что все МКА выявляли G-белок РСВ в цитоплазме клеток культуры, наибольшее количество зараженных клеток детектировали с помощью МКА 5D4 (80%). Полученные МКА направлены к двум неперекрывающимся эпитопам на G-белке РСВ.
Заключение. Полученные МКА могут использоваться для обнаружения РСВ в клиническом материале методами ИФА и ИФл. Активность в РБН создает предпосылки для получения на основе МКА гуманизированных рекомбинантных антител и возможность их использования для терапии РСВ-инфекции.
Список литературы
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8. Boyoglu-Barnum S., Todd S.O., Chirkova T., Barnum T.R., Gaston K.A., Haynes L.M., et al. An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice. Virology. 2015; 483: 117-25. DOI: http://doi.org/10.1016/j.virol.2015.02.035
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11. Power U.F., Nguyen T.N., Rietveld E., de Swart R.L., Groen J., Osterhaus A.D., et al. Safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (BBG2Na) in healthy young adults. J. Infect. Dis. 2001; 184(11): 1456-60. DOI: http://doi.org/10.1086/324426
12. Haynes L.M., Jones L.P., Barskey A., Anderson L.J., Tripp R.A. Enhanced disease and pulmonary eosinophilia associated with formalin-inactivated respiratory syncytial virus vaccination are linked to G glycoprotein CX3C-CX3CR1 interaction and expression of substance P. J. Virol. 2003; 77(18): 9831-44. DOI: http://doi.org/10.1128/JVI.77.18.9831-9844.2003
13. Boyoglu-Barnum S., Todd S.O., Meng J., Barnum T.R., Chirkova T., Haynes L.M., et al. Mutating the CX3C motif in the G protein should make a live respiratory syncytial virus vaccine safer and more effective. J. Virol. 2017; 91(10): e02059-16. DOI: http://doi.org/10.1128/JVI.02059-16
14. Cortjens B., Yasuda E., Yu X., Wagner K., Claassen Y.B., Bakker A.Q., et al. Broadly reactive anti-resp i ra tory syncytial virus G antibodies from exposed individuals effectively inhibit infection of primary airway epithelial cells. J. Virol. 2017; 91(10): e02357-16. DOI: http://doi.org/10.1128/JVI.02357-16
15. Boyoglu-Barnum S., Chirkova T., Anderson L.J. Biology of infection and disease pathogenesis to guide RSV vaccine development. Front. Immunol. 2019; 10: 1675. DOI: http://doi.org/10.3389/fimmu.2019.01675
Journal of microbiology, epidemiology and immunobiology. 2020; 97: 7-14
Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus
https://doi.org/10.36233/0372-9311-2020-97-1-7-14Abstract
The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.
Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.
Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.
Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.
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6. Bates J.T., Keefer C.J., Slaughter J.C., Kulp D.W., Schief W.R., Crowe J.E. Escape from neutral ization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein. Virology. 2014; 454-455: 139-44. DOI: http://doi.org/10.1016/j.virol.2014.02.010
7. Jorquera P.A., Tripp R.A. Respiratory syncytial virus: prospects for new and emerging therapeutics. Expert. Rev. Respir. Med. 2017; 11(8): 609-15. DOI: http://doi.org/10.1080/17476348.2017.1338567
8. Boyoglu-Barnum S., Todd S.O., Chirkova T., Barnum T.R., Gaston K.A., Haynes L.M., et al. An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice. Virology. 2015; 483: 117-25. DOI: http://doi.org/10.1016/j.virol.2015.02.035
9. Ueba O. Respiratory syncytial virus. I. Concentration and purification of the infectious virus. Acta. Med. Okayama. 1978; 32(4): 265-72.
10. Xiang K., Cheng Y., Zhou M., Sun L., Ji Y., Wang Y., et al. Production of monoclonal antibody against EP0 protein of pseudorabies virus and determination of its recognized epitope. Monoclon. Antib. Immunodiagn. Immunother. 2014; 33(6): 409-13. DOI: http://doi.org/10.1089/mab.2014.0046
11. Power U.F., Nguyen T.N., Rietveld E., de Swart R.L., Groen J., Osterhaus A.D., et al. Safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (BBG2Na) in healthy young adults. J. Infect. Dis. 2001; 184(11): 1456-60. DOI: http://doi.org/10.1086/324426
12. Haynes L.M., Jones L.P., Barskey A., Anderson L.J., Tripp R.A. Enhanced disease and pulmonary eosinophilia associated with formalin-inactivated respiratory syncytial virus vaccination are linked to G glycoprotein CX3C-CX3CR1 interaction and expression of substance P. J. Virol. 2003; 77(18): 9831-44. DOI: http://doi.org/10.1128/JVI.77.18.9831-9844.2003
13. Boyoglu-Barnum S., Todd S.O., Meng J., Barnum T.R., Chirkova T., Haynes L.M., et al. Mutating the CX3C motif in the G protein should make a live respiratory syncytial virus vaccine safer and more effective. J. Virol. 2017; 91(10): e02059-16. DOI: http://doi.org/10.1128/JVI.02059-16
14. Cortjens B., Yasuda E., Yu X., Wagner K., Claassen Y.B., Bakker A.Q., et al. Broadly reactive anti-resp i ra tory syncytial virus G antibodies from exposed individuals effectively inhibit infection of primary airway epithelial cells. J. Virol. 2017; 91(10): e02357-16. DOI: http://doi.org/10.1128/JVI.02357-16
15. Boyoglu-Barnum S., Chirkova T., Anderson L.J. Biology of infection and disease pathogenesis to guide RSV vaccine development. Front. Immunol. 2019; 10: 1675. DOI: http://doi.org/10.3389/fimmu.2019.01675
