Вопросы вирусологии. 2021; 66: 383-389
Получение вирусоподобных частиц норовируса (Caliciviridae: Norovirus), содержащих белок VP1 энтеровируса Echovirus 30 (Picornaviridae: Enterovirus: Enterovirus B)
https://doi.org/10.36233/0507-4088-79Аннотация
Введение. Энтеровирусная (неполио) инфекция имеет широкое распространение во всём мире, регистрируется в форме спорадической заболеваемости и масштабных вспышек и может быть причиной такого тяжёлого поражения, как серозный менингит. Эпидемиологические исследования показали, что на территории Российской Федерации у больных энтеровирусным менингитом наиболее часто выявляется вариант энтеровируса (ЭВ) (Picornaviridae; Enterovirus) Echovirus 30 (Е30). Однако вакцины для профилактики заболевания, вызванного этим возбудителем, до настоящего времени не разработаны. Одним из перспективных современных направлений в плане создания вакцинных препаратов является использование вирусоподобных частиц (ВпЧ), в т.ч. химерных – содержащих биологические структуры вирусов, принадлежащих к различным видам.
Цель настоящей работы – получение ВпЧ норовируса (Caliciviridae; Norovirus), содержащих на своей поверхности белок VP1 Е30.
Материал и методы. В работе использовали нуклеотидные последовательности генов белков VP1 норовируса генотипа GII.4 и VP1 вируса E30 генотипа h, циркулирующих на территории России. На их основе сконструирован белок SN-VP1E30, в котором оболочечный (S) и шарнирный (hinge) регионы VP1 норовируса слиты в одну молекулу с полноразмерным VP1 E30. Данный белок экспрессировали в E. coli, очищали методом аффинной хроматографии, после чего характеризовали с использованием электрофореза в полиакриламидном геле (ПААГ) и иммуноблоттинга. ВпЧ визуализировали методом электронной микроскопии.
Результаты. Белок SN-VP1E30 экспрессировался в E. coli в нерастворимой форме. Подбор условий для получения его растворимой формы показал, что использование высоких концентраций сахарозы существенно повышает эффективность ренатурации. При сравнении электрофоретической подвижности денатурированного и неденатурированного препаратов SN-VP1E30 установлено, что большинство мономеров образуют соединения со значительной молекулярной массой. С помощью электронной микроскопии показано, что ренатурированный белок SN-VP1E30 самопроизвольно формирует in vitro полые ВпЧ диаметром ~50 нм.
Заключение. Показана возможность получения in vitro химерных ВпЧ норовируса, содержащих на своей поверхности белок VP1 циркулирующего на территории РФ варианта E30. Полученные частицы в дальнейшем могут быть использованы в разработке вакцинных препаратов для профилактики серозного менингита и других заболеваний, вызванных данным ЭВ.
Список литературы
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Problems of Virology. 2021; 66: 383-389
Construction of norovirus (Caliciviridae: Norovirus) virus-like particles containing VP1 of the Echovirus 30 (Picornaviridae: Enterovirus: Enterovirus B)
https://doi.org/10.36233/0507-4088-79Abstract
Introduction. Enterovirus (nonpolio) infection is widespread all over the world, registered as sporadic cases and large-scale outbreaks and can cause severe lesions such as serous meningitis. Epidemiological studies have shown that enterovirus (Picornaviridae; Enterovirus) variant Echovirus 30 (E30) is the most frequently detected variant in patients with enterovirus meningitis in the Russian Federation. However, no vaccines to prevent the disease caused by this pathogen have been developed so far. One of the promising modern trends in terms of creating vaccine preparations is the use of virus-like particles (VLPs), including chimeric ones containing the biological structures of viruses belonging to different species.
The aim of this work was to obtain norovirus (Caliciviridae; Norovirus) VLPs displaying enterovirus Echovirus E30 full-length VP1 on the surface.
Material and methods. The nucleotide sequences of VP1 protein of norovirus genotype GII.4 and VP1 E30 of genotype h circulating in Russia were used. The SN-VP1E30 protein was constructed, in which the shell (S) and the hinge regions of the norovirus VP1 are fused into one molecule with the full-length VP1 of the E30 virus. The protein was expressed in E. coli, purified using affinity chromatography, and characterized by polyacrylamide gel electrophoresis (PAGE) and immunoblotting. VLPs were visualized by electron microscopy.
Results. The S N-VP1E30 protein expressed in E. coli as insoluble form, so the conditions for SN-VP1E30 solublisation were defined. Sucrose has been shown to significantly increase the efficiency of renaturation. Electrophoretic mobility comparison of denatured and non-denatured SN-VP1E30 demonstrated that most monomers form high molecular weight compounds. Electron microscopy showed that renatured SN-VP1E30 spontaneously forms empty virus-like particles about 50 nm in diameter.
Conclusion. Chimeric protein SN-VP1E30 self-assemble into VLPs displaying the VP1 protein of E30 variant that is highly prevalent in Russia. Further immunological research is necessary to characterize VLPs potential for development of the vaccine for enteroviral meningitis prevention.
References
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2. Mao Q., Wang Y., Bian L., Xu M., Liang Z. EV-A71 vaccine licensure: a first step for multivalent enterovirus vaccine to control HFMD and other severe diseases. Emerg. Microbes Infect. 2016; 5(7): e75. https://doi.org/10.1038/emi.2016.73
3. Zell R., Delwart E., Gorbalenya A.E., Hovi T., King A.M.Q., Knowles N.J., et al. ICTV virus taxonomy profile: Picornaviridae. J. Gen. Virol. 2017; 98(10): 2421–2. https://doi.org/10.1099/jgv.0.000911
4. Xu W., Liu C., Yan L., Li J., Wang L., Qi Y., et al. Distribution of enteroviruses in hospitalized children with hand, foot and mouth disease and relationship between pathogens and nervous system complications. Virol. J. 2012; 9: 8. https://doi.org/10.1186/1743-422X-9-8
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10. Lema C., Torres C., Van der Sanden S., Cisterna D., Freire M.C., Gomez R.M., et al. Global phylodynamics of Echovirus 30 revealed differential behavior among viral lineages. Virology. 2019; 531: 79–92. https://doi.org/10.1016/j.virol.2019.02.012
11. Xia M., Huang P., Sun C., Han L., Vago F.S., Li K., et al. Bioengineered norovirus S60 nanoparticles as a multifunctional vaccine platform. ACS Nano. 2018; 12(11): 10665–82. https://doi.org/10.1021/acsnano.8b02776
12. Tan M., Huang P., Xia M., Fang P.A., Zhong W., McNeal M., et al. Norovirus P particle, a novel platform for vaccine development and antibody production. J. Virol. 2011; 85(2): 753–64. https://doi.org/10.1128/JVI.01835-10
13. Kissmann J., Ausar S.F., Foubert T.R., Brock J., Switzer M.H., Detzi E.J., et al. Physical stabilization of Norwalk virus-like particles. J. Pharm. Sci. 2008; 97(10): 4208–18. https://doi.org/10.1002/jps.21315
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16. Mohsen M.O., Gomes A.C., Vogel M., Bachmann M.F. Interaction of viral capsid-derived virus-like particles (VLPs) with the innate immune system. Vaccines. 2018; 6(3): 37. https://doi.org/10.3390/vaccines6030037
17. Anggraeni M.R., Connors N.K., Wu Y., Chuan Y.P., Lua L.H., Middelberg A.P. Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle. Vaccine. 2013; 31(40): 4428–35. https://doi.org/10.1016/j.vaccine.2013.06.087
18. Rivera-Hernandez T., Hartas J., Wu Y., Chuan Y.P., Lua L.H., Good M., et al. Self-adjuvanting modular virus-like particles for mucosal vaccination against group A streptococcus (GAS). Vaccine. 2013; 31(15): 1950–5. https://doi.org/10.1016/j.vaccine.2013.02.013
19. Tacket C.O., Sztein M.B., Losonsky G.A., Wasserman S.S., Estes M.K. Humoral, mucosal, and cellular immune responses to oral Norwalk virus-like particles in volunteers. Clin. Immunol. 2003; 108(3): 241–7. https://doi.org/10.1016/s1521-6616(03)00120-7
20. Zhao H., Li H.Y., Han J.F., Deng Y.Q., Zhu S.Y., Li X.F., et al. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice. Sci. Rep. 2015; 5: 7878. https://doi.org/10.1038/srep07878
21. Zhang C., Zhang X., Zhang W., Dai W., Xie J., Ye L., et al. Enterovirus D68 virus-like particles expressed in Pichia pastoris potently induce neutralizing antibody responses and confer protection against lethal viral infection in mice. Emerg. Microbes Infect. 2018; 7(1): 3. https://doi.org/10.1038/s41426-017-0005-x
22. Le D.T., Müller K.M. In vitro assembly of virus-like particles and their applications. Life (Basel). 2021; 11(4): 334. https://doi.org/10.3390/life11040334
