体外研究脊髓灰质炎病毒Ⅰ型疫苗株结构蛋白对人呼吸道上皮细胞内天然免疫反应系统的作用及其免疫学意义
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摘要
尽管针对脊髓灰质炎病毒已经有长达数百年的研究并成功研制出了预防性疫苗,但是对于该病毒如何启动机体天然免疫反应,进而激活获得性免疫反应这一过程的具体分子机理仍有诸多空白。本研究通过体外分析脊髓灰质炎病毒Sabin减毒株Ⅰ型的结构蛋白对人呼吸道上皮细胞内天然免疫反应信号分子的影响,了解脊髓灰质炎病毒在上皮细胞中启动免疫信号的具体分子机制。采用PCR方法扩增脊髓灰质炎病毒四种结构蛋白VP1、VP2、VP3和VP4,经酶切纯化后与pcDNA3.1+真核表达载体连接,分别构建含目的基因的四种重组质粒。将重组质粒分别转染至人呼吸道上皮细胞16HBE中,48h后收集培养上清、细胞。提取细胞的总RNA,利用Q-PCR法分别检测四种结构蛋白表达所诱导的天然免疫信号分子的mRNA表达量。同时,上清、细胞裂解物分别与人T细胞共培养,检测其对T细胞增殖的影响。体外在16HBE细胞中分别表达SabinⅠ病毒VP1～VP4四种结构蛋白均可刺激如LTα3、NIK等NF-κB天然免疫信号通路相关分子的表达,并且表达这四种蛋白的细胞裂解物与T细胞共培养后均表现出刺激T细胞非特异性增殖的效应。但是,仅有VP1、VP2表达所诱导分泌相关因子的培养上清才表现出间接刺激T细胞增殖的能力。SabinⅠ毒株的四种结构蛋白可激活NF-κB信号通路,促进T细胞的非特异性增殖,从而激活适应性免疫应答。
To explore the specific molecular mechanism of the poliovirus that triggers immune signals,we evaluated the effect of structural proteins of the attenuated poliovirus Sabin I strain on signaling molecules in the innate immune response(IIR)in human respiratory epithelial cells. The four fragments of structural proteins from the poliovirus(VP1,VP2,VP3 and VP4)were amplified by polymerase chain reaction(PCR),purified and digested. The recombinant plasmid containing these target genes was constructed with the pcDNA3.1+eukaryotic expression vector. Supernatants and cells were obtained 48 hrs after the plasmids had been transfected into a human respiratory epithelial cell line(16 HBE). Total RNA was extracted from cells and then,using these RNA templates,expression of IIR signaling molecules induced by the four structural proteins were tested by quantitative-PCR. Meanwhile,the supernatants and cell lysates were co-cultured with T cells to measure the effect on T-cell proliferation. Four successfully constructed recombinant plasmids containing VP1～VP4 of the Sabin-I strain could upregulate the expression of nuclear factor-kappa B(NF-κB)signaling pathway-related molecules(e.g. LTα3,NIK)in 16 HBE cells. In addition,the lysates from 16 HBE cells transfected by four recombinant plasmids stimulated T-cell proliferation. However,only the supernatants from cells transfected by VP1 and VP2 recombinant plasmids stimulated T-cell proliferation. The structural proteins of the Sabin-I strain can activate the NF-κB signaling pathway and promote non-specific proliferation of T cells to activate the adaptive immune response.
To explore the specific molecular mechanism of the poliovirus that triggers immune signals,we evaluated the effect of structural proteins of the attenuated poliovirus Sabin I strain on signaling molecules in the innate immune response(IIR)in human respiratory epithelial cells. The four fragments of structural proteins from the poliovirus(VP1,VP2,VP3 and VP4)were amplified by polymerase chain reaction(PCR),purified and digested. The recombinant plasmid containing these target genes was constructed with the pcDNA3.1+eukaryotic expression vector. Supernatants and cells were obtained 48 hrs after the plasmids had been transfected into a human respiratory epithelial cell line(16 HBE). Total RNA was extracted from cells and then,using these RNA templates,expression of IIR signaling molecules induced by the four structural proteins were tested by quantitative-PCR. Meanwhile,the supernatants and cell lysates were co-cultured with T cells to measure the effect on T-cell proliferation. Four successfully constructed recombinant plasmids containing VP1～VP4 of the Sabin-I strain could upregulate the expression of nuclear factor-kappa B(NF-κB)signaling pathway-related molecules(e.g. LTα3,NIK)in 16 HBE cells. In addition,the lysates from 16 HBE cells transfected by four recombinant plasmids stimulated T-cell proliferation. However,only the supernatants from cells transfected by VP1 and VP2 recombinant plasmids stimulated T-cell proliferation. The structural proteins of the Sabin-I strain can activate the NF-κB signaling pathway and promote non-specific proliferation of T cells to activate the adaptive immune response.
引文
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[2] Conrad D A and Jenson H B. New recommendations for poliovirus vaccination. Combination regimen captures best effects of available vaccines[J]. Postgrad Med,1997,102(5):45-48,51-43,59-60 passim.
[3] Arita M. Poliovirus studies during the endgame of the po-lio eradication program[J]. Jpn J Infect Dis,2017,70(1):1-6.
[4] Bandyopadhyay A S,Garon J,Seib K,Orenstein W A. Polio vaccination:past,present and future[J]. Fu-ture Microbiol,2015,10(5):791-808.
[5] Garon J R,Cochi S L and Orenstein W A. The chal-lenge of global poliomyelitis eradication[J]. Infect Dis Clin North Am,2015,29(4):651-665.
[6] Hamidi A and Bakker W A. Innovative IPV from attenu-ated Sabin poliovirus or newly designed alternative seed strains[J]. Pharm Pat Anal,2012,1(5):589-599.
[7] Dotzauer A and Kraemer L. Innate and adaptive immune responses against picornaviruses and their counterac-tions:an overview[J]. World J Virol,20121(3):91-107.
[8] Lei X,Xiao X and Wang J. Innate immunity evasion by enteroviruses:insights into virus-host interaction[J]. Vi-ruses,2016,8(1):22.
[9] Vijay K. Toll-like receptors in immunity and inflammato-ry diseases:past,present,and future[J]. Int Immuno-pharmacol,2018,59:391-412.
[10]Zhao J,He S,Minassian A,Li J,Feng P. Recent ad-vances on viral manipulation of NF-kappaB signaling pathway[J]. Curr Opin Virol,2015,15:103-111.
[11]Couderc T,Barzu T,Horaud F,Crainic R. Poliovirus permissivity and specific receptor expression on human endothelial cells[J]. Virology,1990,174(1):95-102.
[12]Tucker S P,Thornton C L,Wimmer E,Compans R W. Bidirectional entry of poliovirus into polarized epithe-lial cells[J]. J Virol,1993,67(1):29-38.
[13]Racaniello V R. One hundred years of poliovirus patho-genesis[J]. Virology,2006,344(1):9-16.
[14]Ren R and Racaniello V R. Poliovirus spreads from mus-cle to the central nervous system by neural pathways[J]. J Infect Dis,1992,166(4):747-752.
[15]Saito T and Gale M J. Principles of intracellular viral rec-ognition[J]. Curr Opin Immunol,2007,19(1):17-23.
[16]Hayden M S and Ghosh S. NF-kappaB in immunobiolo-gy[J]. Cell Res,2011,21(2):223-244.
[17]Hayden M S,West A P and Ghosh S. NF-kappaB and the immune response[J]. Oncogene,2006,25(51):6758-6780.
[18]Wang X,Li M,Zheng H,Muster T,Palese P,Beg A A,Garcia-Sastre A. Influenza A virus NS1 protein pre-vents activation of NF-kappaB and induction of alpha/be-ta interferon[J]. J Virol,2000,74(24):11566-11573.
[19]Onoguchi K,Yoneyama M,Takemura A,Akira S,Taniguchi T,Namiki H,Fujita T. Viral infections acti-vate types I and III interferon genes through a common mechanism[J]. J Biol Chem,2007,282(10):7576-7581.
[20]Karin M and Greten F R. NF-kappaB:linking inflamma-tion and immunity to cancer development and progres-sion[J]. Nat Rev Immunol,2005,5(10):749-759.
[21]Hayden M S and Ghosh S. Signaling to NF-kappaB[J].Genes Dev,2004,18(18):2195-2224.
[22]Kato A and Schleimer R P. Beyond inflammation:air-way epithelial cells are at the interface of innate and adap-tive immunity[J]. Curr Opin Immunol,2007,19(6):711-720.
[23]Hammad H and Lambrecht B N. Barrier epithelial cells and the control of type 2 immunity[J]. Immunity,2015,43(1):29-40.
[24]Peterson L W and Artis D. Intestinal epithelial cells:reg-ulators of barrier function and immune homeostasis[J].Nat Rev Immunol,2014,14(3):141-153.
[25]Heath W R and Carbone F R. The skin-resident and mi-gratory immune system in steady state and memory:in-nate lymphocytes,dendritic cells and T cells[J]. Nat Immunol,2013,14(10):978-985.
[26]Fearon D T and Locksley R M. The instructive role of innate immunity in the acquired immune response[J].Science,1996,272(5258):50-53.
[27]Kutubuddin M,Simons J and Chow M. Identification of T-helper epitopes in the VP1 capsid protein of poliovirus[J]. J Virol,1992,66(5):3042-3047.
[28]Leclerc C,Deriaud E,Mimic V,van der Werf S. Identi-fication of a T-cell epitope adjacent to neutralization anti-genic site 1 of poliovirus type 1[J]. J Virol,1991,65(2):711-718.
[29]Emini E A,Jameson B A and Wimmer E. Identification of a new neutralization antigenic site on poliovirus coat protein VP2[J]. J Virol,1984,52(2):719-721.
[30]van der Marel P,Hazendonk T G,Henneke M A,van Wezel A L. Induction of neutralizing antibodies by polio-virus capsid polypeptides VP1,VP2 and VP3[J]. Vac-cine,1983,1(1):17-22.