HIV-1广谱中和活性感染者病毒膜蛋白基因与中和表型分析

发布时间：2018-01-25 23:22

本文关键词： 人类免疫缺陷病毒膜蛋白广谱单克隆中和抗体序列分析中和表型　出处：《中国疾病预防控制中心》2017年硕士论文　论文类型：学位论文

【摘要】：背景鉴于中和抗体在预防HIV-1感染中的重要作用,设计能够诱导高效广谱中和抗体应答的免疫原是HIV疫苗研究的重要目标。膜蛋白是HIV-1中和抗体的靶位,因此对具有广谱中和活性感染者病毒膜蛋白基因(env)特征的研究有助于HIV疫苗免疫原的设计。本研究通过探究HIV-1感染者膜蛋白序列的进化特征及假病毒的中和表型,为广谱中和感染者中毒株进化逃逸研究及设计诱导广谱中和抗体的膜蛋白免疫原提供参考信息。目的分析广谱中和活性感染者HIV-1膜蛋白基因的进化特征;分析基于不同时间点膜蛋白基因的假病毒对自体血浆和代表性广谱单克隆中和抗体(bmNAbs)的中和敏感性。方法对 HIV-1 广谱中和活性感染者(CBJC515)20050816、20060418、20070424、20081118和20090519等5个连续时间点的血浆样本进行RNA提取、逆转录、单基因组扩增(SGA)和测序,使用Sequencer、MEGA、BioEdit等软件和Geno2pheno(coreceptor)、Weblogo等在线工具分析env基因的序列特征。从20050816、20060418和20081118等3个时间点序列中挑选代表性env序列,克隆至pcDNATM3.1 Directional TOPO表达载体并筛选鉴定,将env表达质粒与HIV-1骨架质粒(pSG3△env)共转染293T/17细胞制备假病毒,并分别与各时间点自体血浆和代表性bmNAbs进行中和实验,分析假病毒对自体血浆和bmNAbs的中和表型。结果1.从CBJC515的5个连续时间点血浆中共扩增获得env基因SGA序列120条,对20050816、20060418和20081118等3个时间点序列分别构建获得11、4和13个功能性env克隆。所获得的env基因均与中国RL42参考株聚集,并进化为优势组和劣势组两个分支,相同时间点序列多彼此聚集,少数不同时间点序列呈交叉分布。2.不同年份SGA序列的各可变环基因距离持续变化,并随时间具有增长趋势。V3环氨基酸长度和N-糖基化位点数目高度保守,V1V2可变环长度和N-糖基化位点数随时间具有增长趋势。X4型辅助受体病毒占比随病程延长具有增高趋势,优势毒株组X4型辅助受体的占比一般高于劣势组。顶端四肽以GPGR为主且其占比随病程有增高趋势,优势组顶端四肽均为GPGR,劣势组中有GLGR和GQGR两种。3.本研究构建的假病毒对8个连续时间点自体血浆的中和敏感性与使用不同亚型假病毒进行的中和实验结果相似,中和敏感性均呈现先升高(第0-15月),后下降(第15-32月),之后再上升(第32-45月)的波动。假病毒对当前时间点和该时间点之前的血浆中和敏感性低,对之后时间点血浆中和敏感性增强。4.10E8和VRC01能中和该研究样本3个时间点构建的所有假病毒,PGT135对上述病毒无中和活性;12A21、PGT121、2G12可中和大部分假病毒;X4型假病毒对2G12、12A21较R5型假病毒敏感,对VRC01较R5型假病毒抵抗;优势组病毒对VRC01、10E8较劣势组抵抗,对2G12、12A21、PGT121较劣势组敏感。5.假病毒对VRC01的中和敏感性与V1V2长度和gp120上N-糖基化位点数负相关;对2G12和12A21的中和敏感性与V1V2氨基酸数正相关;对2G12与PGT121的中和敏感性与N-糖基化位点数正相关。结论1.该感染者体内包含优势和劣势两组病毒,病毒通过增加V1V2可变环长度和N-糖基化位点数、辅助受体转换等方式逃避中和选择压力,向不同方向进化和变异。2.病毒对连续时间点自体血浆的中和敏感性呈现先升高(第0-15月),后下降(第15-32月),之后再上升(第32-45月)的波动,提示感染者体内病毒与中和抗体的动态进化过程。3.在本研究测试的6个bmNAbs中,假病毒对10E8、PGT121和VRC01的敏感性较高,全部对PGT135抵抗;10E8对假病毒的中和能力强于12A21、2G12和VRC01;PGT121对假病毒的中和能力强于12A21和2G12。4.劣势组毒株对VRC01和10E8的中和敏感性高于优势组病毒;优势组毒株对PGT121、12A21和2G12的敏感性高于劣势组毒株;假病毒乃至同一时间点假病毒对特定bmNAbs的敏感性差异明显,提示感染者准种毒株间中和表型的复杂性。
[Abstract]:In view of the background of neutralizing antibodies in the prevention of an important role in HIV-1 infection, immune design can induce efficient broad-spectrum neutralizing antibody response of the original is an important target of HIV vaccine. The membrane protein is HIV-1 neutralizing antibody targets, so the broad-spectrum neutralizing activity of infected virus membrane protein Bai Jiyin (Env) study on the characteristics of design in HIV vaccine immunogens help. This study through the evolution characteristics of HIV-1 infection of membrane protein sequences and viral neutralization phenotype for broad-spectrum neutralization infected strains escape immune evolutionary membrane protein research and design induce broad-spectrum neutralizing antibodies to provide reference information. Objective to analyze the evolution characteristics of the HIV-1 membrane protein gene of neutralizing activity at different time points of infection; analysis of membrane protein gene pseudotyped virus based on monoclonal autologous plasma and representative broad-spectrum neutralizing antibody (bmNAbs) and the sensitivity method. The infection of HIV-1 broad-spectrum neutralizing activity (CBJC515) and 20050816200604182007042420081118 20090519 plasma samples of 5 consecutive time points for RNA extraction, reverse transcriptase, single genome amplification and sequencing (SGA), Sequencer, MEGA, BioEdit software and Geno2pheno (coreceptor), Weblogo and other online tools to analyze the sequence characteristics of env gene of the selected representative. Env sequence from 2005081620060418 and 20081118 to 3 time points in the sequence, Directional TOPO was cloned into pcDNATM3.1 expression vector and screening and identification, the env expression plasmid and HIV-1 plasmid (pSG3 Env) were transfected into 293T/17 cells for preparing false virus, respectively and each time point of autologous plasma and representative bmNAbs neutralization experiment analysis of autologous plasma and bmNAbs pseudotyped virus neutralization phenotype. The results of 5 consecutive time points of the 1. plasma from CBJC515 env gene was amplified SGA sequence 12 0, 2005081620060418 and 20081118 of the 3 time sequences were constructed to obtain 11,4 and 13 functional env clones. Env gene were obtained by clustering and China RL42 reference strains, and evolutionary advantage and disadvantage group group of two branches, at the same time sequence with each other together, a few different time sequence is the variable cross distribution of.2. in different years SGA loop sequence gene distance continues to change with time, and with the growth trend of.V3 loop amino acid length and N- glycosylation sites are highly conserved and variable length V1V2 ring and N- glycosylation sites over time with the growth trend of.X4 auxiliary receptor virus accounted for with the extension of the course of the increasing tendency, the dominant strains were type X4 coreceptor proportion is generally higher than the inferior group. The top four peptides based on GPGR and the proportion with the course of disease has increased, the dominant group of top four peptides were GPGR, GLGR inferior group Two GQGR and.3. constructed in this study pseudovirion of 8 consecutive time points of autologous plasma neutralization sensitivity with different subtypes of the virus neutralization test false similar results, and sensitivity were increased (0-15 months), then decreased (15-32 months), and then on the rise (32-45 month) fluctuations. Plasma pseudovirus of prior to a current time point and the point of time and the sensitivity is low, for all time points after the virus in plasma and increased sensitivity to.4.10E8 and VRC01 and the study sample of 3 time points of PGT135, no neutralizing activity of the virus; 12A21, PGT121,2G12 and most of the false virus; X4 pseudotyped virus pseudotyped virus with 2G12,12A21 R5 on VRC01 sensitive than R5 pseudotyped virus resistance; virus of VRC01,10E8 was the dominant group of group 2G12,12A21, inferior resistance, PGT121 is sensitive to.5. pseudovirion disadvantage group VRC01 and sensitive And the length of V1V2 and gp120 on N- glycosylation sites are negatively correlated; positive correlation of neutralization sensitivity and V1V2 amino acid number 2G12 and 12A21; on 2G12 and PGT121 and N- sensitivity and glycosylation sites are related to the infection in vivo. Conclusion 1. contains the advantages and disadvantages of the two group of viruses, the virus through the increase of V1V2 variable length and ring N- glycosylation sites, coreceptor conversion of neutralization escape selection pressure to evolve in different directions and the variation of the.2. virus on the continuous time autologous plasma neutralization sensitivity increased (0-15 months), then decreased (15-32 months), then increased (32-45 1) fluctuations, suggesting that virus infection in vivo and the dynamic evolution process of.3. neutralizing antibody in 6 bmNAbs test in this study, the false virus on 10E8, PGT121 had higher sensitivity and VRC01, all of PGT135 resistance; 10E8 on false virus neutralizing ability is stronger than that of 12A2 1,2G12 and VRC01 PGT121; the false virus neutralizing ability is stronger than that of 12A21 and 2G12.4. strains were sensitive to VRC01 and inferior and 10E8 higher than that of the dominant group virus; sensitivity of PGT121,12A21 and 2G12 were the dominant group was higher than that of inferior group strains; sensitivity and false virus at the same time point of pseudotyped virus specific bmNAbs, suggesting that infection those kind of quasi complexity strains and phenotype.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

【参考文献】