基于深圳男男同性性行为人群的HIV-1广谱中和抗体筛选分析

发布时间：2018-08-03 13:03

【摘要】：背景:人免疫缺陷病毒(Human Immunodeficiency Virus,HIV)引起的获得性免疫缺陷综合征(Acquired immune deficiency syndrome,AIDS,又称艾滋病)自20世纪80年代被发现以来迅速蔓延全球。近年来我国艾滋病新发病例上升的势头依然未能得到扭转,其中男男同性性行为人群(Men who have sex with men,MSM)疫情增长异常迅速,引起国内外广泛关注。中和抗体是机体针对HIV感染产生的主要免疫应答,如何诱导正常人体产生可中和不同毒株的广谱中和抗体(Broadly neutralizing antibodies,bNAbs)是设计、研发有效疫苗的基石和关键环节。新近研究表明,我国MSM人群中HIV-1流行毒株不断重组进化和扩散,正在给相关抗体、药物研究和应用带来重大挑战。目的:深圳作为国内MSM主要集散地和我国最大的口岸城市之一,建立bNAbs筛选分析平台并应用于本地MSM人群样本,为下一步开展基于本地流行毒株的bNAbs机制和应用研究奠定基础。方法:第一部分为搭建HIV-1广谱中和抗体筛查技术平台。培养扩增含标准HIV-env基因的系列质粒和骨架质粒(含env以外的HIV基因),测序、酶切鉴定,然后分别将两种质粒共转染293T细胞,制备具有单次感染能力的假病毒;通过TZM-bl细胞实验检测假病毒滴定度,计算半数细胞感染量(TCID50)。第二部分为所建平台在本地MSM人群筛查中的初步应用和验证。包括建立小型MSM队列,按计划分别定期随访、留样,测序分析人群中HIV-1病毒流行亚型;利用平台筛选队列人群样本,检测平台的可行性;并对具有一定广谱中和能力的代表性血样进行抗体竞争实验初步分析其结合位点。结果:近年来,在深圳MSM人群中流行的HIV-1亚型CRF07_BC(43.4%)和CRF55_01B(15.4%)占比增长迅速。将来自该人群队列的CRF07_BC感染者34人88份血样进行广谱中和抗体检测,筛选出具有一定广谱中和抗性的血样10份(ID50 Titer"g25),其中2例显示了较佳中和宽度,可作用于全部12种假病毒中的7种(58.3%)。初步分析其结合机制均非靶向gp120。结论:随着深圳地区MSM人群中的HIV-1毒株不断进化,其流行特性日益复杂,对其中增长迅速重组亚型的bNAbs相关研究亟待加强。通过本研究已成功建立小型MSM队列和TZM-bl检测分析技术平台,并应用于实践,初步筛选结果提示部分具有中和活性的患者血清内存在bNAbs。
[Abstract]:Background: acquired immunodeficiency syndrome (Acquired immune deficiency syndrome) caused by human immunodeficiency virus (Human Immunodeficiency) has spread rapidly all over the world since its discovery in 1980s. In recent years, the rising trend of new AIDS cases in China has not been reversed. The epidemic situation of men who have sex with men (Men who have sex with) has been increasing rapidly, which has attracted wide attention both at home and abroad. Neutralizing antibody is the main immune response to HIV infection. How to induce (Broadly neutralizing antibodiesbNAbs, which can neutralize different strains of virus, is the cornerstone and key link in the design and development of effective vaccine. Recent studies show that the continuous recombination, evolution and diffusion of HIV-1 epidemic strains in MSM populations in China are bringing great challenges to the research and application of related antibodies, drugs and drugs. Objective: as one of the main distribution centers of MSM in China and one of the largest port cities in China, Shenzhen established a bNAbs screening and analysis platform and applied it to local MSM samples, which laid a foundation for further research on the mechanism and application of bNAbs based on local epidemic strains. Methods: the first part is to set up HIV-1 wide-spectrum neutralizing antibody screening platform. A series of plasmids containing standard HIV-env gene and skeleton plasmids (including HIV gene other than env) were amplified, sequenced and identified by enzyme digestion. Then the two plasmids were co-transfected into 293T cells to prepare pseudovirus with single infection ability. The pseudoviral titer was detected by TZM-bl cell experiment, and the half cell infection dose (TCID50) was calculated. The second part is the preliminary application and validation of the platform in local MSM population screening. Small MSM cohort was set up, followed up regularly according to plan, samples were kept, HIV-1 virus epidemic subtypes were sequenced, and the platform was used to screen the samples of the cohort population to test the feasibility of the platform. The binding sites of representative blood samples with certain broad-spectrum neutralization ability were preliminarily analyzed by antibody competition test. Results: in recent years, the proportion of HIV-1 subtypes CRF07_BC (43.4%) and CRF55_01B (15.4%) in Shenzhen MSM population increased rapidly. Ten blood samples (ID50 Titer "g25) with certain broad-spectrum neutralization resistance were screened from 88 blood samples of 34 patients with CRF07_BC infection from this cohort. Two of them showed better neutralization width. It can act on 7 out of 12 pseudoviruses (58.3%). The binding mechanism of GP120 was preliminarily analyzed. Conclusion: with the continuous evolution of HIV-1 strains in MSM population in Shenzhen area, its epidemic characteristics are becoming more and more complicated, and the related studies on bNAbs subtypes, which are increasing rapidly, need to be strengthened. The small MSM cohort and TZM-bl detection and analysis platform have been successfully established and applied to practice. The preliminary screening results suggest that there is bNAbs in some patients with neutralizing activity.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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