HIV-1 B’亚型感染者Nef蛋白CTL表位变异与病毒载量的相关性

目的探讨HIV-1 B’亚型毒株感染者Nef蛋白细胞毒性淋巴细胞(cytotoxic T lymphocyte,CTL)表位变异及其与病毒载量的相关性。方法从137例HIV-1 B’亚型毒株感染者的血浆样本中提取病毒RNA,经逆转录及巢式PCR扩增获得Nef基因并测序,将获得的Nef基因序列翻译为蛋白序列,然后与HIV免疫学数据库中经过实验证实的CTL表位进行比对,分析Nef蛋白CTL表位变异与感染者病毒载量的关系。结果与HIV免疫学数据库中CTL表位的比较发现,本研究137例HIV-1 B’亚型毒株感染者病毒Nef蛋白大部分CTL表位相对保守,只有5个表位的变异频率超过10%,CTL表位旁序列的变异程度较锚着位点变异大,表位旁上下游第一位氨基酸的突变频率较高,表位氨基酸总变异频率、锚着位点和表位旁序列的氨基酸变异频率与病毒载量呈正相关,非锚着位点氨基酸的变异与病毒载量无相关性。结论 HIV-1 B’亚型毒株感染者Nef蛋白大部分CTL表位较保守,CTL表位旁序列变异程度较锚着位点大,表位旁上下游第一位氨基酸的突变频率较高,推测表位旁序列氨基酸的变异可能是Nef蛋白CTL逃逸突变的主要方式,Nef蛋白CTL表位变异与病毒适应性损伤的关系还需进一步研究。     

人类免疫缺陷病毒-1B和C亚型Nef蛋白特异性CD8+T细胞交叉应答反应的研究

目的：探讨中国人群人类免疫缺陷病毒-1B（HIV-1B亚型）Nef蛋白特异性CD8^＋T细胞应答在B、C亚型间的交叉反应性。方法：选取51例HIV-1B亚型感染者，采取外周血单个核细胞（PBMC），用合成的54个HIV-1B、C亚型Nef全基因序列肽库作为抗原，酶联免疫斑点吸附试验（ELISpot）方法检测HIV-1B亚型Nef蛋白特异性CD8^＋T细胞对B、C亚型抗原的应答反应。结果：在产生特异性应答效应的个体中，82．9％（29／35）感染个体同时识别B和C亚型肽段。感染个体在对两个亚型肽段的识别数量及应答强度上无显著差异（P=0．529和P=0．754）。HIV-1B亚型特异性CD8^＋T细胞能针对70．4％无论B还是C亚型的Nef肽段产生应答反应。被特异性CD8^＋T细胞同时识别的B、C亚型Nef肽段间氨基酸序列的同源性显著高于仅能被识别的B亚型或C亚型肽段的氨基酸序列（P=0．01）。结论：HIV-1B亚型Nef蛋白特异性CD8^＋T细胞应答在B、C亚型间具有良好的交叉反应性，能产生交叉反应的氨基酸序列具有较高的亚型间同源性。     

HIV-1感染者全基因多肽特异性T细胞免疫应答的初步研究

目的 探讨HIV-1感染者人群分泌γ干扰素(IFN-γ)的HIV-1特异性T细胞反应的特征.方法 对HIV-1感染者进行6个月和12个月随访,采用ELISPOT试验检测HIV-1特异性T细胞反应,采用流式细胞术计数CD4+ T细胞.结果 6个月随访,CD4+T细胞计数与病毒载量呈负相关,具有统计学意义;分泌IFN-γ的HIV-1特异性T细胞反应最强的是Nef肽库;高强度特异性T细胞反应频度最高的是Nef(72%)肽库.12个月随访,CD4T细胞计数与病毒载量呈负相关,具有统计学意义;分泌IFN-γ的HIV-1特异性T细胞反应最强的是Nef肽库;高强度特异性T细胞反应频度最高的是Nef(74.1%)肽库.结论 HIV-1 B亚型的所有蛋白均可以诱导产生分泌IFN-γ的HIV-1特异性T细胞反应.6个月和12个月两次随访的研究结果均表明,针对Nef、Gag表位的HIV-1特异性T细胞反应最强、频度最高.     

中国HIV-1感染者病毒特异性细胞毒性T细胞定量研究

目的对8例HIV长期感染不进展(LTNP)及病程进展缓慢(SP)患者的病毒特异性细胞毒性T细胞(CTL)特征进行研究。方法设立队列研究,从中随机选出8例患者(4例LTNPs,4例SP)。通过采用重叠肽技术组建HIV-1B亚型全序列肽段,组建成三维肽库进行ELISPOT分析8例患者的HIV-1特异性细胞免疫反应。结果在大多数患者中存在较强的T细胞反应,特别对HIV-1病毒的pol、gag、nef蛋白的免疫反应比其他蛋白强。结论病毒特异性CTL免疫反应在HIV-1 LTNPs有较强并且很广泛的反应,这对于有效抑制病毒在人体内的生存有可能起到很大的作用,很可能是这些LTNPs及SP病程进展缓慢的主要原因之一。     

我国 HIV-1 B''''亚型感染长期存活者病毒分离及体外复制的研究

目的从HIV-1B’亚型感染的长期存活者分离HIV-1病毒,观察HIV-1病毒分离与CD4淋巴细胞水平和病毒载量的相关性。方法采用外周血单核细胞共培养法,从感染者外周血分离HIV-1毒株,测定其复制动力学,并比较HIV分离率与CD4细胞计数和病毒载量的关系。结果从190例HIV感染者中分离到101株HIV-1病毒,平均病毒分离率为53%;载量为<103、103～104、104～105和>105拷贝/ml时,病毒分离率分别为3.7%、36%、68%和73%;CD4细胞计数为<200、200～500和≥500个/μl时,病毒分离率分别为66%、59%和28%。结论HIV病毒培养阳性率与CD4细胞计数呈负相关,与病毒载量呈正相关;HIV体外复制力与病毒载量呈正相关关系。     

我国HIV-1 B’亚型感染长期存活者病毒分离及体外复制的研究

目的从HIV-1B’亚型感染的长期存活者分离HIV-1病毒,观察HIV-1病毒分离与CD4淋巴细胞水平和病毒载量的相关性。方法采用外周血单核细胞共培养法,从感染者外周血分离HIV-1毒株,测定其复制动力学,并比较HIV分离率与CD4细胞计数和病毒载量的关系。结果从190例HIV感染者中分离到101株HIV-1病毒,平均病毒分离率为53%;载量为<103、103～104、104～105和>105拷贝/ml时,病毒分离率分别为3.7%、36%、68%和73%;CD4细胞计数为<200、200～500和≥500个/μl时,病毒分离率分别为66%、59%和28%。结论HIV病毒培养阳性率与CD4细胞计数呈负相关,与病毒载量呈正相关;HIV体外复制力与病毒载量呈正相关关系。     

人类免疫缺陷病毒Ⅰ型 B′亚型感染者人类白细胞抗原Ⅰ类基因对病毒载量的影响

目的探讨人类免疫缺陷病毒Ⅰ型（HIV-1）B＇亚型感染者人类白细胞抗原（HLA）-A、-B、-C位点等位基因的分布及其对病毒载量的影响。方法对146例HIV-1B＇亚型感染者采用聚合酶链反应-序列特异性引物（PCR-SSP）扩增方法进行HLA-A、-B、-C位点等位基因检测,计算各位点等位基因的频率及其对感染者病毒载量的影响。结果在146例HIV-1B＇亚型感染者中分别检出HLA-A位点14个等位基因,-B位点24个等位基因,-Cw位点12个等位基因,其中频率大于0．1的等位基因为HLA-A＊02,-A＊11,-A＊24,-A＊30,-B＊13,-B＊40,-B＊51,-Cw＊03,-Cw＊06,-Cw＊08;HLA-A、-B、-C等位基因纯合子携带者的病毒载量高于杂合子（P=0．0013）;HLA-A＊03（P=-0．0314）、-A＊30（P=-0．0072）、-B＊13（P=0．0087）、-Cw＊06（P=0．0145）等位基因携带者具有较低病毒载量。结论HIV-1B＇亚型感染者HLA-A＊03、-A＊30、-B＊13、-Cw＊06等位基因与低病毒载量相关。     

gp120蛋白多肽抗体的设计、制备与鉴定

目的:设计多肽免疫原,制备针对中国流行株B’亚型人类免疫缺陷病毒一型(HIV-1)gp120蛋白的抗血清和相应单克隆抗体。方法:通过蛋白抗原设计,设计出免疫原多肽,将多肽欧联载体蛋白免疫小鼠,制备小鼠多克隆抗血清和单克隆抗体;用ELISA实验、蛋白免疫印迹鉴定其活性和表位。结果:成功制备了能结合gp120蛋白的小鼠多克隆抗血清,并且获得4株分泌特异性抗免疫原的IgG1亚型的单克隆细胞株。结论:蛋白抗原设计是成功的,该多肽能诱导结合蛋白的抗体,有可能成为针对艾滋病病毒中国流行株的多肽疫苗。     

HIV-1B’亚型及CRF07_BC重组毒株感染者中和反应特征分析

目的探讨我国主要流行的HIV～1B’和CRF07-BC重组毒株感染者中和反应特征。方法采用基于Env假病毒的以表达CD4及CCR5／CXCR4的TZM-bl细胞系为靶细胞的中和抗体测定方法,测定40例感染时间在3年左右的HIV—CRF07-BC重组毒株感染者及31例感染时间在10年以上的HIV-1B’亚型毒株感染者血浆对11株包括B（4株）、CRF07BC（3株）、CRF01-AE（3株）及中和敏感毒株（1株）Env假病毒的中和反应,并分析感染者血浆的中和宽度和中和强度。结果B’亚型毒株感染者在中和宽度及亚型交叉中和强度方面均高于感染时间相对较短的CRF07BC重组毒株感染者,B’亚型感染者对CRF01-AE亚型病毒的中和活性显著高于CRF07-BC重组型感染者：而CRF07-BC重组型感染者对同亚型病毒的中和强度则显著高于B’亚型感染者。结论HIV-1感染者中和抗体宽度以及对异源病毒的中和活性与感染时间正相关,适度的病毒抗原刺激有助于广谱中和活性的产生。     

1型HIV感染多重PCR检测方法的建立及临床应用

目的建立检测人免疫缺陷病毒1型(HIV-1)的多重巢式PCR(nPCR)和多重反转录PCR(RT-PCR)方法。将其与已经上市的NASBA法试剂盒进行检测敏感性比较;探讨血浆RNA水平对PCR检测敏感性的影响;评价该方法用于我国HIV-1感染者诊断的价值。方法针对HIV-1的gag、pol和gp41区设计3套引物,建立检测HIV DNA的多重nPCR和检测HIVRNA的多重RT-PCR方法;分别以建立的PCR方法和NASBA法对119例HIV阳性患者进行检测,比较2者的检测敏感性;将患者分为不同的病毒载量组,比较PCR方法在各组患者中的检测敏感性差异;使用建立的PCR方法对10例可疑急性感染者进行检测;扩增HIV-1膜区C2-C3段,对nPCR检测阳性的43例DNA样本进行亚型鉴定。结果多重nPCR的检测敏感度为97.5%(116/119),多重RT-PCR的敏感度为78.2%(93/119),2者的特异度均为100%(50/50),多重nPCR和多重RT-PCR的阳性预测值分别为97.5%(116/119)和78.2%(93/119),阴性预测值均为100%(50/50),准确性分别为98.2%和84.6%。经比较,多重nPCR和多重RT-PCR的检测敏感度都高于NASBA法。在病毒载量<103copy/mL的患者,nPCR的检测敏感性高于RT-PCR,在病毒载量在(103～104)copy/mL的患者及病毒载量≥104copy/mL的患者,2者的敏感性比较相近,均接近100%;检测的10例可疑急性感染者中,有5例患者的nPCR和RT-PCR检测结果阳性,抗体在随访过程中阳转,证实为HIV急性感染者;检测的43例DNA样本分属于B’亚型(37例),AE亚型(5例)和BC亚型(1例)。结论本课题组建立的PCR检测方法可以对我国主要流行的B’、AE和BC亚型病毒株得到很好的扩增效果;nPCR的检测敏感性受血浆RNA水平的影响相对较小,RT-PCR的检测敏感性受血浆RNA水平的影响相对较大;PCR方法可以用于HIV急性感染者的早期诊断。     

HIV-specific T cell immunity across the entire HIV genome in Chinese men who have sex with men

Background Man who has sex with man （MSM） is one of the high risk groups for spreading HIV/AIDS. It was reported that the most prevalent human irnmunodeficiency virus type 1 （HIV-1） strain among MSM is subtype B; however, T cell immunity remains unknown across the HIV-1 B genome in this population. Methods Using Elispot assay with synthetic peptides spanning the sequence of HIV-1 consensus B, HIV-l-specific cytotoxic T-cell lymphocyte responses were quantified among 3 treated and 19 untreated HIV-1 infected MSM from Beijing, China. Cross-sectional association between viral loads and cellular immune responses were analyzed. Results Peptide pools corresponding to each HIV-1 protein were used for Env, Gag, Pol, Nef, Tat/Rev, Vpr/Vpu and Vif. The results showed that the magnitude of T cell responses in the 3 treated HIV^＋ MSM group [median, 770 spot forming cells （SFCs） per 106 peripheral blood mononuclear cells （PBMCs）] might be significantly lower than that in the 19 untreated HIV^＋ MSM group （median, 6175 SFCs per 106 PBMCs）. Nef, Gag and Pol are the most frequently targeted HIV-1 antigens; and 16 subjects （73%） were identified with vigorous T cell immunity against each of these three proteins. The overall magnitude of T cell immunity closely related to its breadth （r=-0.72, P〈0.05） and was inversely but weakly associated with viral loads （r=-0.15）. Further analysis showed that both Gag （r=-0.24） and Pol specific T cells （r=-0.12） contributed to this inverse association whereas Nef specific T cells showed no association with viral loads. Conclusions The magnitude of HIV-1 specific T cells is inversely but weakly associated with viral loads among MSM; HIV-specific T cell responses against conservative sequences （Gag and Pol） are the main contributors to this association among Chinese HIV^＋ MSM. These findings have important implications for vaccine design.     

HIV-Specific IL-2+ and/or IFN-γ+ CD8+ T Cell Reponses during Chronic HIV-1 Infection in Former Blood Donors

Objective Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 T-cell responses and HIV-1 viral load or CD4 count over the course of infection.In this study,153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors.Methods The patients were stratified into three groups according to CD4 count:CD4≥500 cells/μL; 350 cells/μL≤CD4<500 cells/μL; CD4<350 cells/μL.PBMCs were isolated from the patients' anticoagulated blood samples.IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay.Results An overall inverse correlation were observed between CD4 count and plasma viral load.Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses,CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/μL,no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/μL to 500 cells/μL,significant correlation was only observed between the response breadth of IL-2+IFN-γ+CD8 T cells and CD4 count; however,as for patients whose CD4 counts were more than 500 cells/μL,direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+CD8 T cells and viral load or CD4 count.Conclusions Universal consistent inverse correlation was only indentified between CD4 count and viral load.The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata,which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.     

CTL responses to regulatory proteins Tat and Rev in HIV-1 B'/C virus-infected individuals

Objective To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B＇/C virus-infected ART-naive individuals. Methods HIV-1-specific CTL responses were analyzed by IFN-7 ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P〈0.05. Results Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. Conclusion Tat and Rev can serve as targets for HIV-l-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV- 1 natural infection and also be useful for the design and testing of candidate vaccines.     

Complete Human Immunodeficiency Virus-1 Specific T Lymphocyte Response to Chinese Human Immunodeficiency Virus-1 B/C Chronic Infectors

Objective To characterize the human immunodeficiency virus （HIV） -specific T lymphocyte responses and identify the immunodominant regions in Chinese HIV-1 recombinant subtype B/C chronic infectors at complete genome level. Methods Twenty-five HIV-I B/C recombinant chronic infectors were screened for their specific T lymphocyte responses to a panel of peptides corresponding to the complete HIV-1 subtype B genome by gamma interferon ELISPOT assay. Kruskal-Wallis nonparametric analysis of variance was used to test significant differences across gene regions, and Tukey pairwise analysis was used to identify differences between gene regions. Spearman rank correlation was used to assess the relation between responses. Results The order of recognized frequencies of specific T lymphocyte responses to HIV proteins was Nef〉Vpr〉Gag〉Pol〉Vpu〉Env〉Rev〉Vif〉Tat. When adjusted for protein length, Nef, Vpr, Gag, and Pol were the most intensely targeted proteins and the central region of Nef, Gag p24, Pol RT, and Vpr was most frequently recognized. No significant correlation was observed between the magnitude of IFN-γ production of HIV-l-specific T lymphocyte responses and plasma viremia, breadth of response and CD4 counts. Conclusion The central region of Nef, Gag p24, Pol RT, and Vpr is most frequently targeted in HIV-1 B/C recombinants chronic infectors. HIV-1-specific T lymphocyte responses and plasma viremia or CD4 counts play no protective role at complete genome level in these infectors.     

甘露糖结合凝集素与人类免疫缺陷病毒感染的关联性研究（英文）

目的探讨甘露糖结合凝集素（mannose-binding lectin,MBL）多态性与中国北方汉族人群HIV-1感染的关联。方法采用病例对照研究,以91例HIV感染者（北京佑安医院）和91例健康人（北京同仁医院）作为研究对象,用焦磷酸测序技术（pyrosequencing）检测病例组和对照组MBL ExonⅠ第52、54、57三个密码子的点突变和启动子＋4、-221、-550三个位点的多态性,用酶联免疫吸附法（ELISA）检测血浆MBL总浓度及血浆中有活性的MBL浓度,用流式细胞术检测HIV感染者的CD4＋T淋巴细胞计数,用bDNA法检测HIV感染者的病毒载量。结果等位基因B在HIV-1感染者中的分布频率显著高于健康对照组（0.18vs0.14）。HIV-1感染者的血浆MBL浓度（0.44）及血浆中活性MBL的浓度（0.61）比健康对照组的浓度要低。携带有A/B、A/C或者B/B基因型的HIV-1感染者与携带有A/A基因型的HIV-1感染者比较,血浆中MBL浓度、血浆中活性MBL浓度以及CD4＋T细胞数量均偏低,但病毒载量偏高。结论 MBL与中国汉族人群的HIV-1感染具有关联作用。携带有B等位基因型、低MBL浓度以及低MBL活性浓度的个体,更容易受到HIV-1病毒的感染。     

长期不进展者与艾滋病患者HIV-1 nef特异性CD8+ T细胞应答的初步研究

目的　探讨我国HIV 1nef特异性CD8 T细胞应答的特点及其免疫保护作用。方法以长期不进展组 (LTNP) 7例和艾滋病组 9例为研究对象 ,以覆盖HIV 1nef全长的 2 6个重叠肽段组成的 3个肽段库作为刺激原 ,用IFN ELISPOT方法检测LTNP组与艾滋病组患者HIV 1nef特异性CD8 T细胞应答 ,并测定其CD4 T细胞、CD8 T细胞和病毒载量 ,观察两组间HIV 1nef特异性CD8 T细胞应答的差异及其与CD4 T细胞和病毒载量的相关性。结果　LTNP组和艾滋病组HIV 1nef特异性CD8 T细胞应答的强度分别为 4 0 4± 334和 5 9± 12 1SFC/ 10 6外周血单个核细胞 (PBMC) ,LTNP组显著高于艾滋病组 ;HIV 1nef特异性CD8 T细胞应答的强度与CD4 T细胞计数呈正相关 ,但与病毒载量没有显著的相关性。结论　HIV 1nef特异性CD8 T细胞应答在艾滋病发病中具有一定的保护作用 ,欧美流行株与我国流行株之间具有免疫交叉反应性     

Identification of HIV-1 specific T lymphocyte responses in highly exposed persistently seronegative Chinese

Background Studies of highly exposed persistently seronegative （HEPS） individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly assessed at the entire viral genome level. Methods Ten HEPS Chinese with a history of frequent penetrative vaginal intercourse （mean frequency, at least once a week）, with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were comprehensively analyzed by an interferon- 7 Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins. Results HIV-1-specific T-cell responses of interferon- 7 secretion were identified in 3 （30%） out of 10 HEPS individuals; the specific cytotoxic T lymphocytes were targeted at Pol （2/10）, Env （2/10）, and Tat （1/10）. HIV-1-specific T-cell responses of interferon- ~ secretion were identified in 20 （80%） out of 25 seropositive intravenous drug users （IDUs）, revealing that all HIV-1 proteins and protein subunits could serve as targets for HIV-1-specific CD8^＋ T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol, 60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive individuals. None of the seronegative healthy individuals gave the positive T-cell responses. Conclusions About 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections.