HIV-1高暴露持续血清阴性者对Nef、Gag肽段库的特异性CTL应答的研究

目的 研究中国HIV-1高暴露持续血清阴性（highly exposed persistently seronegative,HEPS）者的Nef、Gag特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）应答特点,探讨HIV-1特异性CTL应答在这类特殊人群中抵抗感染的作用机制.方法 选取10例HIV-1高暴露持续血清阴性者,11例经性接触感染且从未接受抗病毒治疗的HIV/AIDS患者及4例未经暴露的健康志愿者.以覆盖HIV-1 gag全长和部分nef的14个肽段库为刺激原,应用IFN-γ ELISPOT法测定3组人群的特异性CTL应答,并对3组的应答强度、宽度以及对肽段库识别比例进行比较.结果 50%（5/10）的HEPS,100%（11/11）的HIV/AIDS患者均存在Nef及Gag特异性CTL应答,而4例健康对照均为阴性.存在应答的HEPS者对14个肽段库的平均应答强度和宽度分别是HIV/AIDS患者的4.3%和37.7%.在HEPS者中主要识别的肽段库均为HIV/AIDS患者中识别比例相对较低的肽段库.结论 与HIV/AIDS患者相比,HEPS者中的HIV-1特异性CTL应答存在着不同的特点和规律,可能在保护机体免于HIV-1感染中发挥着重要作用.     

中国人群HIV-1B亚型Nef蛋白特异性细胞毒性T细胞反应的研究

目的探讨中国人群HIV-1B亚型Nef蛋白特异性细胞毒性T细胞(CTL)反应特征及其与病毒载量以及CD4细胞数量间的关系。方法选取33例HIV-1B亚型感染者。用合成的HIV-1B亚型Nef全基因序列肽库作为抗原,ELISPOT方法检测HIV-1B亚型Nef蛋白特异性CTL反应,同时测定病毒载量及CD4细胞数量。结果70％的感染者对Nef产生特异性CTL反应,单一肽段能够被识别的频率不超过40％,应答强度为(1102±2136)SFC(斑点形成细胞数)／106 PBMC。HIV-1B亚型Nef特异性CTL应答的强度和频率之间没有显著的相关性。HIV-1 Neff特异性CTL反应强度与病毒载量间存在显著负相关,与CD4细胞数量间存在显著正相关。结论初步确定了Nef蛋白CTL应答的优势区域。这些区域主要集中在一些高度保守的氨基酸序列。提示HIV-1B亚型Nef特异性CTL应答在疾病进展中对机体具有保护性作用。     

不同疾病进展阶段的HIV-1 B亚型毒株感染者对Gag、Nef肽段库的特异性CTL应答比例的比较和分析

目的 对不同疾病进展阶段的HIV-1B亚型毒株感染者Gag、Nef特异性细胞毒性T淋巴细胞（cytotoxic Tlymphocyte，CTL）应答进行研究，比较和分析不同患者群对不同肽段库应答比例的异同，探讨针对不同肽段库的特异性CTL应答在延缓病程进展中所起的作用。方法 选取56例未经抗病毒治疗的中国HIV-1B亚型毒株感染者。其中包括长期无进展者（long-term nonprogressors，LTNP）、HIV感染早中期患者和AIDS患者3组不同疾病进展阶段患者。以覆盖HIV-1 Gag全长和部分Nef的14个肽段库为刺激原，应用IFN-γ ELISPOT法测定3组患者的特异性CTL应答，并比较3组患者对不同肽段库的应答比例。结果 3组不同进展阶段患者对14个肽段库总体的特异性CTL应答的平均反应宽度和强度间差异均无统计学意义。3组患者对14个肽段库的识别模式可分为两种类型：（1）对Gag-p24-1、Gag-p24-5、Gag-p 2/7/1/6-1以及Gag-p2／7／1／6-3这4个肽段库的识别比例高低与病情进展情况相平行。3组患者对4个肽段库整体的识别比例间差异有统计学意义（P=0．041）；（2）对其他10个肽段库的识别与病情进展不平行，在HTV感染早中期患者中比例高，而在LTNP中低。结论 针对不同肽段库的CTL应答可能在控制病毒复制过程中发挥不同的作用，对疾病进展的控制可能需要针对多个抗原表位的有效CTL应答。     

HIV/AIDS患者特异性细胞毒性T细胞功能的研究

目的 了解中国HIV／AIDS患者HIV特异性细胞毒性T细胞(CTL)功能。方法 将覆盖HIV-1 P15、P17和P24 Gag全长的94个重叠多肽作为抗原，用IFN-γ ELISPOT方法检测HIV／AIDS患者HIV-1特异性CTL功能。结果 HIV-1抗原多肽P17-15、P17-16、P24-7、P17-8，P24-28最易被HIV／AIDS患者特异性CTL识别。HIV感染者识别HIV-1多肽的数量和强度均高于AIDS患者。结论 我国HTV／AIDS患者体内存在识别不同HIV-1 Gag多肽的特异性CTL，且HIV特异性CTL功能与疾病进展相关。     

中国HIV-1 C/B'重组毒株和B'毒株感染者Nef特异性T细胞免疫应答的研究

目的 研究中国主要流行的HIV-1 C/B'重组毒株和B'亚型毒株感染者Nef特异性T细胞反应特征,确定两种亚型感染者共同识别的免疫优势区.方法 本研究以59名HIV-1 C/B'重组毒株、27名B'亚型毒株感染者为研究对象,用ELISPOT检测针对HIV-1型C/B'Nef重叠多肽产生IFN-γ的特异性T细胞反应.结果 44例(74.58%)HIV-1 C/B'重组毒株感染者产生Nef特异性T细胞反应,主要识别EVA7081.1、5、6、7、43、44、45、47、48、49这10条多肽,氨基酸序列为Nef63～115和117～139的区域.20例(74.07%)的HIV-1 B'毒株感染者产生Nef特异性T细胞反应,主要识别EVA7081.1、2、43、49这4条多肽,氨基酸序列为Nef 63～77和87～119的区域.两种亚型感染者特异性T细胞反应的强度和广度与病毒载量和CIM细胞数不相关.结论 中国HIV-1 C/B'重组毒株和B'亚型毒株感染者共同识别氨基酸序列为Nef63～77和87～115的免疫优势区,提示此区域可用于疫苗的设计.     

CD8+ T细胞对HIV-1合成表位的免疫主导应答研究

目的研究CD8^＋ T细胞对人免疫缺陷病毒1型（HIV-1）表位的免疫主导应答。方法分别采用酶联免疫斑点技术（ELtSPOT）和羧乙基锗倍半氧化物（CFSE）标记流式分析技术，以覆盖HIV-1 Env、Pol、Gag、Vif、Nef、Tat区的701个重叠肽段组成的34个肽段库及其部分单肽段作为刺激表位，对一例感染HIV-1的长期不进展者（LTNP）的外周血单个核细胞（PBMC）中CD8^＋ T细胞的了γ-干扰素（IFN-γ）分泌细胞频率和细胞增殖率进行了测定研究。结果HIV-1 Gag区域肽段诱导产生的CD8^＋ T细胞的IFN-γ分泌细胞频率最高，Nef、Tat、Vif区域依次顺减，Env和Pol区域不能诱导产生显著性应答；在IFN-γ ELISPOT实验中，肽段和相应肽段库刺激产生的结果一致；CD8^＋ T细胞在单肽段刺激下，用ELISPOT技术测定的IFN-γ分泌细胞频率和CFSE标记流式分析技术测定的细胞增殖率显示出较好的相关性。结论CD8^＋ T细胞能特异性识别某些HIV-1抗原表位，诱导出免疫主导应答；当进行HIV-1特异性CD8^＋ T细胞反应增殖测定和免疫主导应答研究时，ELISPOT是值得称道的标准实验，同时，推荐一种新颖的CFSE标记流式分析技术。     

HIV-1B’/C重组病毒感染者Vif、Vpr和Vpu特异性细胞免疫应答研究

目的 探讨我国HIV-1 B'/C重组病毒感染者针对HIV-1调节蛋白的细胞免疫反应特征及其与病毒复制控制的关系.方法 以覆盖HIV-1 C亚型Vpr、Vpu和Vif蛋白全长的重叠肽段作为刺激抗原,利用ELISPOT方法检测新疆HIV-1 B'/C重组病毒感染者的特异性细胞免疫反应.使用SIGMAPLOT 10.0和SIGMASTAT 3.5进行统计分析,用双尾t检验比较组间差异,用Spearmam秩相关分析免疫反应与病毒载量及CD4细胞计数的关系.结果 在检测的60名HIV-1 B'/C重组病毒感染者中,能够识别Vif、Vpr和Vpu蛋白产生CIL应答者分别为68%、52%和8%,Vpr和Vif蛋白存在多个强CIL反应的免疫优势区域.研究中还发现针对Vpr、Vif和Vpu蛋白的CTL反应强度和广度与HIV感染者的病毒载量及CD4细胞数量无明显的相关性.结论 HIV-1 Vpr和Vif蛋白包含多个可被机体免疫系统特异性T细胞识别的免疫优势区域.对这些免疫优势区所包含的CIL表位进行鉴定并探讨其在自然感染过程中的作用,对新一代的HIV疫苗设计有重要的参考意义.     

塞姆利基森林病毒翻译增强序列对HIV Gag DNA疫苗抗原表达和免疫原性的调节

目的 研究塞姆利基森林病毒（Semliki forest virus，SFV）衣壳蛋白5’翻译增强区对基于该病毒复制子的HIV Gag DNA疫苗抗原表达水平和免疫原性的影响：方法将SFV衣壳蛋白（capsid，C蛋白）基因的5’端102bp翻译增强区插入到SFT复制子DNA疫苗载体pCMV-Rep的SFV亚基因启动子下游，得到DNA疫苗载体pCMV-RepC。将删除ATG的HIV-1 gag基因插入pCMV-RepC，使gag编码区与翻详增强区融合，得到DNA疫苗质粒pCMV-RepC-gag。同时，构建携带未融合翻译增强序列的DNA疫苗质粒pCMV-Rep-gag。Western blot检测翻译增强序列对Gag表达水平的影响。用上述两种DNA疫苗分别免疫BALB／c雌性小鼠，ELISA检测Gag特片的抗体反应，ELISPOT和细胞内因子染色技术检测细胞免疫应答。结果衣壳蛋白5’翻译增强区增强了Gag表达水平，对体液免疫应答没有显著影响，但显著增强了特异性细胞免疫应答水平。结论SFVC蛋白翻译增强区能显著提高SFV复制子DNA疫苗的抗原表达和抗原特异性细胞免疫反心。     

HIV-1蛋白诱导T细胞产生IFN-γ反应特征及对HIV-1感染者病情进展的影响

目的 探讨HIV-1特异性T细胞反应特征及对HIV-1感染者病情进展的影响.方法 通过合成重叠肽技术及ELISPOT技术,采用队列研究方法对37名HIV-1感染者的病毒特异性T细胞免疫反应进行分析.结果 83.78%(31/37)的HIV-1感染者对1个或多个合成肽反应(反应强度大于50 SFU/106 PBMCs).HIV-1B型病毒不同蛋白均可激发HIV-1感染者的特异性T细胞反应,其中HIV-1 Gag蛋白被识别的频率最高,81%的HIV-1感染者识别HIV-1 Gag而且HIV-1 Gag蛋白诱导的T细胞反应强度最高,相对强度达到28.25%,明显高于其他蛋白,差异具有统计学意义(F=17.969,P<0.001);重叠肽诱导T细胞分泌IFN-γ反应频率、反应强度在HIV-1感染无症状期和艾滋病期无明显区别,但是HIV-1 Gag蛋白诱导的T细胞反应强度在无症状期明显高于艾滋病期.结论 HIV-1B型病毒不同基因编码蛋白激发T细胞分泌IFN-γ反应小同,其中HIV-1 Gag蛋白特异性T细胞在控制病情进展方面发挥了重要作用.     

我国HIV-1 CRF07_BC流行重组毒株Tat蛋白第一外显子CTL表位预测

目的 通过测定和分析我国HIV-1 CRF07_BC毒株Tat蛋白第一外显子的基因序列预测其CTL表位的分布情况.方法 236份来自第3次全国HIV分子流行病学调查中感染CRF07_BC毒株的患者的血浆样本,应用巢式聚合酶链反应(nested-PeR)扩增tat基因第一外显子基因区并测序,使用BIMAS HLA Peptide Binding Predictions在线软件和统计学软件对CTL表位进行预测分析.结果 236份CRF07_BC毒株来自16个省份,主要为静脉吸毒传播(58.9%),其次为性传播(25.0%).236条CRF07_BC Tat蛋白第一外显子区共分布有12种CTL表位,主要分布于脯氨酸富集区、半胱氨酸富集区和疏水核心区,碱性区和谷氨酰胺富集区没有发现已知的CTL表位分布,这些表位主要受5种HLA基因型(A * 2501、A * 2902、B * 15、B * 5301和Cw * 1203)以及6种HLA血清型(B53、B58、B57、A3、A68和Cw12)限制,其中5种表位的单氨基酸变异频率大于50%.结论 我国HIV-1 CRF07_BC毒株Tat蛋白第一外显子CTL表位分布于不同功能区,并且存在氨基酸变异.     

The Mamu B 17-restricted SIV Nef IW9 to TW9 mutation abrogates correct epitope processing and presentation without loss of replicative fitness

CD8(+) cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A()01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B()17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4(+)T cells infected with an engineered SIV(mac)239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4(+)T cells, the TW9 mutant virus failed to induce IFN-gamma expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.     

HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.     

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-γ ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = −0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.     

Detection of HIV-1-specific CTL responses in Clade B infection with Clade C Peptides and not Clade B consensus peptides

The variability of HIV-1 sequences within and between persons in vivo complicates immunologic screening with a fixed sequence, and using peptides based on consensus sequences therefore has become a common practice for pathogenesis and vaccine studies. Here, we screen a cohort of HIV-1-infected persons in the United States for CD8+ T lymphocyte (CTL) responses using Gag peptides based on the Clade C primary isolate DU422 and the consensus sequence for Clade B. Surprisingly, the DU422 and Clade B consensus peptides are similar in sensitivity, but many responses are detected only by one set or the other. About equal numbers of discordantly detected responses are specific to consensus Clade B peptides as DU422 peptides. A minority of discordant detection is due to the varying frames of the peptide sets and therefore a technical artifact; the majority is due to sequence differences. This lack of superiority of the Clade B consensus peptides to detect CD8+ T lymphocyte responses is an unexpected finding that suggests that detection of HIV-1-specific cellular immunity with these peptides may be significantly insensitive and raises questions as to whether screening with a single sequence adequately reflects responses to the viral swarm in vivo.     

Cross-Clade-Specific Cytotoxic T Lymphocytes in HIV-1-Infected Children

We studied cytotoxic T lymphocyte (CTL) cross-reactivity between human immunodeficiency virus type 1 (HIV-1) subtypes within a group of infants infected with either HIV-1 B or non-B clade. Fifteen children were infected with a clade B virus. Nine were infected with non-B virus, including two clade A, four clade D, two clade F, and one clade G. CTL activities fromin vitroactivated peripheral blood mononuclear cells were tested against autologous cell line infected with recombinant vaccinia viruses encoding for Env, Gag, Pol, or Nef proteins from a clade A or B isolate. HIV-1-specific CTL elicited from infection with clade B virus could lyse targets expressing clade A proteins, and vice versa. In infants with positive CTL responses, cross-clade recognition was predominant and was detected within 88% of the Pol, 83% of the Nef, 67% of the Gag, and 55% of the Env responders. Longitudinal studies showed that CTL cross-reactivity to both B and A targets was stable for several years. Elicitation of CTL reactivities capable of elimination of virus-infected cells is an important goal for the development of an efficient AIDS vaccine. The significant cross-reactivity of CTL shown in this study supports the concept that vaccines developed using a single-clade immunogen may be applicable to induce broadly reactive T cell responses.     

Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein.

HIV-1 replication depends on the expression of trans-regulatory genes (tat, rev) encoded in the 3' part of the retroviral genome. HIV-1 Rev trans-activator protein allows the cytoplasmic translocation of incompletely spliced retroviral mRNA which is required for the translational switch from regulatory (Tat, Rev, Nef) to structural proteins (Gag, Pol, Env). The HIV-1 Rev regulatory protein comprises an activation domain (RAD) and a RNA binding domain (RBD). Both functional domains are not well defined and the RBD appears to overlap with the nuclear localization signal (NLS). Our mutational analysis localized the Rev protein domain important for RRE (nucleotide 7781 to 8000) binding in vitro to amino acid residues 31 to 50. Mutations in this domain always resulted in exclusion from the nucleoli. Furthermore, these mutants did not support Rev-dependent p24 Gag production in vivo. Sequences immediately upstream of this domain (RevM4, RevM19) were attenuated in their in vivo activity possibly indicating a role in Rev protein oligomerization. The observed tight correlation between subcellular localization and RNA binding in vitro indicates that this short stretch of amino acids supports two essential functions required for HIV-1 replication.     

Development of a whole blood intracellular cytokine staining assay for mapping CD4(+) and CD8(+) T-cell responses across the HIV-1 genome

A whole blood peptide mapping intracellular cytokine staining (ICS) assay was developed that allows the direct comparison, at the individual peptide level, of CD4(+) and CD8(+) T-cell responses that span every encoded protein, in patients infected with HIV-1. Whole blood samples from HIV-1 infected patients were stimulated with overlapping synthetic peptides spanning nine subtype C HIV-1 gene regions (Gag, Pol, Nef, Env, Tat, Rev, Vif, Vpu, Vpr). Following stimulation and permeabilization, cells were stained with fluorochrome labelled antibodies to CD3, CD8 (CD4(+) cells were defined as CD8 negative cells), and IL-2 and IFN-gamma. A total of 396 overlapping peptides were arranged in pools with a matrix design which allowed the identification of individual peptide responses from multiple pool responses. HIV-1 infected patients screened using this method showed a broad range of peptide responses across the entire HIV-1 genome with CD8 T-cell responses being higher in frequency in magnitude than CD4(+) T-cell responses. The advantages of this whole blood ICS assay include the following: (1) the response to all potential HIV-1 epitopes across the genome can be examined, (2) the responding cell type can be monitored in the same reaction, and (3) considerably less blood is required than would be necessary if peripheral blood mononuclear cells (PBMC) were first isolated prior to peptide stimulation.     

Rev activity determines sensitivity of HIV-1-infected primary T cells to CTL killing

The HIV Nef protein is thought to promote HIV immune evasion by downmodulating MHC-I and protecting infected cells from CTL killing. In addition, we demonstrated that Rev, an HIV regulatory protein needed for expression of the HIV late genes, can influence CTL killing. When Rev activity level was reduced by virtue of amino acid alterations in the Rev protein sequence, infected cells were more resistant to anti-Gag and anti-Env CTL killing. A screen of primary viral isolates revealed that viruses derived from asymptomatic, infected people had lower Rev activity, lower Gag levels, and greater resistance to anti-Gag CTL killing. Thus, rev alleles with low activity may have a selective advantage in infected people with effective immune responses.     

Temporal loss of Nef-epitope CTL recognition following macaque lipopeptide immunization and SIV challenge

To address the subtle interactions between antiviral cytotoxic T-cell (CTL) immune responses and the evolution of viral quasispecies variants in vivo, we performed a longitudinal study in a simian immunodeficiency virus (SIV)-infected rhesus macaque that had a long experimental SIV infection before developing simian AIDS. Before being infected with SIV, this animal was immunized with a mixture of seven lipopeptides derived from SIV Nef and Gag proteins and showed a bispecific antiviral CTL response directed toward Nef 169-178 and 211-225 peptides. After SIV infection, CTL activity against the Nef 169-178 epitope was no longer detectable, as assessed from peripheral blood mononuclear cells stimulated by autologous SIV. CTL activity against the 211-225 epitope was lost after 3 months, and an additional CTL response to the amino acids 112-119 Nef epitope emerged. Analysis of the Nef proviral sequence revealed the presence of immune escape variants first in the 211-225 epitope and much later in the 112-119 epitope. In contrast, epitope 169-178 showed only two mutations among all viral sequencing performed. We conclude that in this macaque, bispecific CTL exerted a strong selective pressure and escape virus mutants finally emerged. We identified CTL recognizing a conserved Nef epitope 112-119 (SYKLAIDM), essential for viral replication, which could be associated with a prolonged AIDS-free period. These results stress the importance of the induction of broader multispecific CTLs directed against highly conserved and functional T-cell epitopes by vaccination, with the aim of keeping HIV infection in check.