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。     

艾滋病痴呆综合征病人HIV-1tat基因多态性及氨基酸序列分析

目的 研究艾滋病痴呆综合征患者体内HIV-1 tat第一外显子基因序列的特征和变异情况,为艾滋病痴呆综合征发病机制的研究提供依据.方法 提取1例艾滋病痴呆综合征病例尸检标本的外周组织(淋巴结、脾脏)和中枢神经组织(脑膜、额叶灰质、额叶白质、颞叶、基底核)共7个部位的基因组DNA,巢式PCR扩增HIV-1 tat第一外显子基因,与克隆载体pGEM-T连接,经转化、氨苄青霉素和蓝白斑筛选出阳性克隆,每个部位挑取5个菌落测序,测序结果利用BioEdit、MEGA4软件比对并生成系统进化树、计算基因距离和同义/非同义替换值(ds/dn),分析氨基酸位点的改变.结果 该病例感染的病毒是HIV-1 B亚型,分离自不同组织的HIV-1 tat第一外显子基因序列存在差异;与标准序列相比,该病例的HIV-1tat第一外显子基因编码的氨基酸序列有16个位点发生了变异,并且中枢各部位部分氨基酸位点的变异与外周不同,特别是中枢基底核5个序列及颞叶1个序列Q54R的变异值得关注.结论 艾滋病痴呆综合征患者体内,HIV-1 tat第一外显子序列与标准序列存在差异,并且在外周和中枢不同部位中存在的变异不同,这些变异是否与艾滋病痴呆综合征的发病机制有关,还有待进一步研究.

Abstract：

Objective To study the variation and characteristics of HIV-1 tat exon 1 gene from a patient with AIDS dementia complex( ADC), so as to research the pathogenesis of ADC. Methods The tat gene was amplified with nested PCR from genomic DNA which was extracted from lymph node, spleen and different brain tissues( meninges, grey matter from frontal cortex, white matter from frontal cortex, temporal cortex and basal ganglia) of a patient who died of ADC. PCR products were cloned into the pGEM-T vector,after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, Neighbor-Joining tree, p-Distances, values of ds/dn, and analysis of amino acid motifs were all done. Results The samples were all identified as HIV-1 B and genetic variation exists in HIV-1 tat isolated from different tissue;Compared with HXB2, sixteen sites of the amino acid seque nce coded by the HIV-1 tat gene which was isolated from the patient changed. In addition, part of the changes were different between periphery and brain,especially, the five Q54R changes from basal ganglia and one Q54R change from temporal cortex are deserve to follow with interest. Conclusion Variations exist in the HIV-1 tat genes extracted from the ADC patient and the variations from peripheral and central nerve tissues were different, whether the variations concerned with the pathogenesis of ADC need more research.     

广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型的关系

目的研究广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型间的关系。方法从广西主要流行区收集来的50份HIV-1感染者血液样本中提取前病毒DNA，使用巢式聚合酶链反应（nested-PCR）扩增HIV-1 env基因片段并进行亚型鉴定，选择38份CRF01-AE重组型HIV-1毒株env，基因V3环及邻近区域的序列进行系统树和氨基酸变异分析。结果38份CBF01-AE重组毒株中36份与分离于广西地区的CRF01-AE.97CNGX2f和泰国代表毒株THCM240接近，另外2份与中非共和国代表株90CF402聚成一簇；CRF01-AE重组毒株V3环顶端四肽存在着4种类型：CPCQ、GPGR、GPGH和GPGA；根据V3环关键氨基酸推测辅助受体使用情况，结果显示：71．05％的CRF01-AE重组毒株可能使用CCR5作为辅助受体，28．95％不能对其辅助受体的使用情况做出预测。结论广西HIV-1 CRF01-AE重组毒株V3顶端四肽变异较大，而且大部分毒株可能为NSI型。这可为广西该毒株的防治和诊断试剂的更新提供参考。     

HIV-1 nef基因的克隆及原核表达研究

目的:构建HIV-1 Nef基因的原核表达载体并在大肠杆菌中进行表达,初步优化其表达条件。方法:利用特异性引物PCR扩增获得HIV-1 Nef基因片段,PCR产物经限制性内切酶双酶切后连接载体pET24a(+),构建重组质粒pET24a(+)-Nef。经双酶切鉴定及序列测定后的重组质粒转化入E.coliBL21,IPTG诱导表达目的蛋白,优化表达条件,SDS-PAGE及Western blot分析鉴定表达产物。结果:PCR扩增获得618bpDNA片段,酶切鉴定结果表明HIV Nef基因已克隆入原核表达载体pET24a(+)中,测序证明序列正确。SDS-PAGE和Western blot分析证实重组菌诱导后可表达相对分子质量(Mr)符合预期的融合蛋白。0.1mmol/LIPTG在37℃诱导6h为最佳表达条件,重组蛋白表达量可由15.8%增加到43.9%。结论:在大肠杆菌中成功地表达了HIV Nef蛋白,并初步获得了最优化的表达条件。     

DC-SIGNR基因多态性与人类免疫缺陷病毒-1易感性的关联研究

目的 探索DC-SIGNR基因多态性对中国汉族人群人类免疫缺陷病毒-1(human immunodeficiency virus-1,HIV-1)病毒易感性的影响.方法 用聚合酶链反应、琼脂糖凝胶电泳结合测序对345例HIV-1血清阳性感染者及468例H1V-1血清阴性高危人群的DC-SIGNR基因绞链区重复序列分型,然后用卡方检验检测DC-SIGNR基因各亚型在不同人群组中出现的频率.结果 共发现DC-SIGNR基因14个基因型和5种等位基因,其中等位基因7出现的频率最高(67.1%),明显高于美国白人(67.1% vs.46.0%,P<0.01).HIV-1感染组7/7基因型的出现频率明显低于高危人群组(38.55% vs.48.29%,P=0.0057),而HIV-1感染组的9/5基因型的出现频率明显高于高危人群组(4.35% vs.1.07%,P=0.0029).结论 中国人群DC-SIGNR基因多态性分布有着独特的遗传学特征,中国人群中DC-SIGNR基因型9/5可能增加对HIV-1的易感性.     

中国HIV-1感染者KIR基因多态性与疾病进展关系的研究

目的 通过对中国HIV-1感染缓慢进展者(TP)和典型进展者(TP)杀伤细胞免疫球蛋白样受体(KIR)基因频率的研究,探讨中国人群K/R基因多态性与HIV-1感染者疾病进程的关系.方法 采用聚合酶链反应-序列特异性引物(PCR-SSP)技术,分析43例HIV-1感染SP和38例TPKIR基因位点的多态性.结果 SP组和TP组的KIR2DS3等位基因频率分别为3.6%和14.2%,SP组显著低于TP组,两组的KIR2DS3等位基因频率差异具有统计学意义(P=0.018,OR=0.210,95%CI=0.053-0.833);SP组的活化性KIR基因数目低于TP组,但差异无统计学意义(P=0.208).结论 低表达KIR2D53基因可能是延缓中国HlV-1感染者疾病进程的保护因素之一.     

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.     

中国HIV-1型B、C亚型主要流行株外膜蛋白基因V3-V4区序列的变异分析

目的　研究我国人类免疫缺陷病毒 1型 (HIV 1)B、C亚型主要流行株在感染过程中基因变异的特点及其与选择压力的关系。方法　应用巢式聚合酶链反应 (nested PCR)对 2 5 8例HIV 1感染者血样中的HIV 1外膜蛋白 (env)基因进行扩增 ,并使用ABI 377型测序仪对扩增产物测序后 ,选择其中 37份B亚型和 35份C亚型HIV 1毒株env基因包括V3～V4区的序列进行比较分析 ,并计算和分析氨基酸同义替换与非同义替换的比值 (Ks Ka)。结果　B亚型毒株V3～V4区的基因离散率高于C亚型毒株。无论B亚型 ,还是C亚型毒株 ,其V4区基因序列较V3区变异更大。在C亚型毒株中 ,V3区基因序列变异甚至比V3上游区和C3区小。B和C亚型毒株整个V3～V4基因区的Ks Ka比值均 <1,差异有非常显著性 (P <0 0 0 1) ,其中B亚型毒株以V3区的Ks Ka比值最小 ,而C亚型毒株则以V4区的Ks Ka比值最小。结论　B和C亚型毒株env基因的变异主要发生在V4区而不是V3区。C亚型毒株V3区较V3上游区和C3区还要保守 ,是本研究的特殊发现。这两种亚型在我国快速流行中发生的变异是在选择压力下发生的 ,而不是随机进化的结果 ,而且选择压力对这两种亚型毒株V3、V4区的作用程度也不一样。这将为我国艾滋病防治策略的制定和疫苗研究提供科学的依据。     

中国HIV-1感染人群中和抗体水平与疾病进展关系研究

目的 研究中国HIV感染者、AIDS患者及疾病长期不进展者（LTNP）HIV-1中和抗体水平,探讨中和抗体对HIV-1感染疾病进程的影响.方法 提取正常人外周血单个核细胞（PBMC）,应用HIV-1 SF33毒株进行Single-Round PBMC中和试验,流式细胞仪分析CD4＋T细胞内p24-Ag表达.Aleast-squares regression计算中和抗体滴度.结果 HIV感染者、AIDS患者及LTNP中和抗体平均滴度分别为1：29.09、1：22.07、1：154.37.LTNP中和抗体滴度显著高于HIV感染者（t=-2.220,P＜0.05）及AIDS患者（t=-3.812,P＜0.01）;HIV感染者与AIDS患者中和抗体滴度差异无统计学意义（t=0.424,P＞0.05）.结论 中和抗体水平影响中国HIV感染者疾病进展,高水平中和抗体有利于延缓病情进展.     

Variability of the HIV-1 nef regulatory gene and its association with different HIV stages

Biological properties of HIV-1 laboratory strains and isolates were studies and compared with research results of the nef gene fragment obtained from their proviral DNA and RNA as well as from RNA and HIV-1 DNA isolated immediately from the blood of patients at early HIV stages. The electrophoresis pattern as well as the results of determination of nucleotide sequences showed that the HVI-1 RNA nef gene of rapid/high laboratory strains of HIV isolates and RNA obtained from patients at late infection stages had a 135-nucleotide inner deletion. The phenomenon can be regarded is proof of that nef gene structure, if violated, causes an enhanced virulence of and an intensified multiplication of the virus (according to laboratory markers) observed at late HIV stages, which triggers, at least in a number of cases, the infection aggravation.     

Deleterious genetic influence of CX3CR1 genotypes on HIV-1 disease progression

We previously reported that patients homozygous for a specific mutation (M280) in the chemokine receptor CX3CR1 progressed to AIDS more rapidly than those with other genotypes. This deleterious effect would predict that a cohort of prevalent patients would be depleted in M280 carriers, because these patients would have disappeared before recruitment. This hypothesis is confirmed in this new study based on the French SEROCO cohort showing that patients homozygous for the M280 allele were rare among the seroprevalent group. These results may explain the conflicting results published on the impact of CX3CR1 polymorphism in seroconverters.     

中国HIV-1 B'/C亚型感染者对异体病毒中和作用与疾病进展关系研究

目的 探讨中国HIV-1 B'/C亚型感染者对异体病毒中和作用与疾病进展的关系.方法 根据CD4 T淋巴细胞数量和有尤临床症状将HIV-1 B'/C亚型感染者分为HIV慢性感染组和AIDS组.将HIV-1感染者血清稀释(1/10～1/320)后,与在基因结构特点上同源性很低的3株HIV-1作用,以检测其中和作用.同时以正常人血清加病毒悬液为对照孔,能够抑制对照孔50%病毒复制的血清为中和作用阳性.将某个HIV-1感染者血浆能够中和异体病毒的个数占3个异体病毒的百分率定义为HIV-1感染者中和异体病毒的宽度;将某个HIV-1感染者血浆中和3个异体病毒抗体滴度的几何平均滴度定义为HIV-1感染者中和异体病毒的强度.结果 HIV-1慢性感染组与AIDS组之间中和异体病毒的宽度和强度差异有统计学意义,HIV-1慢性感染组显著高于AIDS组.HIV-1慢性感染组中和异体病毒的宽度和强度与病毒载量呈正相关,而AIDS组巾和异体病毒的宽度和强度与病毒载量没有显著的相关性.HIV-1慢性感染组和AIDS组中和异体病毒的宽度和强度与CD4 T淋巴细胞数均没有显著的相关性.结论 中国HIV-1B'/C亚型感染者不同疾病进展阶段针对异体病毒中和作用能力不同,HIV慢性感染组显著高于AIDS组,当疾病进展到AIDS期时,失去对异体病毒的中和作用,提示针对异体病毒的中和抗体与疾病进程有关.     

Analysis of the HIV-1 nef gene in five intravenous drug users with long-term nonprogressive HIV-1 infection in Italy

Great variability in the course of human immunodeficiency virus type 1 (HIV-1) infection results from a complex interplay between host and virus factors. Some of the patients with prolonged nonprogressive infection have been reported to harbor virus variants with gross deletions in the accessory nef gene that has been implicated in in vivo pathogenicity in simian and mouse models. To investigate the role of nef-deleted HIV-1 in long-term nonprogressor (LTNP) drug addicts in Italy the nef sequence from proviral DNA was analyzed from five LTNPs and five rapid progressor controls. Only small (2-12 amino acids) in-frame deletions and insertions were detected in the N-terminal polymorphic and variable regions obtained from three LTNPs and one rapid progressor. There was no evidence of premature termination of the Nef protein and all of the identified functional motifs were well conserved in both groups. Phylogenetic analysis showed interdigitation of nef sequences obtained from LTNPs and rapid progressors. The nef sequence of one LTNP, however, diverged significantly from those of the other patients. Availability of two additional blood DNA samples obtained previously from this subject allowed to detect evolution of nef at 14-17 years of HIV-1 infection, including progressive deletions. Although alterations of nef may be relatively frequent and continue to evolve in LTNPs, this study of a small number of patients does not indicate that gross deletions or loss of functional motifs play a major role in delaying or halting disease progression in infected drug abusers in Italy.     

HIV-1 IgA specific serum antibodies and disease progression during HIV-1 infection

OBJECTIVE: To evaluate the role of serum human immunodeficiency virus type 1 immunoglobulin A (HIV-1 IgA) antibodies in the progression of HIV-1 infection in relation to viral load and CD4 cell counts. METHODS: Sequential serum specimens were obtained from 218 homosexual men: 123 HIV-1 seropositives, 24 HIV-1 seroconverters, and 71 HIV-1 seronegatives. HIV-1 IgA antibodies were tested blindly by enzyme-linked immunosorbent assay and Western blot. T-lymphocyte subsets were measured by flow cytometry. Viral plasma load was determined by a sensitive branched DNA assay. RESULTS: HIV-1 IgA antibodies with a titer greater than or equal to 50 were detected among 50% of the seroconverters, 27% of the HIV-1-seropositive asymptomatic subjects, 25% of lymphadenopathy, and 23% of HIV-1-related symptomatic subjects. Among patients with the acquired immune deficiency syndrome, the prevalence of virus-specific IgA antibodies (55%) was significantly higher (p < 0.03) as compared with the HIV-1-seropositive asymptomatic subjects, lymphadenopathy and HIV-1-related symptomatic patients, but not versus the seroconverters (p = 0.8). IgA antibodies to HIV-1 gP160 were the most prevalent among all subjects tested. A significant decrease in CD4 cell counts was observed after HIV-1 seroconversion. Viral load was slightly higher among the seroconverters who demonstrated higher (> or =50) HIV-1 IgA levels. CONCLUSIONS: HIV-1 IgA serum antibodies did not predict the progression of the disease. Correlation between HIV-1 IgA antibodies titer, viral load, and CD4 cell counts was not detected.