中国HIV-1B''/C亚型感染者对自身病毒中和作用与疾病进展关系的研究

目的：探讨中国HIV-1 B’/C亚型感染者对自身病毒中和作用与疾病进展的关系。方法：将24例HIV-1 B’/C感染者自身原代病毒与同期和6个月自身血浆作用后,感染正常PBMC,培养7天测定p24抗原浓度,以正常人血浆加病毒悬液为对照。以抑制50%对照孔p24浓度的血浆最高稀释倍数的倒数计算中和抗体滴度,中和抗体滴度≥8倍为具有中和作用。结果：在同期血浆中和自身病毒试验中,3例缓慢进展者（SP）均具有中和作用,HIV组仅4例（4/21）具有中和作用,SP组中和抗体滴度明显高于HIV组。在6个月血浆中和自身病毒试验中,SP组中和抗体滴度明显增加,HIV组12例具有中和作用,SP组中和抗体滴度明显高于HIV组。中和抗体滴度与病毒载量呈明显的负相关。结论：疾病缓慢进展的HIV-1 B’/C亚型感染者对自身病毒中和作用明显高于HIV组,提示中和抗体在延缓疾病进程中发挥重要作用。     

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.     

中国92例HIV/AIDS患者HIV-1 gp41 2F5和4E10中和抗体表位变异研究

目的 探讨92例HIV/AIDS患者HIV-1病毒近膜端(membrane proximal external re-gion,MPER)中和抗体2F5和4E10保守表位ELDKWA、NWFDIT氨基酸变异特点,为中国HIV/AIDS患者免疫治疗以及疫苗设计提供数据.方法 Nest-PCR扩增HIV-1 env区gp41段基因,核酸序列测定,翻译为氨基酸与HIV-1 Sequence Database HXB Ⅱ参考株中和抗体表位数据比对,分析2F5、4E10中和表位氨基酸变异情况.结果 92例HIV/AIDS患者HIV-1外膜蛋白env gp41段中和抗体2F5、4E10保守表位氨基酸均存在突变;2F5中和抗体表位主要有E662A(14.1%)、K665S(17.4%)、A667K(16.3%)突变;4E10中和抗体表位主要有N671S(13.0%)、D674S(3.3%)、T676S(16.3%)突变;CRF_B'C亚型与B'亚型的2F5和4E10表位氨基酸突变差异具有统计学意义(P<0-05);CRF_B'C与CRF01_AE亚型2F5表位突变差异具有统计学意义(P<0.05);B'亚型缓慢进展者、HIV感染者和AIDS患者的4E10表位氨基酸突变差异具有统计学意义(P<0.05).结论 92例HIV/AIDS患者HIV.1包膜蛋白env gp41段中和抗体2F5、4E10中和表位氨基酸存在突变,且变异多样化;不同亚型中和抗体保守表位氨基酸位点变异有差异;B'亚型4E10中和抗体表位变异可能与疾病进展有一定联系.     

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 B'亚型毒株nef基因多态性及其对疾病进展影响的研究

目的 研究我国北方地区B'亚型HIV-1感染者病毒基因组中nef基因多态性、重要功能区的保守程度,探索其与疾病进展的关系.方法 HIV-1 B'亚型感染长期不进展者(LTNP)30例,典型进展者(TP)42例,从全血标本中提取全前病毒DNA,经nested-PCR扩增nef基因全长,扩增产物纯化后直接测序,对测得的序列进行系统进化和氨基酸变异分析,并计算比较LTNP组与TP的HIV/AIDS组氨基酸突变位置和频率的差异.结果 nef氨基酸序列长度可变区R21K/E/H/I/Q取代,TP组突变频率(59.52%)低于LTNP组(93.33%,P<0.005,OR=0.11).功能区外S15R/K/N取代,TP组突变频率(64.29%)高于LTNP组(33.33%,P<0.01,OR=3.60);K39R/E/N取代,TP组突变频率高于LTNP组(P<0.005).其他功能区内有少数序列发生变异,但在两组间无差异.结论 未发现中国北方地区HIV-1 B'亚型nef基因与疾病长期不进展相关联的明显缺失或缺陷,但nef基因序列长度可变区R21位K/E/H/I/Q取代R可能与疾病缓慢进展有关,S15R/K/N取代、K39R/E/N取代可能与疾病进展相关.nfe氨基酸序列的重要功能区较为保守.     

慢性HIV-1感染人群血浆不同中和能力及与有关因素的研究

目的 探讨安徽省既往献血的HIV慢性感染人群血浆中和能力及其与CD4、病毒载量的关系.方法 从安徽省既往献血的294位HIV慢性感染人群静脉抽血,EDTA抗凝后进行血浆和外周血单个核细胞(PBMC)的分离、CD4和病毒载量的检测,采用拥有共享序列的临床分离病毒1597株和实验室株SF33病毒两种病毒分别做血浆的中和试验.用200TCID50病毒感染TZM-bl细胞,通过加入不同稀释度血浆,用Luciferase Assay System检测血浆的中和能力并计算中和50%的病毒进入细胞的血浆浓度(EC50).结果 294位患者血浆针对HIV-1实验株SF33和临床分离株1597的中和能力间差异有统计学意义(P<0.05),针对实验株的中和能力明显高于临床分离株,91%患者血浆对HIV-1实验株SF33具有一定的中和抗体,其中EC50>500的抗体水平较高的血浆占33%,对临床分离株1597的中和抗体阳性率为79%,其中EC50>500的占7%.方差分析显示,血浆中和能力在CIM不同水平之间没有显著差异.294位HIV-1B'亚型感染的血浆病毒载量(1g)平均为4.40(2.60～6.46).方差分析显示,血浆对SF33的中和能力在不同病毒载量水平之间有显著差异,病毒载量不同的各组血浆针对1597病毒的中和能力之间差异无统计学意义.经Spearman's B相关分析,对SF33株的中和水平与病毒载量呈正相关,对1597株的中和水平与病毒载量的相关性没有统计学意义.在病毒载量1g<4的患者中,10.53%血浆样品的EC50高于500;在病毒载量1g≥5的患者中,6.46%血浆样品的EC50高于500.结论 HIV-1慢性感染人群的血浆对实验株SF33的中和能力随着病毒复制能力的提高而增加,但对临床分离株1597的中和能力未见相关性.     

HIV-1感染疾病缓慢进展者CD8+T淋巴细胞非细胞毒性免疫应答作用的研究

目的 探讨HIV-1感染疾病缓慢进展者CD8+T淋巴细胞非细胞毒性抗病毒应答功能(CNAR)的变化.方法 应用密度梯度离心法、免疫磁珠法纯化健康人CD4+T淋巴细胞和HIV感染者CD8+T淋巴细胞,用HIV毒株SF-33感染健康人CD4+T淋巴细胞,并加入不同疾病进程HIV感染者CD8+T淋巴细胞共培养,收集培养上清,应用ELISA方法测定上清中HIV-1 p24含量.结果 我们研究发现缓慢进展组(slow progressors,SP)、HIV典型进展组(typical progressors,TP)、健康对照组及AIDS组中CNAR功能依次下降(89%>77%>73%>61%),各组间的下降差异均有统计学意义(P<0.05);在HIV感染者中,CNAR功能与CD4+T细胞绝对计数呈显著正相关;与病毒载量无显著相关性.结论 CNAR功能对HIV感染疾病不进展可能具有保护作用.     

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。     

32例有偿献血感染者HIV-1膜区序列测定及V3环氨基酸变异特点分析

目的 了解我国有偿献血人群中HIV-1B'亚型流行株的膜蛋白V3环序列特征.方法 巢式PCR扩增前病毒DNAc2-c3区后测序,进行病毒亚型鉴定和V3环序列特点分析.结果 所检的32个HIV-1B'亚型株V3环顶端四肽有五种形式.AIDS组与无症状感染组相比前者呈现更多的T/SI型毒株V3环变异特点.结论 我国有偿献血人群中HIV-1 B'亚型流行株V3环顶端四肽有多种形式.处于病程不同时期的患者体内病毒V3区呈现不同的氨基酸变异特点和电荷积累趋势,提示可能具有不同的生物学表型特征.     

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.     

High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination

Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC₅₀ V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.     

Presence of neutralizing antibodies to heterologous human immunodeficiency virus type 1 isolates in sera of infected individuals is not predictive of rate of disease progression.

These studies were undertaken to examine whether the presence of human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies in sera of infected individuals would alter the rate of disease progression. HIV-1-infected individuals (n = 87) were initially examined for neutralizing activity in vitro against both laboratory and tissue culture-adapted clinical heterologous HIV-1 isolates. The neutralizing activities of sera were determined by a 90% or greater reduction in HIV-1 p24 levels in vitro. In a cross-sectional analysis of all infected individuals, we observed that sera from asymptomatic individuals neutralized a significantly greater number of heterologous HIV-1 isolates than sera from symptomatic patients. Patients who could be followed up longitudinally (n = 24) were then studied to determine the impact of neutralizing antibodies on the rate of disease progression. We observed no significant difference between the numbers of HIV-1 isolates neutralized in vitro by sera from patients who remained clinically stable and by those from patients who progressed rapidly. Our data indicated that the presence or absence of neutralizing antibodies to heterologous HIV-1 isolates was not associated with the rate of disease progression.     

Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens

The genetic diversity of HIV-1 envelope glycoproteins (Env) remains a major obstacle to the development of an antibody-based AIDS vaccine. The present studies examine the breadth and magnitude of neutralizing antibody (NAb) responses in rhesus monkeys after immunization with DNA prime-recombinant adenovirus (rAd) boost vaccines encoding either single or multiple genetically distant Env immunogens, and subsequently challenged with a pathogenic simian-human immunodeficiency virus (SHIV-89.6P). Using a standardized multi-tier panel of reference Env pseudoviruses for NAb assessment, we show that monkeys immunized with a mixture of Env immunogens (clades A, B, and C) exhibited a greater breadth of NAb activity against neutralization-sensitive Tier 1 viruses following both vaccination and challenge compared to monkeys immunized with a single Env immunogen (clade B or C). However, all groups of Env-vaccinated monkeys demonstrated only limited neutralizing activity against Tier 2 pseudoviruses, which are more characteristic of the neutralization sensitivity of circulating HIV-1. Notably, the development of a post-challenge NAb response against SHIV-89.6P was similar in monkeys receiving either clade B, clade C, or clade A+B+C Env immunogens, suggesting cross-clade priming of NAb responses. In addition, vaccines encoding Env immunogens heterologous to SHIV-89.6P primed for a rapid anamnestic NAb response following infection compared to vaccines lacking an Env component. These results show that DNA/rAd immunization with multiple diverse Env immunogens is a viable approach for enhancing the breadth of NAb responses against HIV-1, and suggest that Env immunogens can prime for anamnestic NAb responses against a heterologous challenge virus.     

Extensive cross-reactive neutralizing antibody response in Indian patients with limited genetic diversity of HIV-1

Genome sequence analysis of HIV-1 subtype C viruses from India shows monophyletic lineage and relatively limited genetic diversity. To understand its immunological implication, cross-reactivity of neutralizing antibody response was investigated. In primary screening, neutralizing antibody response to single heterologous primary HIV-1 subtype C isolate was assessed in plasma samples from 235 HIV-1 infected, anti-retroviral treatment naive individuals from Pune, India. Plasma samples that showed > or =90% neutralization and two randomly selected plasma samples that showed 50-60% neutralization were tested against a panel of primary HIV-1 subtype C isolates obtained from epidemiologically unlinked individuals from India. The neutralizing antibody response showed extensive cross-neutralization, suggesting presence of shared neutralization determinants among circulating HIV-1 subtype C viruses in India.     

Vertical HIV-1 transmission: importance of neutralizing antibody titer and specificity

Neutralization analyses were carried out with plasma from 132 volunteer human immunodeficiency virus (HIV)-1 infected women (76% pregnant, 24% with infants suspected for HIV-1 infection) collected between 1994 and 1998, against autologous and heterologous primary- and the reference HIV-1 MN isolates. A significantly lower percentage of HIV-1 transmissions was observed after 1996, parallel to a more intense antiretroviral treatment of infected pregnant women. HIV-1 isolation was significantly more frequent from peripheral blood mononuclear cells of mothers of infected children than mothers of uninfected children (P = 0.0065). Neutralization of autologous HIV-1 isolates was comparable for HIV-1 transmitters and nontransmitters' plasma, whereas neutralization of the reference isolate HIV-1 MN was more frequent at high titers for pregnant women who did not transmit HIV to their offspring compared to pregnant women who did. Although neutralization of heterologous primary HIV-1 isolates from HIV transmitters and non transmitters by transmitter plasma occurred with similar frequency, neutralization of isolates from transmitters was much more frequent when heterologous plasma from nontransmitters were used. Macrophage-tropic heterologous HIV-1 isolates were neutralized more frequently at higher titers by plasma from nontransmitters than from transmitters. The results obtained indicate that antiretroviral treatment, lack of success of HIV-1 isolation and high titers of antibodies able to neutralize macrophage-tropic viruses appear to be of importance for protection against HIV-1 vertical transmission for the group of patients studied.     

Antibody-dependent cellular cytotoxicity and neutralizing activity in sera of HIV-1-infected mothers and their children.

The prognostic and protective role of antibodies mediating cellular cytotoxicity (ADCC) and neutralization was evaluated in sera of HIV-1-infected mothers and their consecutively followed children. The presence and titres of ADCC mediating and/or neutralizing antibodies in maternal sera did not predict HIV-1 infection in their respective children. No significant difference in the sera from the children was seen when comparing the presence of neutralizing antibodies between the uninfected and infected children. Stratification of the infected group according to clinical status revealed differences. Only one of 24 AIDS patients had a high neutralizing titre against IIIB. Four patients had a very low titre and the remaining had no detectable neutralizing antibodies at all. In contrast, 10/17 infected non-AIDS children had neutralizing antibodies. Similarly, no significant difference was seen when comparing the presence of ADCC-mediating antibodies between the uninfected and the infected group of children. However, a significantly higher frequency of ADCC was seen in the seropositive non-AIDS children compared with the AIDS children. This study clearly shows that the presence of antibodies mediating ADCC and neutralization in infected children, 0-2 years old, is associated with a better clinical status and delayed disease progression.     

Virologic,immunologic, and clinical follow-up of a couple infected by the human immunodeficiency virus type one,group O

The pathogenic course (virologic, immunologic, and clinical changes) of infection due to human immunodeficiency virus type one (HIV-1) group O viruses is unknown at present. To address this issue, serial HIV-1 isolates from a married couple (patients A and B) infected with a group O virus were analyzed to determine the temporal association between disease status and alterations in several parameters including plasma viral burden as measured by semiquantitative polymerase chain reaction, changes in CD4+ T cells, presence of neutralizing antibodies, and the ability to induce syncytia on the MT2 cells. For patient A who has been asymptomatic for at least 8 years, both the absence of syncytium-inducing (SI) variants and the presence of autologous and heterologous neutralizing antibodies correlated with a clinically healthier status. In contrast, a switch from NSI to SI variants was observed in patient B in 1990, followed by an expanded in vitro host range, increased viral burden, and a sharp decrease in CD4+ T cells 4 years later. Moreover, plasma obtained from this patient uniformly failed to neutralize both autologous and heterologous viruses. These observations in patient B correlated with a slightly unfavorable clinical status. Based on our preliminary results, it appears that the pathogenic course of infections due to group O viruses is similar to that reported previously for infections due to group M viruses. J. Med. Virol. 51:202–209, 1997. © 1997 Wiley-Liss, Inc.     

HIV-1自然感染中的中和抗体反应

中和抗体(Nab)可以防止I型人类免疫缺陷病毒(HIV-1)侵入靶细胞.HIV-1感染数周后即可诱导产生Nab,这些早期抗体只能特异性地中和自体病毒但不能中和异源性病毒.在一些慢性感染者体内则可以检测到可同时中和同源性和异源性病毒的广谱中和抗体(BNab).BNab的靶点通常位于包膜蛋白的保守区域.HIV-1 BNab的产生还受到病毒变异及结构遮盖等因素的限制,同时Nab的中和广度与病毒载量具有相关性.     

中国HIV/AIDS患者保守表位的2F5抗体与疾病进展关系

目的 研究中国HIV/AIDS患者gp41保守表位中和抗体2F5与疾病进程的关系.方法 应用合成4(ELDKWA)-C24肽及gp41抗原肽包被96孔酶标板,ELISA方法检测特异性抗体水平.提取正常人外周血单个核细胞(PBMC),应用HIV-1 SF33毒株进行病毒多轮感染PBMC中和试验.结果 缓慢进展者(slow progressor,SP)的2F5样中和抗体水平显著高于典型进展者(typical progressor,TP)(P<0.05);2F5样中和抗体水平与gp41特异性抗体水平正相关(r=0.406,P<0.05).结论 中国HIV.-缓慢进展者体内具有高水平的2F5样抗体,2F5抗体可能是疾病不进展的保护性因素之一.