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HIV/AIDS典型进展者与疾病长期不进展者中和抗体保守表位氨基酸变异比较研究

目的 研究中国HIV/AIDS典型进展者及疾病长期不进展者(LTNP)中和抗体保守表位氨基酸变异,探讨中和抗体表位变异与疾病进展关系,为开展中和抗体免疫治疗和疫苗设计奠定理论基础.方法 RT-PCR及巢式PCR扩增HIV/AIDS典型进展者及LTNP的HIV-1 gp120 C2～C3区基因,双脱氧终止法进行核酸序列测定,翻译为氨基酸序列,与HIV-1 Sequence Database参考毒株比对识别中和抗体保守表位氨基酸变异.结果 HIV/AIDS典型进展者CD4结合位点(CD4BS)、CD4诱导(CD4i)、2G12中和抗体保守表位氨基酸均存在变异,LTNP 2G12中和抗体保守表位氨基酸存在变异;LTNP各表位突变率较典型进展的HIV感染者/AIDS患者有降低趋势,但差异无统计学意义(P＞0.05);CD4BS、CD4i、2G12变异表位构成比分别为25.0%、22.9%、52.1%,2G12表位变异明显高于CD4BS和CD4i表位,差异有统计学意义(P＜0.01);CD4BS和CD4i保守表位变异多见于E370Q(10.8%),2G12保守表位变异多见于N295V(18.9%)和T297I(9.5%).结论 中国HIV感染人群中,LTNP中和抗体保守表位氨基酸构成相对稳定,变异较典型进展的HIV感染者/AIDS患者少见,目前发现2G12中和抗体表位存在较低水平变异.中和抗体表位中,2G12表位变异较CD4BS和CD4i表位多见,各类型中和抗体保守表位氨基酸位点的变异程度存在差异.     

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。     

基于gp41的HIV亚单位疫苗研究进展

艾滋病疫苗是当今时代最具挑战性的科学难题之一,现有的以诱导体液免疫为目的的HIV-1抗原均难以诱导产生有效的、持续的广谱中和抗体.近年来,一些具有广谱中和活性的HIV单克隆抗体(mAb)的发现及其相应抗原表位的阐明,给以诱导HIV体液免疫的疫苗研究带来了新的希望.相比于gp120,HⅣgp41的序列更为保守,糖基化位点更少,更适合作为疫苗的抗原.本文综述了靶向gp41的广谱中和抗体及其抗原表位,并阐述了对目前国际上基于gp41的HⅣ亚单位疫苗设计思路及进展.     

艾滋病毒的中和性单克隆抗体

自1985年报告HIV感染患者血清中存在阻止艾滋病毒感染的中和抗体以来,对其确切的作用尚不清楚。中和抗体的作用靶点是HIV被膜蛋白gp120,其通过病毒表面或病毒感染细胞表面的gp41的中介作用,在病毒的病原性和感染性上起着重要作用。作者已制备出与gp120反应的HIV中和性单克隆抗体,本文对其性质和今后应用做一介绍,     

HIV抗体纳米免疫磁珠快速检测试剂的研制

目的 研制一种用纳米免疫磁珠层析技术用以检测HIV抗体的快速诊断试剂.方法 运用碳二亚(EDC)将重组的HIV抗原gp41、gp36偶联到200 nm的超顺磁纳米颗粒上,在硝酸纤维素(NC)膜上包被gp41、gp36抗原,制备成免疫层析检测卡,然后对检测卡进行性能分析评估.结果 对HIV抗体国家参考品血清盘(胶体金类)检测,符合要求;对20份HIV抗体阳性和600份抗体阴性临床血清检测,灵敏度为100%,特异性为98.5%.检测卡室温保存12个月性能稳定.结论 研制出一种纳米免疫磁珠HIV抗体检测试剂,具有检测简便、快速、稳定性好和适于现场检测等特点.     

HIV抗体纳米免疫磁珠快速检测试剂的研制

目的 研制一种用纳米免疫磁珠层析技术用以检测HIV抗体的快速诊断试剂.方法 运用碳二亚(EDC)将重组的HIV抗原gp41、gp36偶联到200 nm的超顺磁纳米颗粒上,在硝酸纤维素(NC)膜上包被gp41、gp36抗原,制备成免疫层析检测卡,然后对检测卡进行性能分析评估.结果 对HIV抗体国家参考品血清盘(胶体金类)检测,符合要求;对20份HIV抗体阳性和600份抗体阴性临床血清检测,灵敏度为100%,特异性为98.5%.检测卡室温保存12个月性能稳定.结论 研制出一种纳米免疫磁珠HIV抗体检测试剂,具有检测简便、快速、稳定性好和适于现场检测等特点.     

人类免疫缺陷病毒-1 gp41免疫原设计、免疫策略及免疫效果的研究进展

人类免疫缺陷病毒(human immunodeficiency virus type,HIV-1)包膜蛋白gp41序列相对保守,其近膜区(MPER)具有一些已知广谱中和抗体(bNAbs)的表位.在HIV疫苗研究中,尽管利用gp41的MPER中和表位进行疫苗设计,但其免疫效果仍不理想.近年发现,一些靶向于gp120和gp41交界面的bNAbs为HIV疫苗的研制提供了新的靶标.如何更好的模拟天然状态的gp41结构以诱导bNAbs的产生仍是学者们的主要研究方向,有大量研究关于靶向于gp41的bNAbs的表位、gp41免疫原的修饰、免疫策略及免疫效果等,因而探讨gp41作为免疫原候选的可行性及提高其免疫效果的方法具有重要意义.     

在鸡痘病毒中共表达HIV-1 gp120与IL-2基因

外膜蛋白gp120是HIV—1主要结构蛋白之一，它的V3区存在着中和抗体决定簇、CTL决定簇，以及对巨噬细胞和脑组织纤维特异性识别的区，与HIV—1可诱导中和抗体应答、特异性细胞毒反应及分子致病机制密切相关。针对V3环的抗体可干扰gp120和CCR5之间的结合，这一发现为艾滋病的预防和治疗提供了新的途径。现已证明，多数细胞因子具有良好的免疫佐剂性能，对免疫应答具有刺激作用。本研究拟利用我国鸡痘病毒疫苗株为载体，构建可共表达HIV-1 gp120与IL-2基因的重组鸡痘病毒载体，为开发HIV重组病毒活载体疫苗提供一种安全的新途径。     

Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope

Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 is targeted by broadly-reactive neutralizing antibodies 2F5 and 4E10, making it an attractive target for vaccine development. To better assess immunogenic properties of gp41, we generated five soluble glutathione S-transferase fusion proteins encompassing C-terminal 30, 64, 100, 142, or 172 (full-length) amino acids of gp41 ectodomain from M group consensus envelope sequence. Antibody responses in HIV-1-infected patients were evaluated using these proteins and overlapping peptides. We found (i) antibody responses against different regions of gp41 varied tremendously among individual patients, (ii) patients with stronger antibody responses against membrane-proximal external region exhibit broader and more potent neutralizing activity, and (iii) several patients mounted antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10. These soluble gp41 fusion proteins could be an important source of antigens for future vaccine development efforts.     

Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1

Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.     

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-β in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-β (aa128–134) and HIV-1 gp41 (aa586–595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-β antibody levels by ELISA. The levels of anti-IFN-β antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-β in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-β recognized rsgp41 (recombinant soluble gp41, Env amino acid 539–684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-β and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128–134 of human IFN-β and the immunosuppressive domain (aa583–599) of HIV-1 gp41. The increased levels of antibodies against interferon-β in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-β and HIV-1 gp41.     

Rational Design of Membrane Proximal External Region Lipopeptides Containing Chemical Modifications for HIV-1 Vaccination

The inability to generate broadly neutralizing antibody (bnAb) responses to the membrane proximal external region (MPER) of HIV-1 gp41 using current vaccine strategies has hampered efforts to prevent the spread of HIV. To address this challenge, we investigated a novel hypothesis to help improve the anti-MPER antibody response. Guided by structural insights and the unique lipid reactivity of anti-MPER bnAbs, we considered whether amino acid side chain modifications that emulate hydrophilic phospholipid head groups could contribute to the generation of 2F5-like or 4E10-like neutralizing anti-MPER antibodies. To test this hypothesis, we generated a series of chemically modified MPER immunogens through derivatization of amino acid side chains with phosphate or nitrate groups. We evaluated the binding affinity of the chemically modified peptides to their cognate monoclonal antibodies, 2F5 and 4E10, using surface plasmon resonance. The modifications had little effect on binding to the antibodies and did not influence epitope secondary structure when presented in liposomes. We selected five of the chemically modified sequences to immunize rabbits and found that an immunogen containing both the 2F5 and 4E10 epitopes and a phosphorylated threonine at T676 elicited the highest anti-peptide IgG titers, although the high antipeptide titers did not confer higher neutralizing activity. These data indicate that side chain modifications adjacent to known neutralizing antibody epitopes are capable of eliciting antibody responses to the MPER but that these chemically modified gp41 epitopes do not induce neutralizing antibodies.     

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.     

HIV-1 gp41 and type I interferon

HIV-1 gp41-like human type I interferon (IFN) could inhibit lymphocyte proliferation and up-modulate MHC class I and II and ICAM-1 molecule expression. Sequence comparison indicates that a similar epitope RILAV-YLKD exists between N-domain of gp41 and two regions in IFN-alpha(aa29-35 and 113-129), IFN-beta (aa31-37 and 125-138) and IFN-omega (aa29-35 and 123-136), which was shown to form IFN-alpha/beta-receptor binding site. Weak sequence similarity was also found to exist in both regions on gp41 and type I IFN of murine and bovine. Experimental studies indicated that a common immunological epitope exists between gp41 and IFN-alpha and -beta. Antibodies against human IFN-alpha and -beta recognized the common immunological epitope and inhibited gp41-binding to the potential cellular receptor protein p45. Moreover, the polyclonal antibody to IFN-beta completely inhibited gp41-binding to human T, B cells and monocytic cells, while IFN-alpha could only inhibit this binding incompletely. It was interestingly observed that human IFN-beta after preincubating with cells could incompletely inhibit the binding of gp41 to human B cells and monocytic cells, and very weakly inhibit the binding to human T cells, indicating that the receptor for IFN-beta-binding may be involved in gp41 binding. This potential relationship may be based on the amino acid sequence homology in the receptor binding region between gp41 and IFN-beta. It was observed that the increased levels of antibodies against human IFN-alpha and -beta exist in HIV-1-infected individuals and are associated with the common epitope on gp41. Besides, several studies provided experimental evidence that the common immunological epitope could induce protective activity against HIV-1. The IFN-alpha-based vaccine has showed a significant reduction of disease progression in IFN-alpha-vaccine-treated HIV-infected patients. Recent experimental evidence indicates that gp41 and IFN-beta were involved in downregulation of CCR5 expression and induction of cell activation or signal transduction. Whether it may be performed by a similar mechanism is still to be investigated.     

Maternal antibodies to HIV-1 envelope domains: No correlation with HIV-1 vertical transmission in patients from Argentina

The role of the maternal antibody response in relation to vertical human immunodeficiency virus type 1 (HIV-1) transmission was investigated in HIV-1-infected mothers from Argentina. Sera from 23 transmitting and 18 nontransmitting HIV-1-infected mothers were tested for the presence of antibodies to V3 loop gp120 peptides representing both Argentinian sequences and several well-characterized viral isolates from different geographic areas. Argentinian sera from transmitting mothers had significantly higher capacity to react with four of 14 V3 loop peptides tested than sera from nontransmitting mothers. Frequency of reactivity against the other peptides did not differ between the two maternal groups. Furthermore, no differences in antibody affinity were found between transmitting and nontransmitting mothers. Sera were also tested against overlapping peptides covering a neutralizing epitope of the HIV-1 MN gp41 (amino acids 648-677). Statistical analysis indicated that no correlation between anti-gp41 antibodies and vertical transmission exists. Although we used V3 loop peptides based on local HIV-1 sequences, our data showed that maternal antibodies to these peptides, as well as to gp41 peptides, are not correlated with protection against HIV-1 vertical transmission.     

Epitope-vaccines: a new strategy to induce high levels of neutralizing antibodies against HIV-1

Based on the experimental evidence that gp120 subunit vaccine did not protect individuals from HIV-1 infection, we suggested that epitope-vaccines of HIV-1 gp41 may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, and characterised immunogenicity of epitope-vaccines. Two epitopes, RILAVERYLKD-epitope (aa586-596) on the N-domain and ELDKWA-epitope (aa669-674) on the C-domain of gp41, were demonstrated by us and others to induce protective activity. After vaccination course, the RILAVERYLKD-dimer epitope-vaccine [C(RILAVERYLKDG)2-BSA] induced strong epitope-specific antibody response by about 1:25,600 dilution, and the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAG)4-BSA] could yet induce strong antibody response to ELDKWA-epitope by 1:12,800-25,600 dilution of antisera in mice, while rgp41 subunit vaccine induced very weak antibody response to both epitopes (1:400). In rabbit experiments, the titres of ELDKWA-epitope-specific antibody induced by ELDKWA-epitope-vaccine [C-(ELDKWAG)4-BSA] reached to 1:6,400, while rgp41 subunit vaccine induced very weak antibody response to this epitope and to P1 and P2 peptides (1:400). Moreover, the ELDKWA-epitope-specific antibodies in mice and rabbit antisera induced by epitope-vaccine could very strongly interact with P2 peptide sequence-corresponding to the C-domain of gp41 (dilution by 1:25,600), and the RILAVERYLKD-epitope-specific antibodies in mice antisera induced by epitope-vaccine could also very strongly interact with P1 peptide sequence-corresponding to the N-domain of gp41 (dilution by 1:102,400). All these results provided experimental evidence that epitope-vaccine may be a new general strategy to induce high levels of neutralizing antibodies against HIV-1 or other viruses.     

Induction of high levels of antibodies recognizing the neutralizing epitope ELDKWA and the D- or K-position-mutated epitopes by candidate epitope vaccines against HIV-1

Monoclonal antibody 2F5 recognizing the ELDKWA epitope on HIV-1 gp41 has a significant neutralization potency against 90% of the investigated viruses of African, Asian, American, and European strains, but the antibody responses to the epitope 2F5 in HIV-1-infected individuals were very low. We attempted to induce high levels of epitope-specific antibodies to ELDKWA and its three mutated epitopes by candidate epitope vaccines. The four candidate epitope vaccines all induced strong antibody responses at dilutions from about 1:6,400 to 1:25,600. We tested the cross-reactions between these antisera and four epitope peptides. The ELDKWA-specific antisera showed strong cross-reactivity with three neutralizing-resistant mutated epitopes which contain changes in the D or K positions of the epitope sequence. Virus variants containing these changes could escape neutralization by monoclonal antibody 2F5. In immunoblotting analysis, the ELDKWA, ELDEWA, and ELEKWA epitope specific antibodies all recognized rsgp41 which confirms that the antibodies against both mutated epitopes, ELDEWA and ELEKWA, could cross-react with the native epitope on rsgp41. Although it is not clear whether the polyclonal antibodies induced by the ELDKWA epitope vaccine could neutralize the mutated viruses containing these mutated epitopes, it is conceivable that epitope vaccines based on mutated epitopes could induce strong antibody responses with predefined epitope specificity to neutralize mutated viruse containing the mutated epitope. An epitope vaccine, using different epitopes including mutated epitopes, could provide a new concept for developing a new vaccine against HIV-1.