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.     

A Neutralizing Antibody Recognizing Primarily N-Linked Glycan Targets the Silent Face of the HIV Envelope

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Antibodies of HIV-1 positive subjects and experimentally immunized primates and rodents bind to sequence divergent regions of the third variable domain (V3) of gp120.

Several motifs have been found to be the target of the neutralizing antibody response to HIV, the human immunodeficiency virus. One of the well characterized motifs maps to a loop within the third hypervariable region (V3) of the exterior envelope glycoprotein gp120 at amino acid positions 308-331 and is referred to as the principal neutralizing determinant (PND). The sequence of this V3 loop raises the question of the immunogenicity and the degree of diversity of the antibody response to the PND. We show here that this neutralization-related motif is highly immunogenic in HIV-positive subjects and in experimentally immunized primates and rodents submitted to various anti-HIV immunization regimens. In probing the diversity of the antibody response to PNDs corresponding to 11 HIV sequence-divergent isolates in serum samples of 101 HIV-positive individuals we found that human antibodies exhibit binding affinity to up to nine PND synthetic peptides. This antibody binding was in all cases tested inhibitable by the homologous PND synthetic peptide. We additionally demonstrate that this antibody cross-reactivity towards sequence-divergent PNDs is detectable in the sera of mice and chimpanzees experimentally immunized against a single HIV-1 isolate. Finally, we noticed that there is a hierarchy of reactivity among the various PNDs wherein the synthetic peptide corresponding to the MN isolate was generally the most prominently recognized by antibodies of human, non-human primate, and rodent origins. Based on these findings and on features of the sequences analyzed we suggest that, despite its overall sequence variability, the PND encompasses conserved amino acid positions or epitopes that are the targets of antibodies recognizing sequence-divergent isolates. We also propose that the high positive charge density of the most frequently recognized PNDs and the high antigenicity value of some of their residues are critical to the broad immunoreactivity of this neutralization-related motif.     

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization

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三种初筛方法检测HIV抗体的结果比较及评估

目的比较酶联免疫吸附试验（ELISA）、明胶颗粒凝集试验（PA）、胶体硒免疫层析法（ICA）检测人类免疫缺陷病毒抗体的结果，了解这三种方法的优劣，探讨临床实验室初筛诊断HIV方法的灵敏性和准确性。方法用卫生部HIV质控血清和免疫印迹法（WB）确认的HIV抗体阳性标本和阴性标本。分别用ELISA法、PA法及ICA法检测；用免疫印迹法确认的HIV阳性的标本作倍比稀释后，再分别用PA法、ELISA法及ICA法检测。结果80例经免疫印迹法确认的HIV阳性患者，ELISA法、PA法、ICA法的检出符合率分别为100％、98．8％、97．5％；经稀释法检测。ELISA法较PA法和ICA法敏感。其检测的准确性分别为97．5％、97．5％、92．5％。结论筛查HIV抗体方法最好选用ELISA法或PA法。以防漏检。     

Neutralizing antibodies mechanism of neutralization and protective activity against HIV-1

The role of the humoral immune response in prevention against HIV-1 infection is still incompletely understood. However, neutralizing antibodies to certain epitopes on HIV-1 envelope glycoproteins inhibit HIV-1 infection in vitro and in vivo. Passive administration of these antibodies by themselvesor in combination completely protected hu-PBL-SCID mice or macaques from intravenous, vaginal, as well as maternal-fetal mucosal transmission. All these studies provide direct experimental evidence that neutralizing antibodies are potentenough to prevent HIV infection, and strongly suggest that neutralizing-antibody-based vaccines could provide effective protection against HIV-1, despite the potent action of CTLs. Some neutralizing epitopes have been defined in vitro and in vivo. Unfortunately, none of the neutralizing-antibody-based candidate vaccines has been demonstrated to induce enough protective activity. Weak antigenicity and immunogenicity of neutralizing epitopes on native or recombinant proteins and other factors made it difficult to induce neutralizing-epitope-specific antibody responses in vivo enough to prevent against primary isolates. Recent studies indicated that HIV-1 variations resulted in escape from neutralization or the CTL responses, which may be the principal challenge for HIV-1 prevention. Epitope vaccine as a new strategy activating both arms of the immune system, namely, using the “principal neutralizing epitopes” and the CTL epitopes in combination, should provide new hope for developing an effective vaccine to halt the HIV-1 epidemic.     

Differential functional phenotypes of two primary HIV-1 strains resulting from homologous point mutations in the LLP domains of the envelope gp41 intracytoplasmic domain

We previously reported that selected mutations of highly conserved arginine residues within the LLP regions of HIV-1(ME46) gp41 had diverse effects on Env function. In the current study, we sought to test if the observed LLP mutant phenotypes would be similar in HIV-1(89.6). The results of the current studies revealed that the LLP-1 mutations conferred reduced Env incorporation, infectivity, and replication phenotypes in both viruses, while homologous LLP-2 mutations had differential phenotypical effects between the two strains. In particular, several of the 89.6 LLP-2 mutant viruses were replication defective in CEMX174 cells despite having increased levels of Env incorporation, and with both strains, there were differential effects on infectivity. This comparison of homologous point mutations in two different strains of HIV supports the role of LLPs as determinants of Env function, but reveals for the first time the influence of virus strain on LLP mutant phenotypes.     

A Phase I Study of the Pharmacokinetics and Safety of Passive Immunotherapy with Caprine Anti-HIV Antibodies,PEHRG214, in HIV-1--Infected Individuals

Abstract

Purpose: To establish the pharmacokinetics and safety of single-dose polyclonal caprine anti-HIV antibodies (^PEHRG214)in HIV-1--infected individuals. Design: A phase 1, open-label, nonrandomized, dose-escalating study. Method: HIV-1--infected patients with CD4+ T-cell counts of 200 cells/µL and plasma HIV viral load (VL)of 5,000 copies/mL received a single intravenous dose of HRG. Dosing began at 6,000 U/kg HRG with proposed step-wise escalation to 96,000 U/kg. Results: Eleven males were enrolled; median CD4+T-cell count and VL were 96 cells/µL and 126,200 copies/mL, respectively. HRG exhibited linear pharmacokinetics across the dosing range studied. The mean terminal elimination half-life (t_1/2)was 136.6 ± 44.6 hours (range, 52.6-198 h).Serum sickness occurred in one 48,000 U/kg HRG recipient. One 6,000 U/kg and two 24,000 U/kg HRG recipients developed a mild rash. Between baseline and day 60,VL remained unchanged (n = 6), increased by 0.67 log₁₀ copies/mL (n = 1), or declined by 0.34-1.55 log₁₀ copies/mL (n = 4). Conclusion: Single-dose HRG exhibited linear kinetics and a long half-life. Although numbers in each dosing group were very small (n = 3), HRG was generally well tolerated in doses below 48,000 U/kg. Multiple dosing with HRG in the HIV-salvage setting may be complicated by immune-complex formation.     

四川省肿瘤医院2005~2018年肿瘤住院患者HIV抗体筛查结果分析

目的 了解四川省肿瘤医院2005~2018年肿瘤住院患者HIV的感染状况和流行特征。方法 采用酶联免疫吸附（ELISA）法对我院237472例住院患者进行HIV抗体检测,同时对HIV抗体阳性患者的临床资料进行回顾性分析。结果 住院肿瘤患者HIV抗体的总体阳性率为1.42‰（338/237472）,其中男性患者HIV抗体阳性检出率为2.18‰（244/111885）,女性患者HIV抗体阳性检出率为0.75‰（94/125587）,性别差异有统计学意义（P＜0.05）;年龄组以21~30岁检出率最高2.18‰;病种以头颈病区的头颈部肿瘤所占构成比最大21.89%;职业分布较广,且以农村居民为主（48.57%）。结论 住院肿瘤患者HIV感染例数呈逐年上升趋势,以中老年男性为主,并存在病区、病种、职业构成差异的特点,为确保医患安全和防止院内感染,应对住院肿瘤患者HIV进行长期监测。     

An Antigenic Atlas of HIV-1 Escape from Broadly Neutralizing Antibodies Distinguishes Functional and Structural Epitopes

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Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.     

Characterization of human IgG repertoires in an acute HIV-1 infection

All known broadly neutralizing antibodies (bnAbs) are highly somatically mutated and therefore significantly differ from their germline predecessors. Thus although the mature bnAbs bind to conserved epitopes of the HIV-1 envelope glycoprotein (Env) with high affinity their germline predecessors do not or weakly bind Envs failing to initiate an effective immune response. The identification of less somatically mutated bnAbs and/or antibody maturation intermediates that are clonally related to bnAbs may be useful to circumvent the major problem of initiating immune responses leading to elicitation of bnAbs. Here, we describe the identification of IgG antibodies from an acutely HIV-1-infected patient using a combination of phage display and high-throughput sequencing. We found two antibodies with only a single point mutation in the V region of their heavy chain variable domains compared to their putative germline predecessors which bound with high affinity to several Envs. They targeted the Env gp41 and did not neutralize HIV-1. Using high-throughput sequencing, we identified several highly abundant CDR3s, germline-like as well as somatically mutated V genes in the VH/VL repertoires of the patient which may provide antibody intermediates corresponding to known bnAbs as templates for design of novel HIV-1 vaccine immunogens.     

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.     

共表达HIV-1 gag-gp120与白细胞介素6重组鸡痘病毒的筛选

目的构建共表达中国株HIV1gaggp120与白细胞介素6(IL6)的重组鸡痘病毒(FPV)。方法分别将HIV1gaggp120基因和IL6基因插入到FPV表达质粒pUTAL复合启动子和单一启动子下游,构建重组FPV表达质粒pUTAGEIL6。利用脂质体法将重组质粒和FPV282E4株共转染鸡胚成纤维细胞,经BUdR加压筛选,重组病毒分别用SDSPAGE和WesternBolt进行鉴定,观察病毒样粒子的形成和重组病毒在哺乳动物细胞中的表达,并分析其免疫原性。结果阳性重组病毒基因组点膜处有显色斑点,WesternBolt结果显示重组病毒表达了gaggp120嵌合蛋白和IL6,在病毒感染细胞中有反转录病毒样粒子形成,且重组病毒可在哺乳动物细胞中表达目的蛋白。小鼠免疫指标检测表明该重组病毒具有很好的免疫原性。结论成功构建了共表达中国株HIV1gaggp120与IL6的重组FPV,为HIV-1基因工程活载体疫苗和巨分子颗粒化疫苗的制备奠定了基础。     

中国株HIV-1外膜蛋白基因疫苗诱导免疫小鼠免疫应答的实验研究

目的 :构建中国流行株HIV 1外膜蛋白 (gp12 0 )基因疫苗并接种小鼠 ,评价其诱导的体液和细胞免疫应答。方法 :将HIV 1gp12 0基因插入到真核表达载体pVAX1中 ,构建重组真核表达质粒pVAX1 GP12 0 ,并经EcoRI和PstI双酶切以及测序鉴定。同时以pVAX1 GP12 0和空载体pVAX1分别免疫BALB/c小鼠 ,采用ELISA检测免疫小鼠的特异性抗体和IFN γ水平 ,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖 ,用乳酸脱氢酶 (LDH)试验检测小鼠特异性细胞毒性T淋巴细胞 (CTL)的应答。结果 :酶切及测序结果表明 ,成功地构建了HIV 1gp12 0基因疫苗。与空载体pVAX1组相比较 ,pVAX1 GP12 0免疫组小鼠血清抗HIV 1gp12 0抗体的滴度和IFN γ的水平均升高 ,两者差异显著 (P <0 .0 1)。pVAX1 GP12 0免疫组小鼠脾淋巴细胞增殖的刺激指数 (SI)及特异性CTL的杀伤活性 ,均高于空载体pVAX1组 (P <0 .0 1)。结论 :构建了针对我国HIV 1流行株的gp12 0基因疫苗。以其免疫BALB/c小鼠可诱导特异性体液和细胞免疫应答 ,为进一步将其用于我国的HIV的治疗奠定了基础。     

Cell-surface-expressed HIV-1 envelope induces the death of CD4 T cells during GP41-mediated hemifusion-like events

Cells expressing the HIV-1 envelope glycoprotein complex (gp120/gp41, Env) induce the death of target cells either after cell-to-cell fusion or after cell-to-cell contact in a fusion-independent fashion. Here, we demonstrate that Env-induced death of single cells (including primary CD4 T cells) required gp120 and gp41 function. The gp41 peptide C34, which blocked syncytium formation, completely inhibited the death of single target cells by specifically acting on gp41 function. Moreover, Env-induced single cell death was exclusively observed in CD4 cells and was associated with specific gp41-mediated transfer of lipids from the membrane of Env-expressing cells to the target cell but not with detectable cytoplasm mixing (complete fusion). We conclude that after gp120 function, gp41 mediates close cell-to-cell contacts, thereby triggering cell death in single uninfected cells in the absence of detectable cell-to-cell fusion.     

携带中国株HIV-1 gp120基因的重组腺病毒的构建及其感染巨噬细胞的形态学研究

目的: 构建携带中国株HIV-1 gp120基因并可感染小鼠骨髓来源巨噬细胞(BMM)的重组腺病毒。方法: 利用AdMax腺病毒构建系统,以双质粒共转染人胚肾转化细胞293Ad5⁺细胞,完成重组腺病毒载体包装,并进一步扩增和纯化。通过ELISA测定gp120的蛋白产量,以确认目的基因重组与表达成功。以Karber法对病毒进 行TCID₅₀滴度测定,利用BMM进行感染复数(MOI)流式细胞术测定,并利用荧光显微镜观察细胞活化形态。结果: 成功构建AdMax-HIV-1 gp120(简称Ad-gp120)及其对照病毒(Ad-GFP),其滴度分别为10^8.3和10^8.1 TCID₅₀/mL。这些病毒可感染BMM,MOI测定结果表明2种重组腺病毒感染BMM和293Ad5⁺细胞的能力接近,验证了上述TCID₅₀滴度结果。Ad-gp120可在293Ad5⁺细胞中表达gp120蛋白,并可感染和诱导BMM相关形态改变。结论: 成功构建Ad-gp120,其感染可致BMM出现形态发生改变。     

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviralna?ve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.     

HIV-1 gp120蛋白对人血-视网膜屏障细胞的毒性作用

目的 探索HIV-1 gp120蛋白侵犯人血.视网膜屏障的机制.方法 原代培养人血-视网膜屏障细胞(HBRBC),包括人视网膜微血管内皮细胞(HRCEC)、人视网膜微血管周细胞(HRCPC)、人视网膜色素上皮细胞(HRPE),以培养基作为对照.用MTT法观察7种不同浓度(0.01～0.15 mg/L)的HIV-1 gp120蛋白作用24 h和0.08 mg/L HIV-1 gp120蛋白作用不同时间(4～72 h)对3种细胞的生长抑制作用.0.08、0.1、0.12、0.15 mg/L的HIV-1 gp120蛋白作用24 h后,用流式细胞仪检测其对3种细胞凋亡率和线粒体膜电位(△ψm)的影响;用Western免疫印迹检测Cleaved cagpase-9蛋白的活化情况.用透射电镜观察经0.08 mg/L HIV-1 gp120蛋白处理24 h前后3种细胞超微结构的变化.结果 HIV-1gp120蛋白作用24 h,低浓度(<0.08mg/L)对3种细胞的活性均没有明显影响,而当浓度超过0.08mg/L时,对细胞的增殖活性有明显的抑制作用,呈浓度依赖性(HRCEC:r=-0.763,P<0.01;HRCPC:r=-0.804,P<0.01;HRPE:r=-0.698,P<0.01).HIV-1 gp120蛋白(0.08mg/L)作用12 h即可显著抑制细胞的增殖活性.24、48和72 h抑制效应更明显,相对增殖率分别为HRCEC:84%、70%、41%、22%,HRCPC:80%、69%、38%、18%,HRPE:86%、73%、45%、26%,抑制效应呈时间依赖性(HRCEC:r=0.833.P<0.01;HRCPC:r=-0.784,P<0.01;HRPE:r=-0.701,P<0.01).HIV-1 gp120蛋白作用24 h后与对照组相比,各浓度组3种细胞凋亡率增加、△ψm明显降低以及Cleaved cagpase-9蛋白表达增强,均呈浓度依赖性.透射电镜示0.08mg/LHIV-1 gp120蛋白处理24h后,3种细胞均出现了线粒体肿胀、溶酶体增多等早期凋亡的微观改变.结论 HIV-1 gp120蛋白能够抑制人血-视网膜屏障细胞的增殖并具有诱导凋亡的作用,破坏线粒体的结构和功能是其可能的机制.