抗HIV-1中和抗体的研究进展

自1982年美国疾病预防与控制中心(CDC)对那些与免疫缺陷和卡氏肺囊虫肺炎等症状有关的病例命名为获得性免疫缺陷综合征(AIDS)以来,研究者们对预防和治疗性AIDS疫苗的研究进行了不懈的努力,实验中的疫苗包括env亚单位疫苗、减毒活疫苗、灭活疫苗、DNA疫苗等等,但至今尚没有一种疫苗能获得理想的免疫效果.最近研究者们对中和抗体(Neutralization antibody, nAb.)在HIV-1感染中的作用的研究结果让人们看到一些新的曙光.     

HIV-1中和抗体和基于抗体的疫苗设计

研制一种安全有效并能广泛使用的HIV疫苗对于预防和控制HIV的流行具有重要的意义。尽管人类在HIV-1病原学和免疫学方面的认识不断取得新的进步,对于HIV-1而言,普遍认为诱导保护性中和抗体的科学障碍很难逾越。在抗击HIV-1的感染中传统的疫苗策略不能提供保护。然而,近来的研究揭示在小部分HIV-1感染病人的血清中存在的某些抗体能够中和大多数的HIV-1毒株,对这些血清抗体的深入分析有助于人们揭示抗体识别的病毒表位,这些研究表明自然产生的能够中和HIV-1的中和抗体的发现可能引导未来疫苗设计的思路。高分辨率的结构信息将揭示Env 和中和抗体(Nab)结合区原子水平的结构,这些信息能够帮助设计更好的免疫原。     

利用细胞筛选法从噬菌体抗体库鉴定人源HIV-1单克隆中和抗体

以细胞法从HIV-1CRF07_BC特异性噬菌体抗体库中筛选和鉴定针对病毒包膜蛋白(Envelope,Env)的人源单克隆中和抗体。将pCH064.2-Env质粒转染293T细胞,以表达Env的活细胞淘洗噬菌体抗体库;利用细胞ELISA方法筛选Env特异性噬菌体阳性克隆,并测定抗体重链可变区(VH)和轻链可变区(VL)序列;以Ni+2-NTA层析柱纯化抗体Fab,SDS-PAGE分析抗体纯度;采用基于转染细胞和重组蛋白的ELISA以及流式细胞技术分析抗体的结合活性;以HIV-1假病毒系统评价抗体的中和活性。四轮淘洗使细胞特异性噬菌体得到约650倍的高度富集,细胞ELISA筛选到28个抗HIV Env的阳性克隆,序列分析发现五个具有不同VH和VL序列的抗体Fab(2801、2837、2863、2870和2920)。这些抗体与转染env的293T细胞反应,但不与重组表达的可溶性gp120蛋白反应,说明它们有可能针对依赖于Env复合物的空间构象表位。我们发现2801、2863和2870三个Fab抗体对HIV-1CRF07_BC亚型CH120.6毒株具有较强的中和活性,其IC50值分别为2.24μg/mL、0.89μg/mL和3.09μg/mL。其中,2801和2863对B亚型HIV-1毒株SF162具有较强的交叉中和作用,其IC50值分别为0.69μg/mL和3.52μg/mL。提示表达于293T细胞表面的HIV-1 Env可以有效富集和筛选噬菌体抗体库,并能成功分离和鉴定依赖于Env空间构象表位的HIV-1中和抗体。因此本研究为探讨HIV-1中和抗体的反应机制和研发艾滋病疫苗提供了新的技术平台。     

HIV-1广谱中和抗体筛选技术的研究进展

HIV-1广谱中和抗体(HIV-1 bNAbs)是一类可以中和大多数流行株的抗体。HIV-1 bNAbs的研究可以为抗艾滋病药物提供候选分子和为艾滋病疫苗设计提供指导,是评估艾滋病疫苗效果的重要指标之一。HIV-1 bNAbs可通过传统筛选技术获得,如杂交瘤技术、EBV转化和展示库技术等。近年来,随着单细胞克隆和分选技术的发展,HIV-1 bNAbs的筛选效率和抗体特异性显著提高。多项技术结合的筛选手段和新型筛选技术LIBRA-seq,以及生物信息学辅助的筛选技术将抗体序列和功能信息统一起来,为HIV-1 bNAbs筛选和疫苗设计提供技术支持。除了HIV-1,这些筛选技术和方法也可用于其他病毒bNAbs的筛选,为疫苗设计和抗病毒药物开发提供了有益启示。综述了广泛应用于HIV-1 bNAbs的筛选技术和最新进展,为后续HIV-1或其他病毒bNAbs的筛选提供参考。     

HIV-1中和抗体疫苗研究进展

由于HIV具有与其它微生物极为不同的生物学特点,HIV疫苗的研究面临着前所未有的困难和挑战。20多年来,艾滋病疫苗研究主要采用了诱发中和抗体为主或细胞免疫为主两种策略,然而至今尚无实质性突破。诱发有效中和抗体一直是传统疫苗研发的重要策略,但HIV的高变异、多亚型等特点,使该策略在HIV疫苗研发中的应用成效甚微。近年来,一些具有广谱中和活性的HIV单抗的发现及其相应抗原表位的阐明,给HIV中和抗体疫苗的研究带来了新的希望。综合分析与评述这些进展,对于重新思考艾滋病疫苗和采用更好的策略进行艾滋病疫苗研究会衣纸帮助。     

抗HIV-1中和抗体的研究进展

自1982年美国疾病预防与控制中心(CDC)对那些与免疫缺陷和卡氏肺囊虫肺炎等症状有关的病例命名为获得性免疫缺陷综合征(AIDS)以来,研究者们对预防和治疗性AIDS疫苗的研究进行了不懈的努力,实验中的疫苗包括env亚单位疫苗、减毒活疫苗、灭活疫苗、DNA疫苗等等,但至今尚没有一种疫苗能获得理想的免疫效果。最近研究者们对中和抗体(Neutralizationantibody,nAb.)在HIV1感染中的作用的研究结果让人们看到一些新的曙光。尽管对nAb是否在预防HIV1感染过程中具有保护作用存在争议,但是仍然有不少的研究者在nAb方面展开了大量的工作,以期…     

HIV-1的表型及其感染的细胞嗜性

HIV-1的表型分为合胞体诱导型(syncytium-inducing,SI)和非合胞体诱导型(non-syncytium-inducing,NSI)。依据所用辅助受体和感染靶细胞的不同,HIV-1又被分为R5、X4和R5X4型。R5和X4型病毒分别利用CCR5和CXCR4作为辅助受体,而R5X4型病毒可利用这两种辅助受体。在病毒的复制力、细胞嗜性以及合胞体诱导能力上,SI型与X4型病毒一致,NSI型与R5型病毒一致。在HIV-1感染过程中,疾病的发展伴随着病毒从NSI型向SI型、及R5型向X4型的转变。HIV-1的表型影响和决定着HIV-1的感染、传播及AIDS的疾病进程。HIV-1的表型和细胞嗜性主要由病毒gp120的V3区(特别是第11和25位的氨基酸)决定。V3区的氨基酸序列信息,将为预测HIV-1的表型,以及病毒感染后的疾病进程提供生物信息学的依据。     

HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

利用报告基因比较悬浮细胞与贴壁细胞对HIV-1假病毒感染效率的差异

【目的】研究用人免疫缺陷病毒(Human immunodeficiency virus,HIV)-1假病毒感染带有β-半乳糖苷酶(β-galactosidase,β-gal)报告基因和HIV受体CD4+CCR5+的Tzmbl细胞,分析悬浮状态与贴壁状态对HIV-1假病毒感染Tzmbl细胞的影响,为进一步进行HIV生物学研究与中和抗体实验室评价提供实验基础。【方法】通过将pNL43 R-E-与编码HIV膜蛋白的质粒共转染293T细胞,收集上清,获得HIV假病毒。该假病毒感染悬浮的和贴壁的Tzmbl细胞后可表达β-gal报告蛋白,通过X-gal染色和仪器分析可测定表达β-gal报告基因的细胞数与细胞感染率。【结果】HIV假病毒感染悬浮细胞的效率高于其对贴壁的Tzmbl细胞感染的效率,且细胞的感染率的改变与病毒的型相关。【结论】该研究结果可为进一步利用具有单轮感染活性的HIV假病毒进行生物研究和中和抗体实验提供研究方法。     

SDF-1基因对HIV-1核酸疫苗诱导的免疫应答

研究SDF-1基因对HIV-1核酸疫苗诱导免疫应答的影响,以探求治疗性HIV-1核酸疫苗的新策略。将pCIneoGAG联合SDF1基因或者pCIneoGAG单独免疫Balb/c小鼠,采用ELISA检测免疫小鼠的特异性抗体和IFNγ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。研究结果提示:与pCIneoGAG免疫组比较,pCIneoGAG联合SDF-1基因免疫组小鼠血清的抗HIV-1p24抗体滴度降低,有显著性差异(p<0.01);而与pCIneoGAG免疫组比较,pCIneoGAG联合SDF-1基因免疫组小鼠血清的IFNγ升高,差异显著(p<0.01);pCIneoGAG联合SDF-1基因免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCIneoGAG免疫组,有显著性差异(p<0.01)。因此,SDF-1基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,SDF-1基因对体液免疫有抑制作用。SDF-1基因对于治疗性HIV-1核酸疫苗是具有较好应用前景的免疫佐剂。     

High level serum neutralizing antibody against HIV-1 in Chinese long-term non-progressors

NAb have been considered to be an important component of a protective immune response to HIV-1, yet the relationship between the capacity of HIV-1 NAb, the conserved neutralization epitopes and disease progression has been unclear. To gain a better understanding of the protective roles that NAb and conserved neutralization epitopes could play in LTNP, twenty-eight HIV-1-infected subjects were investigated by evaluation of the concentrations of HIV-1 NAb and conserved neutralization epitopes, using single-round PBMC neutralization assay and sequencing. Our study revealed that the concentration of NAb in LTNP was significantly higher than that in subjects with asymptomatic HIV (P < 0.05) and AIDS (P < 0.01). No amino acids substitutions were found in the conserved epitopes of the HIV-1 gp120 region in LTNP, whereas the viruses circulating both in persons with asymptomatic HIV and those with AIDS had amino acid substitutions in their conserved neutralization epitopes. This study suggests that high levels of NAb and stable epitopes in gp120 could play a crucial role in protection against disease progression.     

Natural killer cell function in HIV-1 infected patients

A cross-talk between dendritic cells (DC) and resting natural killer (NK) cells leads to the activation of both cell populations, a process requiring cell-cell contact. When the number of activated NK cells overwhelms surrounding DC, they became able to kill specifically immature DC, a feedback mechanism to shut off DC-mediated immune responses. DC, at the mucosal site, can capture HIV and transfer it to CD4+ T lymphocytes present in the regional lymph node thus giving rise to a productive infection; on the other hand, NK cells represent the first line of defence against viral infection. Our preliminary results suggest that during the early phases of an HIV infection, NK cell activity is not functionally compromised, but that infected cells might escape natural immune surveillance through several mechanisms, including a reduced lysis of autologous DC.     

Similar neutralizing activity in the HIV-1 infected long term non-progressors(LTNPs) and typical progressors(TPs)

Neutralizing antibodies are considered to be an important protective parameter used in HIV-l vaccine evaluation.However,the exact role that neutralizing antibodies plays in controlling the disease prog...     

Early and prolonged antiretroviral therapy is associated with an HIV-1-specific T-cell profile comparable to that of long-term non-progressors

Background

Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).

Methodology/Principal Findings

We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4⁺ and CD8⁺ T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-γ, IL-2, TNF-α production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4⁺ and CD8⁺ T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8⁺ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.

Conclusions

Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption.     

Development of broadly neutralizing antibodies in HIV-1 infected elite neutralizers

Broadly neutralizing antibodies (bNAbs), able to prevent viral entry by diverse global viruses, are a major focus of HIV vaccine design, with data from animal studies confirming their ability to prevent HIV infection. However, traditional vaccine approaches have failed to elicit these types of antibodies. During chronic HIV infection, a subset of individuals develops bNAbs, some of which are extremely broad and potent. This review describes the immunological and virological factors leading to the development of bNAbs in such “elite neutralizers”. The features, targets and developmental pathways of bNAbs from their precursors have been defined through extraordinarily detailed within-donor studies. These have enabled the identification of epitope-specific commonalities in bNAb precursors, their intermediates and Env escape patterns, providing a template for vaccine discovery. The unusual features of bNAbs, such as high levels of somatic hypermutation, and precursors with unusually short or long antigen-binding loops, present significant challenges in vaccine design. However, the use of new technologies has led to the isolation of more than 200 bNAbs, including some with genetic profiles more representative of the normal immunoglobulin repertoire, suggesting alternate and shorter pathways to breadth. The insights from these studies have been harnessed for the development of optimized immunogens, novel vaccine regimens and improved delivery schedules, which are providing encouraging data that an HIV vaccine may soon be a realistic possibility.     

HIV-1-specific B cell activation. A major constituent of spontaneous B cell activation during HIV-1 infection

B cell activation is a well known consequence of HIV-1 infection, and seropositive subjects show high numbers of spontaneously activated Ig-secreting cells in circulation. To better define the importance of the HIV-1-specific response in this phenomenon, we first studied whether in vitro spontaneous anti-HIV-1 antibody production was accompanied by reactivation of memory B lymphocytes. Unstimulated PBL from HIV-1-infected individuals with prior history of hepatitis B and/or EBV infection did not consistently show spontaneous in vitro synthesis of anti-hepatitis B core Ag or anti-EBV antibodies; in addition, PWM-induced synthesis of anti-hepatitis B virus and anti-EBV antibodies was decreased compared to HIV-1-seronegative subjects. Moreover, in comparing the frequencies of activated HIV-1-specific B cell precursors and activated Ig-secreting precursors in limiting dilution experiments, a sizable fraction (20 to 40%) of circulating cells spontaneously secreting Ig produced antibody against HIV-1 determinants. The ratio between the two frequencies fitted in very well with the amount of Ig removed from unstimulated culture supernatants after HIV-1-specific antibody absorption with solid-phase HIV-1. These findings indicate that B cell activation during HIV-1 infection is mainly oriented toward a specific response to HIV-1 determinants; the possible relevance of this phenomenon to lymphomagenesis in AIDS patients is discussed.     

Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses

Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.     

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages.

We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation.

The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages.

Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.     

Modeling breastmilk infectivity in HIV-1 infected mothers

Estimation of breastmilk infectivity in HIV-1 infected mothers is difficult because transmission can occur while the fetus is in utero, during delivery, or through breastfeeding. Since transmission can only be detected through periodic testing, however, it may be impossible to determine the actual mode of transmission in any individual child. In this article we develop a model to estimate breastmilk infectivity, along with the probabilities of in-utero and intrapartum transmission. In addition, the model allows separate estimation of early and late breastmilk infectivity, and individual variation in maternal infectivity. Methods for hypothesis testing of binary risk factors and a method for assessing goodness of fit are also described. Data from a randomized trial of breastfeeding versus formula feeding among HIV-1 infected mothers in Nairobi, Kenya, are used to illustrate the methods.