HIV-1整合酶及其抑制剂的研究进展

HIV-1整合酶是由HIV病毒pol基因编码的分子量为32KD的蛋白质,是HIV病毒复制的必需酶之一,它催化病毒DNA整合入宿主染色体DNA。人类细胞中没有HIV 整合酶的类似物[1],理论上抑制整合酶对人体副作用很小。因此HIV-1整合酶成为继HIV-1蛋白酶,逆转录酶后治疗艾滋病的富有吸引力和合理的靶标。本文综述了HIV整合酶结构,抑制剂的研究以及以HIV-1 整和酶为靶点治疗AIDS方法的最新研究进展。     

抗人类免疫缺陷病毒1型广谱中和抗体的研究进展

现行抗反转录病毒治疗药物的联合应用可有效抑制艾滋病进程并显著延长患者寿命,但由于人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)潜伏库的存在,艾滋病迄今尚无法治愈。近年发现抗HIV广谱中和抗体能有效降低患者体内病毒载量并延缓疾病进程,为研发艾滋病疫苗和治愈策略带来了曙光,尤其是序贯免疫策略的使用极大推进了广谱中和抗体的开发和应用进程。2018年,美国食品药品管理局(Food and Drug Administration,FDA)批准了第1个临床应用的广谱中性单克隆和抗体,无疑为抗HIV单克隆抗体药物的研发注入了一支强心剂。本文围绕近年来抗HIV广谱中和抗体的研究进展进行综述,探讨未来广谱中和抗体研发面临的挑战。     

基于RNA的抗HIV-1基因治疗方法研究进展

艾滋病自发现以来在全球范围内迅速蔓延,危害性极高,目前广泛采用的高效抗逆转录病毒疗法(HAART)虽能够显著提高HIV-1感染者生活质量,但存在着价格昂贵,耐药和副作用的问题经常会导致HAART治疗的中断。要获得长期持续的抗病毒治疗效果还有待于研发新的抗病毒药物和治疗方法。近年来随着分子生物技术、干细胞研究、纳米技术等相关技术的发展,关于抗HIV-1基因治疗方法的研究受到了广泛关注。主要针对基于RNA的抗HIV-1基因治疗方法,包括反义RNA、核酶、RNA诱饵以及RNA干扰技术在抗HIV-1基因治疗方面进行综述。研究表明,以RNA为基础的抗HIV-1基因治疗方法有望成为传统治疗方法的一种有效辅助手段。     

抗人类免疫缺陷病毒广谱中和抗体与疫苗设计

自发现人类免疫缺陷病毒1型（HIV-1）30年来,科学家们不断探索有效的HIV-1疫苗,但至今效果不理想。由于“精英患者”的广谱中和血清能起到有效的抗病毒作用,研究人员希望通过对体液保护免疫机制的研究,推动疫苗的设计和优化,为尽早研发成功HIV疫苗提供关键理论和技术支撑。近几年来,由于技术的突破和改进,从HIV感染者中分离获得广谱中和抗体的概率和数量大大提高。本文针对近几年来分离的有代表性的广谱中和抗体,根据其不同的识别位点分为四大类进行详细介绍,并总结了从研究广谱中和抗体中所获得的疫苗设计创新思路与启示。     

基于深圳男男同性性行为人群的HIV-1广谱中和抗体筛选分析

目的:筛选基于深圳本地男男同性性行为者(Men who have sex with men,MSM)人群队列中HIV-1流行毒株的广谱中和抗体(Broadly neutralizing antibodies,bn Abs),为下一步机制和应用研究奠定基础。方法:建立小型MSM队列,按计划分别定期随访、留样,测序分析人群中HIV-1病毒流行亚型。选取将骨架质粒与系列标准HIV-1 env质粒12款,分别共转染293细胞制备单次感染能力假病毒。建立TZM-bl细胞实验测定并计算中和活性(ID50Titers)技术平台,用于选定病毒亚型样品的筛选。最后取所获具有一定广谱中和抗性的代表性血样,通过抗体竞争实验初步分析其结合位点。结果:近年来,深圳MSM的HIV-1流行亚型分布中,CRF07_BC(43.4%)和CRF55_01B(15.4%)占比快速增长。选择来自该人群队列CRF07_BC感染者34人88份血样进行广谱中和抗性检测,筛选出具有一定广谱中和抗性的血样10份(ID50 Titer≧25),其中2例显示了较佳中和宽度,可作用于全部12种假病毒中的7种(58.3%)。初步分析其结合机制均非靶向gp120。结论:本研究成功建立小型MSM队列和TZM-bl检测分析技术并应用于实践,初步筛选结果提示部分具有中和活性的患者血清内存在bn Abs。     

HIV-1 gp41N端融合肽与脂膜作用的荧光及单分子层膜研究

我们以前的实验证明ＨＩＶ－１ｇｐ４１Ｎ端２３肽即融合肽ＨＩＶｗｔ能促融合,但它的突变体ＨＩＶＧｌｕ（２位Ｖ→Ｅ）却不能诱发融合。为了进一步研究多肽结构与功能的关系,我们用荧光及单分子层膜技术研究ＨＩＶｗｔ及ＨＩＶＧｌｕ与脂的相互作用。荧光淬灭实验表明ＨＩＶｗｔ插入带负电磷脂并插入较深;而ＨＩＶＧｌｕ虽然能与带负电脂结合但并不插膜。单层膜实验进一步证实了上述结果：ＨＩＶｗｔ对带负电磷脂ＰＯＰＧ的临界插膜压达到４３ｍＮ／ｍ,而ＨＩＶＧｌｕ的临界插膜压只有３１ｍＮ／ｍ。这提示ＨＩＶｗｔ与单层膜不仅存在静电作用还有较强的疏水作用。结合上述实验结果推测ＨＩＶｗｔ能插入脂膜的酰基链因而容易促发融合;而ＨＩＶＧｌｕ插膜很浅或根本不能入膜从而不能促融合     

HIV疫苗研究进展

自从1983年发现人类免疫缺陷病毒(Human immunodeficiency virus,HIV)以来,HIV一直以惊人的速度在全球蔓延,感染HIV的人数也日益增多。到目前为止,因患艾滋病死亡的人数已达到2500万,到2010年这一数字可能会突破8000万,因此研究预防和治疗艾滋病的药物也正日益迫切地摆在人们面     

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.     

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包膜蛋白gp41优势抗原的构建及可溶性表达

目的:筛选1型人免疫缺陷病毒(HIV-1)中国流行株中包膜蛋白gp41的优势抗原片段,构建具有区域流行代表性的HIV-1 gp41重组抗原,为改进现有HIV-1初筛试剂盒中使用的同类抗原奠定基础。方法:利用免疫斑点杂交和生物信息学方法,从收集自重庆、广州、上海的区域代表性150份HIV-1感染者血清标本中筛选gp41抗原性强的候选样本,利用RT-PCR及巢式PCR方法扩增包含重要抗原表位决定蔟的gp41基因片段,与原核表达载体pQE30连接,转化大肠杆菌M15构建gp41重组抗原表达菌株,表达后经亲和层析纯化、SDS-PAGE和Western印迹鉴定。结果:兔源HRP标记的gp41多抗能识别标本中gp41抗原性差异,得到候选样本,扩增包含gp41主要抗原表位片段;构建了包含gp41抗原表达簇的重组原核表达质粒,表达、纯化后经His标签抗体Western印迹鉴定为阳性。结论:高纯度的重组优势gp41抗原的构建和鉴定,为进一步改进现有HIV初筛诊断奠定了基础。     

Recognition of HIV‐inactivating peptide triazoles by the recombinant soluble Env trimer,BG505 SOSIP.664

Peptide triazole (PT) antagonists interact with gp120 subunits of HIV‐1 Env trimers to block host cell receptor interactions, trigger gp120 shedding, irreversibly inactivate virus and inhibit infection. Despite these enticing functions, understanding the structural mechanism of PT‐Env trimer encounter has been limited. In this work, we combined competition interaction analysis and computational simulation to demonstrate PT binding to the recombinant soluble trimer, BG505 SOSIP.664, a stable variant that resembles native virus spikes in binding to CD4 receptor as well as known conformationally‐dependent Env antibodies. Binding specificity and computational modeling fit with encounter through complementary PT pharmacophore Ile‐triazolePro‐Trp interaction with a 2‐subsite cavity in the Env gp120 subunit of SOSIP trimer similar to that in monomeric gp120. These findings argue that PTs are able to recognize and bind a closed prefusion state of Env trimer upon HIV‐1 encounter. The results provide a structural model of how PTs exert their function on virion trimeric spike protein and a platform to inform future antagonist design. Proteins 2017; 85:843–851. © 2016 Wiley Periodicals, Inc.     

Antigenic analysis of the HIV-1 envelope trimer implies small differences between structural states 1 and 2

The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host–receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface–expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.     

Structural Survey of Broadly Neutralizing Antibodies Targeting the HIV-1 Env Trimer Delineates Epitope Categories and Characteristics of Recognition

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Pharmacokinetics of an HIV-1 gp120-specific chimeric antibody in patients with HIV-1 disease

The pharmacokinetics of mouse V/human C (1,) chimeric monoclonal antibody CGP47 439 specific for the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) was studied in patients with stage IV HIV-1 disease in an open-labeled phase I/IIA trial. Twelve male patients were enrolled and nine completed the study. Patients were divided into three groups according to the extent of CGP 47 439 to bind to gp120 from their viral isolates: undetectable for group 1, modestly reactive for group 2, and strongly reactive for group 3. A first dose of 1, 10, or 25 mg was administered by intravenous infusion to group 1, group 2 and group 3 patients, respectively. The patients then received seven doses of 50, 100, or 200 mg, respectively, every three weeks. CGP 47 439 serum concentrations were determined by an ELISA using monoclonal antibody AB19-4 specific for the idiotope of CGP 47 439. Half an hour after infusion only 25.5–36.1% of the administered antibody was found in the serum, reflecting its rapid distribution in the extravascular space and possibly binding to gp120 antigen in some of the patients. The terminal elimination half-life (T_1/2) was 16.2 days in group 1 patients, 9.7 days in group 2 and in group 3 patients 7.5 days and 9.1 days. An antibody response to CGP 47 439 was not a factor in determining elimination rates, because only very low and transient responses were found in three patients. These results suggest that the reactivity of CGP 47 439 with HIV-1 gp120 contributed to its elimination in HIV-1 infected patients.Abbreviations AIDS aquired immune deficiency syndrome - ARC AIDS-related complex - HIV-1 human immune deficiency virus type 1 - gp120 envelope glycoprotein with 120 KD molecular weight - V3 variable domain of gp120 - PND principle neutralizing determinant of gp120 - IgG immunoglobulin G - CD4⁺ lymphocytes: lymphocytes expressing the CD4 marker V_H and V_L variable heavy and variable light chain region of an antibody - C₁ and C_K constant heavy chain region of gamma l and constant K light chain region of an antibody - anti-id anti-idiotypic - AUC area under curve - T_1/2 terminal elimination half-life - ELISA enzyme-linked imuno sorbent assay - PBS phosphate buffered saline - NP-40 detergent - CDC center of disease control - GMP good manufacturing practice     

Roles of the interactions between Env and Gag proteins in the HIV-1 replication cycle

The Env and Gag proteins of HIV-1 are the two major structural proteins of this retrovirus. The interactions between Env and Gag proteins and their regulation in HIV-1 are required for several steps of the replication cycle, involving not only virus assembly, specifically Env incorporation, but also entry steps after virus maturation. A large number of host factors and certain membrane microdomains appear to engage both in transport/trafficking of Env and/or Gag proteins, and in the interactions of these two proteins. The present review briefly summarizes our current knowledge regarding the roles of the interactions between Env and Gag proteins in the virus replication cycle.     

Sequence variations of Env signal peptide alleles in different clinical stages of HIV infection

The human immunodeficiency virus has been shown to increase its infectivity throughout the course of infection. This virus selection property has been associated with genome mutations and recombinations among virus variants, causing amino acid residue alterations in important viral proteins. In order to explore the contribution of Env signal peptide (Env-sp) to Env glycoprotein expression and its possible relationship to increased virus infectivity observed at late stages of infection, we characterized Env-sp sequences derived from twelve patients at “early” and “late” stages of HIV infection without antiretroviral therapy use. In spite of the remarkable overall similarity between both stages, we observed the deletion of a sequence of neutral and basic residues at the Env-sp amino terminus in virus from early stage specimens and the insertion of basic residues in the hydrophobic region on late-stage viral isolates. The Env-sp sequence alterations may have viral adaptive functions during HIV infection.     

中国株HIV-1外膜蛋白真核表达载体的构建与表达

获得性免疫缺陷综合征(Acquired immunode- ficiency syndrome,AIDS)是由人类免疫缺陷病毒(Human immunodeficiency virus,HIV)引起的世界广泛流行、严重危害人类健康的疾病.     

中国株HIV-1外膜蛋白真核表达载体的构建与表达

To construct the eukaryotic expression vector of HIV-1 gp120 gene and observe its expression in vitro, the recombinant expression vector pVAX1GP120 was constructed by inserting the gp120 gene into the eukaryotic expression vector pVAX1. The pVAX1GP120 was transfected into Vero cells by lipofectamine and the expressed product was detected by indirect immunofluore- scence.Restriction enzymes digestion analysis and sequencing results revealed that the recombinant expression vector pVAX1GP120 has been constructed successfully. The indirect immunofluorescence result showed green fluorescence on the membrane of transfected cells. The constructed eukaryotic expression vector of HIV-1 gp120 can be expressed in vitro, which lay the foundation for the further study of HIV-1 DNA vaccine.     

Immunogen design for HIV-1 and influenza

Vaccines provide the most cost effective defense against pathogens. Although vaccines have been designed for a number of viral diseases, a vaccine against HIV-1 still remains elusive. In contrast, while there are excellent influenza vaccines, these need to be changed every few years because of antigenic drift and shift. The recent discovery of a large number of broadly neutralizing antibodies (bNAbs) and structural characterization of the conserved epitopes targeted by them presents an opportunity for structure based HIV-1 and influenza A vaccine design. We discuss strategies to design immunogens either targeting a particular antigenic region or focusing on native structure stabilization. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.