我国HIV-1主要流行株外膜蛋白(env)基因V3～V4区变异及其与生物学特性的关系

目的　研究我国HIV 1主要流行毒株亚型的envV3～V4区变异与生物学特性的关系。方法　应用nested PCR对 1 57份获自我国 1 2个省份的HIV 1毒株env区序列进行扩增 ,并使用ABI 377型测序仪测序 ,然后应用BLAST、GCG和MEGA等生物学软件或程序对env基因V3～V4区序列进行分析。结果　B′亚型毒株V3顶端四肽存在着 4种类型 :GPGR ( 54% )、GPGQ ( 2 8% )、GPGK( 1 6 % )和GPGA( 2 % ) ,B′/C重组毒株全部为GPGQ( 1 0 0 % ) ,CRF0 1 AE重组毒株呈现GPGQ( 95% )和GPGR( 5% )两种类型 ;B′/C和CRF0 1 AE重组毒株V3～V4区及其临近区域N 糖基化位点比B′亚型毒株N 糖基化位点保守。而B′亚型毒株V3环的净电荷分别显著高于B′/C和CRF0 1 AE毒株 (P <0 .0 1 ) ;根据V3环关键氨基酸推测辅助受体使用情况的结果显示 :B′亚型毒株有 9.2 6 %可能使用CCR5,7.4 1 %可能使用CXCR4 ,其余 83.33%不能对辅助受体的使用作出预测。所有B′/C重组毒株被预测可能使用CCR5。CRF0 1 AE重组毒株有 90 .4 8%被预测可能使用CCR5,没有被预测为使用CXCR4的序列 ,9.52 %不能作出预测。结论　B′亚型毒株大部分可能为NSI型 ,少部分可能为SI型 ,而B′/C和CRF0 1 AE重组毒株绝大部分为NSI型。我国主要流行株的V3～V4区尤其是V3环的氨基酸     

北京市男男性接触HIV-1感染者膜蛋白基因序列测定及亚型分析

目的 了解北京市男男性接触人群(MSM)中HIV-1的最新流行趋势及膜蛋白V3环序列特征.方法 巢式聚合酶链式反应(n-PER)扩增2007年提取的北京市男男性接触HIV感染者基因组DNA样品,对膜蛋白基因C2-V3区测序,进行病毒亚型及V3环序列特点分析.结果 11例样本中,4例是欧美B亚型,5例是AE重组亚型,1例是BC重组亚型,1例是01B重组亚型.V3环顶端四肽以GPGQ和GPGR为主.结论 北京市男男性接触HIV-1感染者中重组亚型呈蔓延流行趋势.     

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.     

HIV-1广谱中和性单克隆抗体的研究进展

从HIV感染患者血清中分离的针对HIV-1包膜上保守表位的中和性单克隆抗体(mAb)很多,识别的表位分别是HIV-1gp120上CD4结合位点(CD4bs),gp41的跨膜前域MPER,gp120表面包膜的高甘露聚糖,gp120上V2/V3环,这些抗体对HIV-1疫苗免疫原的研究设计至关重要.本文主要综述了各中和性单克隆抗体的结构特征,识别的表位,抗体与病毒包膜之间的相互作用.     

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。     

人类免疫缺陷病毒1型(HIV-1)糖蛋白gp120部分糖基化位点突变对其抗原性影响

目的 研究HIV-1包膜糖蛋白gp120特定糖基化位点突变后对gp120抗原性的影响,并推测糖基化位点突变对结构的影响.方法 采用连接PCR方法,将获得的野生型gp120中选定N糖基化位点的Asn定点突变为Cln,使该位点去糖基化,构建DNA疫苗,免疫小鼠,ELISA检测鼠血清中的抗体,进行统计学分析,推测糖基化位点突变对gp120抗原性和结构的影响.结果 2组糖基化位点突变的gp120诱发非V3特异性抗体的能力较之野生型gp120有明显提高,但诱发V3特异性抗体能力没有显著提高,7个位点突变的gp120诱发V3特异性抗体能力有很大降低.结论 gp120部分糖基化位点的突变在一定程度上能提高或改变gp120的抗原性,这可能由突变导致的构象变化和/或糖基化寡糖链移除使原来被遮盖的抗原表位暴露引起.     

HIV-1 C亚型密码子优化gp120基因重组腺病毒载体的构建及表达

目的 构建含有HIV-1 C亚型gp120基因重组腺病毒载体,并在293细胞中表达gp120蛋白.方法 PCR扩增,获得HIV-1 C亚型gp120片段,定向克隆入腺病毒转移载体pTrack-CMV,线性化后转化至含有腺病毒骨架载体pAd-easy-1的大肠埃希菌BJ5183,获得重组子prAd-gp120,PacⅠ酶切纯化后转染293细胞,包装成复制缺陷型重组腺病毒vAd-gp120.结果 经PCR、酶切及DNA测序,插入片段大小、方向正确,获得了具有感染力的vAd-gp120重组腺病毒;通过Western 印迹检测,重组腺病毒在293细胞中表达出分子量为120 kD的蛋白.结论 成功构建了含有HIV-1 C亚型gp120基因重组腺病毒载体,并获得该基因的表达.     

含密码子优化型HIV-1 gp120基因重组腺病毒的构建及其免疫效果研究

目的 构建能表达野生型和密码子优化型人免疫缺陷病毒Ⅰ型(HIV-1)B亚型中国流行株gp120基因的非复制型腺病毒。方法 按哺乳动物细胞偏好的密码子对HIV-1B亚型中国流行株Ch gp42的gp120基因进行优化，合成优化基因。将野生型和密码子优化的gp120基因插入穿梭质粒，再与腺病毒骨架质粒pAdEasy-1共转化E．coli BJ5183，获得重组子，转染293细胞后获得重组病毒。分别以两种重组腺病毒疫苗免疫小鼠，ELISA检测小鼠血清中的特异性抗体，乳酸脱氢酶法检测小鼠细胞毒性T淋巴细胞(CTL)反应。结果 获得两株重组腺病毒rAd-wt．gp120和rAd．mod．gp120，能正确表达Gp120。rAd-mod．gp120比rAd-wt．gp120蛋白表达水平明显提高。重组腺病毒免疫小鼠后能产生HIV-1特异性的抗体及CTL反应，rAd-mod．gp120组明显优于rAd-wt．gp120组。结论 成功构建了表达野生型和密码子优化的HIV-1 gp120基因的重组腺病毒，能诱导HIV-1特异性体液和细胞免疫反应。     

HIV-1B/C重组型外膜蛋白env基因序列分析及表型预测

目的 克隆并分析HIV-1B/C重组型外膜蛋白env基因序列,根据其氨基酸序列进行表型预测,为疫苗的抗原设计奠定基础.方法 采集北京地区HIV-1 B/C重组型的抗凝全血标本,分离血浆和提取基因组DNA,采用巢式PCR方法扩增rev-env基因,对扩增产物进行序列测定.根据核苷酸序列推导出相应的氨基酸序列,并对重要的Env功能结构域进行深入的分析和比较.结果 从12例B/C重组型HIV-1感染者中成功克隆到7个rev-env基因,序列分析发现其中6个有完整的开放读码框（ORF）,全部为CRF_07B/C重组型.6个Env蛋白氨基酸N糖基化位点和数目没有显著变化;CD4受体结合位点高度保守;根据V3环氨基酸序列及静电荷数目预测全部使用CCR5辅助受体;GP120/GP41剪切位点高度保守,预测所有GP160前体都能有效剪切;对几个已知中和抗体的中和位点分析推测全部的6个序列都对2G12、2F5中和不敏感;对4E10、PG9及PG16中和敏感.结论 有必要进一步阐明env基因型与相关功能的关系,这将为疫苗和药物研究提供依据.     

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.     

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.     

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to na?ve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.     

原代HIV-1包膜糖蛋白修饰体诱导小鼠产生高滴度gp120抗体

目的 探索1型人类免疫缺陷病毒(HIV-1)包膜糖蛋白修饰对包膜免疫原性的的影响.方法 通过PCR扩增获得原代HIV-1 06044株包膜gp120基因及其突变体gp120/W427S基因,并构建gp120三聚体蛋白真核表达载体pcT-gp120和pcT-gp120/W427S,重组表达载体体外瞬时转染人胚肾HEK2...     

A versatile vector for the production of pseudotyped viruses expressing gp120 antigens from different clades of primary HIV-1 isolates

A novel HIV-1 Env expression vector (SF162-Z) was developed by introducing two new cloning sites on the backbone of an existing vector that produces a full length Env from HIV-1 SF162 isolate. These sites facilitate the swapping of the gp120 portion of the SF162 Env with matching gp120 antigens from HIV-1 isolates of different genetic clades. Final production of functional pseudotyped viruses will express chimeric Env antigens, including gp41 of the parental SF162 and gp120 from other primary isolates. This system is useful for testing the neutralizing sensitivity of partial env gene products frequently identified in viral quasi species in patients infected with HIV or when only partial gp120 gene products are available.     

Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in parallel with increased gp120 net charge

To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations between increased Env net charge and lowered CD4⁺ T cell counts, enhanced viral fitness, reduced sensitivity to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5 HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness.     

The HIV-1 Env protein signal sequence retards its cleavage and down-regulates the glycoprotein folding

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide chain, during its transport, into the lumen of the endoplasmic reticulum (ER). We have analyzed the kinetics of the cleavage of the HIV-1 Env protein signal sequence from gp160 and gp120 in HeLa, BHK, and Jurkat cells. Furthermore, we have determined the effects of this cleavage on the association of the gp160 and gp120 glycoproteins with the ER protein calnexin and the effects of the signal sequence cleavage on protein folding. The cleavage of the HIV-1 Env protein signal sequence on both gp160 and gp120 occurred very slowly in all three cell lines with a t(1/2) of 45-60 min. The core glycosylated and signal-sequence-retained forms of gp160 and gp120 associated with calnexin while the signal-sequence-cleaved forms of gp160 and gp120 had disassociated from calnexin and correctly folded as determined by their ability to associate with the CD4 cellular receptor. Further analysis of the folding state of gp160 and gp120 in nonreducing SDS-PAGE revealed that the signal-sequence-retained and calnexin-associated forms of gp160 and gp120 migrated as broad, diffuse bands, whereas the signal-sequence-cleaved or CD4-associated forms of gp160 and gp120 migrated as single sharper bands. The cause of this retardation in the rate of folding and intracellular transport of HIV-1 glycoproteins was localized to their signal sequences by fusing the vesicular stomatitis virus G protein with the HIV-1 Env protein signal sequence and expressing this chimeric protein in mammalian cells. The HIV-1 Env protein signal sequence on the VSV-G protein also confers a reduced rate of cleavage and slow intracellular transport and folding of the chimeric G protein. These results provide direct evidence that in vivo the HIV-1 glycoprotein signal sequence inhibits the folding of HIV-1 Env protein. Our data also suggest a direct correlation between the rate of the signal sequence cleavage and protein folding.     

艾滋病痴呆综合征患者HIV-1B gp120基因的克隆及真核表达

目的 克隆艾滋病痴呆综合征(ADC)患者体内不同部位的HIV-1B gp120基因,并在人神经胶质瘤细胞U87中表达.方法 分别以一例ADC患者尸检标本的外周(淋巴结)和中枢(脉络丛、大脑枕叶白质)来源的基因组DNA为模板,PCR扩增HIV-1 gp120基因,序列测定后,将目的基因插入表达载体pcDNA3.1(+),构建重组表达载体gp120/pcDNA3.1(+),将所构建的重组表达载体用脂质体法转染人神经胶质瘤U87细胞,间接免疫荧光法测定HIV-IB gp120的表达情况.结果 克隆了ADC患者体内外周淋巴结、脉络丛、大脑枕叶白质3个部位的HIV-1B gp120基因,所构建的表达质粒gp120/pcDNA3.1(+)转染U87细胞后可表达目的蛋白.结论 ADC患者不同部位来源的HIV-1B gp120基因均可在U87细胞中表达,为进一步研究HIV-1B gp120包膜蛋白的神经毒性及其作用机制提供条件.     

不同治疗效果的艾滋病患者抗病毒治疗前HIV-1gp120准种特征差异

目的 探讨HIV-1 gp120准种在不同治疗效果的艾滋病患者抗病毒治疗前的特征差异.方法 回顾性收集治疗方案为AZT+NVP+3TC的艾滋病患者在抗病毒治疗前的血浆样本,包括病毒抑制(VS)组12例,治疗失败(TF)组12例.采用单基因组扩增技术获得gp120准种序列,分析比较遗传多样性、氨基酸长度、潜在糖基化位点及特征性氨基酸的特点.结果 本研究共获得gpl20序列365条序列,其中VS组168条(6-20条),TF组197条(7-28条).TF组的gp120准种复杂度高于VS组,差异有统计学意义(P=0.003);TF组的gp120准种dS及dN中位数均高于VS组(P=0.017;P=0.002).TF组HIV-1准种gpl20氨基酸长度长于VS组(P=0.00l).TF与VS组的gpl20准种相比共有9个氨基酸位点存在明显差异,多数分布在V1/V2区(6/9,66.6％).结论 不同治疗效果的艾滋病患者治疗前的HIV-1 gp120准种基因特征存在差异,TF组患者来源的HIV-1准种遗传多样性更高.     

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.     

N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.