Characterization of immunoreactive epitopes of the HIV-1 p41 envelope protein using fusion proteins synthesized in Escherichia coli

We have identified several immunoreactive epitopes on the human immunodeficiency virus (HIV) type 1 transmembrane envelope protein by synthesizing various regions of the protein as fusions to the trpE gene in Escherichia coli. Ten fusion clones which expressed overlapping peptides were found to contain epitopes reactive with antibodies in sera of North American (NAm) and West African (WAf) patients with acquired immune deficiency syndrome (AIDS). An immunodominant epitope which reacted with all HIV-infected patients' sera was mapped to a 51-amino acid sequence in the N terminus of p41. A novel epitope was also identified in the C terminus of p41 which was reactive with 41% and 48% of the sera tested from NAm and WAf, respectively. In addition, several minor epitopes were identified. We observed that sera from WAf reacted more strongly to minor HIV-1 p41 epitopes than did sera from similarly infected individuals in NAm. We also report on the detection of antibodies from patients with HIV-2 infection in WAf which cross react with HIV-1 p41 recombinant envelope antigens.     

A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1.

A simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described. The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid. A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting. In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C. When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease. Recombinant p24 was purified to near homogeneity by a simple three-step procedure. The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E. coli by the coexpressed protease. The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.     

HIV感染早期病毒p24蛋白的检测

目的:建立敏感、特异的检测血清中HIV-p24蛋白的方法,作为HIV感染早期即窗口期的监测手段。方法:用纯化的p24蛋白免疫小鼠及家兔,获得单克隆及多克隆抗体,经DEAE-52阴离子交换柱纯化后,标记辣根过氧化物酶,建立ELISA双抗体夹心及间接双抗体夹心方法,检测HIV-p24蛋白。结果:包被单抗、标记多抗或包被多抗、标记单抗,均能特异地检出系列稀释的p24蛋白,包被混合单抗较包被多抗更敏感;经标记的抗种属抗体放大可明显提高检测的敏感性。结论:建立了敏感、特异的检测p24蛋白的双抗体夹心法,间接放大方法可检出50pg/mL的HIV-p24蛋白,检测敏感性与国际同类产品相似。     

Expression in yeast of a cDNA clone encoding a transmembrane glycoprotein gp41 fragment (a.a. 591-642) bearing the major immunodominant domain of human immunodeficiency virus.

A cDNA clone corresponding to the gp41 gene fragment nucl. 7573-7730 of the human immunodeficiency virus type 1 (HIV-1) was selected from a random HIV-1 genomic library expressed in yeast. This clone encodes a 52-residue long peptide (amino acid (a.a.)) 591-642) bearing the major immunodominant domain (a.a. 598-609) of the HIV-1 transmembrane glycoprotein gp41. Expression of the recombinant peptide pSE-env591-642 was driven by the alpha-mating factor leader sequence contained in a plasmid pSE-x allowing the synthesis and secretion of foreign gene product in Saccharomyces cerevisiae. Time-course analysis of the secretion into culture medium revealed an optimal production of the glycoprotein fragment at 28-30 h with no observable cytotoxicity. The secreted peptide is highly glycosylated with NH2-terminal heterogeneity probably due to different post-translational modifications. The secreted peptide shows an extreme antigenicity since in ELISA assays, as few as 5 microliters/well of crude supernatant are sufficient to obtain a strong detection by monoclonal antibodies or by 100% of sera from HIV-infected individuals. The purified glycopeptide pSE-env591-642 binds to a monoclonal antibody directed against the immunodominant epitope (a.a. 603-609) with an affinity similar to that of the complete glycoprotein gp160 (Kd values within the 10(-10) M range) and with a 100-fold higher affinity than that of a linear peptide fragment SP-env584-609. These results indicate that overexpression in yeast can efficiently provide an abundant source of highly antigenic gp41 protein fragment pSE-env591-642 which retains the antigenic properties of the native gp160 protein. Such a recombinant peptide should therefore be considered as a good candidate for antigen in HIV detection tests.     

Evaluation of the potency of the anti-idiotypic antibody Ab2/3H6 mimicking gp41 as an HIV-1 vaccine in a rabbit prime/boost study

The HIV-1 envelope protein harbors several conserved epitopes that are recognized by broadly neutralizing antibodies. One of these neutralizing sites, the MPER region of gp41, is targeted by one of the most potent and broadly neutralizing monoclonal antibody, 2F5. Different vaccination strategies and a lot of efforts have been undertaken to induce MPER neutralizing antibodies but little success has been achieved so far. We tried to consider the alternative anti-idiotypic vaccination approach for induction of 2F5-like antibodies. The previously developed and characterized anti-idiotypic antibody Ab2/3H6 was expressed as antibody fragment fusion protein with C-terminally attached immune-modulators and used for immunization of rabbits to induce antibodies specific for HIV-1. Only those rabbits immunized with immunogens fused with the immune-modulators developed HIV-1 specific antibodies. Anti-anti-idiotypic antibodies were affinity purified using a two-step affinity purification protocol which revealed that only little amount of the total rabbit IgG fraction contained HIV-1 specific antibodies. The characterization of the induced anti-anti-idiotypic antibodies showed specificity for the linear epitope of 2F5 GGGELDKWASL and the HIV-1 envelope protein gp140. Despite specificity for the linear epitope and the truncated HIV-1 envelope protein these antibodies were not able to exhibit virus neutralization activities. These results suggest that Ab2/3H6 alone might not be suitable as a vaccine.     

SARS冠状病毒NS融合蛋白的重组表达纯化与免疫学性质分析(英文)

刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequenceoverlappingextension,SOE)构建编码N1227LinkerS450650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

人类免疫缺陷病毒Ⅰ型核心蛋白(p24)在大肠杆菌中的表达、纯化及鉴定

在大肠杆菌中,利用新构建的含T7g-10L RBS以及λ-PR启动子的新型原核表达载体,通过表达gag-pol基因片段,获得了具有天然序列的人类免疫缺陷病毒1型(HIV-1)核心蛋白p24的高效表达。克隆的gag-pol基因片段在其阅读框架移位区域插入了4bp碱基,其表达的病毒蛋白酶在阅读框架上与gag一致,从而实现了对gag-pol融合蛋白的有效加工,产生成熟的核心蛋白p24及其它产物。重组p24以可溶形式存在,可以被抗p24的单克隆抗体特异识别。测定的N端8个氨基酸序列与从病毒纯化的p24完全一致。在使用硫酸铵沉淀后,采用两步离子柱层析,可将重组蛋白纯化到95％以上的纯度。结果表明,纯化的p24可以作为特异性很强的试剂而用于HIV感染的诊断及病情的预后,并可用于p24的生化及结构分析。     

Expression of bovine leukemia virus protein p24 in Escherichia coli and its use in the immunoblotting assay.

The gag gene encoded protein, p24 of bovine leukemia virus (BLV), was cloned and expressed as thioredoxin-6xHis-p24 protein in Escherichia coli. The bacterial cells carrying plasmid pT7THis-p24 expressed the protein of 38 kDa that was detected by immunoblotting analysis using anti-p24 monoclonal antibodies and sera from BLV infected cattle and sheep. The purified p24 fusion protein was shown to be sensitive and specific for detection of BLV antibodies in the infected cattle.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

Oligomerization of the hydrophobic heptad repeat of gp41.

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.     

A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.     

Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies.

In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC.     

Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p24 of B- and C-subtypes

A simple and efficient method for hyperexpression in Escherichia coli and purification of capsid protein, p24, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described. DNA-encoding p24 of C-subtype was cloned from C-subtype gag sequence which was obtained by PCR amplification using DNA extracted from peripheral blood mononuclear lymphocytes (PBMLs) of an HIV-1-infected patient from India. DNA-encoding p24B protein was amplified directly by two-step PCR using genomic DNA obtained from PBMLs of an HIV-infected individual. A T7 promoter-based expression system was optimized for hyperexpression of p24 in the soluble form. Both p24 (B- and C-subtype) were purified to near homogeneity using conventional chromatographic techniques. Purification of p24 (C subtype) was described for the first time with yield of 53 mg from 1 liter of culture. The yield of p24 (B-subtype) was 67 mg from 1 liter of culture, which was severalfold better than reported earlier. The immunoreactivity of both types of p24 to sera from HIV-infected individuals was comparable. This report describes a simple, highly efficient, and reproducible method for obtaining large quantities of highly pure p24 of both B- and C-subtype, which can be used for structural, biochemical, and immunological characterization and, eventually, for diagnostic and prognostic applications.     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。     

Advantage of Dimeric Peptide Antigens in Serodiagnosis of HIV-1 Infection

The reactivity of antibodies with dimeric and monomelic peptide antigens was compared by ELISA. A panel of highly purified synthetic peptides of HIV-1 representing defined regions, 598–609 and 524 533 (fusion domain) of gp41 and 306–320 of gpl20, were used as antigens in the ELISA. These peptides were selected and synthesized taking into account the level of sequence conservation of various strains and hydrophilicity. The analysis included sera from 52 HIV-1 infected individuals and 53 HIV-1 negative controls. Both peptides from gp41 were found to be particularly immunoreactive with sera from HIV-1 infected individuals. The frequency of reactivity to the selected peptide from gp120 (V₃ loop) in infected individuals was 82%. An interesting observation was that the dimeric peptide antigens had a detection rate more than 4-fold higher than the monomeric antigens. We found that lower levels of antibodies could be detected with dimeric antigens. The peptides reacted with few sera other than HIV-1 positive sera. These results implicate the potential dimeric peptide antigens to be utilized in the serodiagnosis of HIV-1 infection.     

Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China

The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560–616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.     

Expression,Purification and Characterization of Hiv-1 Capsid Precursor Protein p41

Human immunodeficiency virus type 1 (HIV-1) has been a global epidemic since 1983; yet, the virology and immunology related to HIV-1 remain elusive. Furthermore, as there is still no effective chemoprophylaxis or vaccine to treat patients with HIV-1, most research focuses on strategies to prevent HIV-1 infection, such as with antiviral drugs, novel therapeutics, or improved diagnostic kits. The HIV-1 Gag precursor protein (p55)—comprising the matrix (MA/p17), capsid (CA/p24), and nucleocapsid (NC/p7) protein domains—is the main structural HIV-1 protein, and is uniquely responsible for virion assembly within the virus life cycle. Recently, the immature and mature capsid structures were solved; however, the precursor protein structure is still unknown. Here, we expressed two subtypes of HIV-1 MA–CA stretch of the Gag protein, referred to as p41, in a bacterial expression system. We characterized the purified p41 protein, and showed its superior antigenicity over that of p24, highlighting the potential influence of the p17 domain on p24 structure. We further showed that p41 has good immunogenicity to induce an antibody response in mice. These results will aid future investigations into the HIV-1 capsid precursor structure, and potentially contribute to improving the design of diagnostic kits.     

A monomeric 3(10)-helix is formed in water by a 13-residue peptide representing the neutralizing determinant of HIV-1 on gp41

The peptide gp41(659-671) (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41(659-671) in water. This peptide forms a monomeric 3(10)-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3(10)-helix is confirmed by its characteristic CD pattern. Studies of the 3(10)-helix have been hampered by the absence of a model peptide adopting this conformation. gp41(659-671) can serve as such a model to investigate the spectral characteristics of the 3(10)-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3(10)-helix. The observation that the 3(10)-helical conformation is highly populated in the peptide gp41(659-671) indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41(659-671) segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41(659-671) is an autonomous folding unit, peptide immunogens consisting of the complete gp41(659-671) sequence are likely to induce antibodies highly cross-reactive with HIV-1.