人类免疫缺陷病毒-1包膜糖蛋白gp41的表达及优化

目的 优化HIV-1本地流行株06044 gp41蛋白表达设计,为gp41免疫原的制备提供实验基础.方法 以含HIV-1 06044 gp41基因的质粒为模板,采用聚合酶链反应(PCR)扩增gp41目的基因,分别构建原核表达载体pET26b-gp41T(含546～683 aa片段)及去掉N-端(NHR)和C-端(CHR)七价重复序列中间部分loop区(582～627aa)的pET26b-gp41 T(△L),经测序确认后,转化至大肠杆菌感受态细胞BL21 (DE3)中,IPTG诱导表达.用聚丙烯酰氨凝胶电泳(SDS-PAGE)及Western blot检测并鉴定重组表达蛋白.优化了重组蛋白的诱导表达条件.结果 pET26b-gp41T和pET26b-gp4 1T (△L)重组表达质粒均能表达gp41蛋白,gp41T (△L)蛋白表达量高于gp41T;不同IPTG浓度诱导的蛋白表达量没有区别;用1 mmol/L IPTG诱导后,37℃条件下表达量最高.Western blot结果显示,gp41T (△L)与His抗体结合性能好.结论 实验获得了稳定表达HIV-1 06044 gp41的原核表达载体以及表达条件,为大量制备gp41蛋白免疫原奠定了基础.     

Downregulation of human immunodeficiency virus type 1 Gag expression by a gp41 cytoplasmic domain fusion protein

The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the Env transmembrane protein, beta-galactosidase (beta-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli beta-gal. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and beta-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein.     

Analysis of genetic variability within the immunodominant epitopes of envelope gp41 from human immunodeficiency virus type 1 (HIV-1) group M and its impact on HIV-1 antibody detection

The serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n = 42), B (n = 62), B' (n = 13), C (n = 38), D (n = 41), E (n = 18), F (n = 27), and G (n = 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B', in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n = 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.     

The effect of human immunodeficiency virus type 1 (HIV-1) gp41 variability on antibody detection

To determine whether genetic variability influences the ability to detect antibody, nine gp41 ectodomain recombinant proteins from human immunodeficiency virus type 1 (HIV-1) CRF07_BC, CRF01_AE and subtype B′ were expressed in a bacterial expression system and purified. An indirect sandwich ELISA was developed with individual purified recombinant proteins. Plasma samples from 26 individuals infected with HIV-1 of different subtypes and four samples from the 1st international antibody reference panel were tested against each recombinant protein by ELISA. Heat-map and two-dimensional hierarchical clustering methods revealed that ELISA reactivity against antigens derived from the same subtypes clustered together. This suggests a similar reactivity pattern among infections of the same subtype, and thus the antigenicity of gp41 recombinant proteins may vary depending on the subtype, and subtype-related serotypes may exist among these antigens. Using association analysis methods, eight signature sites related to the subtype-specific reactivity patterns were identified. This study provides valuable information for the development of diagnostic assays with the ability to detect broadly cross-reactive antibodies induced by infection with different HIV-1 subtypes.     

抗人免疫缺陷病毒1型gp120人源单链抗体的表达

目的研究人源抗人免疫缺陷病毒１型（ＨＩＶ－１）ｇｐ１２０单链抗体（ＳｃＦｖ）。方法以人工合成的ＨＩＶ－ｌｇｐ１２０Ｖ３环多肽为抗原，利用噬菌体抗体库技术，筛选含有抗－ｇｐ１２０ＳｃＦｖ基因的噬菌体，提取质粒，转化大肠杆菌ＨＢ２１５１，表达可溶性ＳｃＦｖ。结果经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ分析，表达产物分子量为２８ｋＤ左右，且具有ｃ－ｍｙｃ活性；ＥＬＩＳＡ和Ｄｏｔｂｌｏｔ结果表明，可溶性ＳｃＦｖ具有较好的抗原结合活性和较强的特异性；竞争性ＥＬＩＳＡ实验结果进一步证明表达产物的特异抗－ｇｐ１２０活性。结论该技术便捷有效，可大量获得人源抗ＨＩＶ抗体片段，为进一步研究抗ＨＩＶ抗体的生物活性和ＨＩＶ感染诊治打下基础     

Monoclonal antibodies to gp110 and gp41 of human immunodeficiency virus.

Six mouse hybridomas secreting monoclonal antibodies specific for the glycoproteins of human immunodeficiency virus were developed. All six antibodies reacted by radioimmunoprecipitation with the glycoprotein precursor of 150,000 daltons as well as one of the proteolytic processing products of 110,000 daltons (gp110) or 41,000 daltons (gp41). Recombinant polypeptides spanning the env coding region were used to locate epitopes on the glycoprotein molecule. The panel of antibodies detected two distinct epitopes of gp41 and one epitope of gp110. We used the antibodies in indirect immunofluorescence assays to evaluate 13 clinical isolates of human immunodeficiency virus from diverse geographic regions, and we found that the gp110 epitope was recognized on all tested isolates, whereas the two gp41 epitopes were detected on 10 of 13 and 4 of 13 isolates.     

Monitoring human immunodeficiency virus type 1-infected patients by ratio of antibodies to gp41 and p24

Antibody responses of 85 patients to human immunodeficiency virus type 1 antigens were quantitated by densitometric analysis of Western blot (immunoblot) assays. All patients had been classified into the following three clinical categories: asymptomatic (ASY), acquired immunodeficiency syndrome (AIDS)-related complex (ARC), or AIDS. Fifty of the patients were monitored for 6 to 29 months. The gp41/p24 antibody ratio was examined in three studies. In the first study, initial specimens from each patient were analyzed. The mean gp41/p24 antibody ratios were 1.5 (ASY), 3.2 (ARC), and 5.4 (AIDS). Of ASY patients, 79% had antibody ratios of less than 2.0. In contrast, 72% of patients with AIDS had ratios of greater than or equal to 2.0. In the second study, serially obtained specimens from ASY, ARC, and AIDS patients were analyzed. These patients were further grouped according to progression of their clinical condition. Of ASY patients whose clinical condition progressed to ARC, 80% consistently had ratios of greater than or equal to 2.0. Of ARC patients whose clinical condition progressed to AIDS, 71% consistently had ratios of greater than or equal to 2.0. Of AIDS patients who died during the study, 100% consistently had ratios of greater than or equal to 2.0. No patients were treated with azidothymidine during the first two studies. In the third study, AIDS patients were monitored before and during treatment with azidothymidine. During treatment, ratios stabilized or improved transiently in five of seven patients. In these three studies, a gp41/p24 antibody ratio of less than 2.0 correlated with a benign clinical state and a ratio of greater than or equal to 2.0 correlated with AIDS or progression to AIDS.     

Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.     

Serologic reactivity of a synthetic peptide from human immunodeficiency virus type 1 gp41 with sera from a Mexican population.

The reactivities of 1,172 serum samples obtained from asymptomatic human immunodeficiency virus type 1 (HIV-1)-positive and HIV-1-negative individuals residing in Mexico to a synthetic disulfide-looped peptide from the HIV-1 gp41 (amino acids 602 to 616 [IWGCSGKLICTTAVP] were examined by an enzyme-linked immunoadsorbent assay (ELISA) procedure. Antibodies to the synthetic peptide were detected in 261 of 268 serum samples from HIV-positive individuals (sensitivity, 97.4%). The peptide also reacted with 12 of 904 serum samples from control HIV-negative individuals (specificity, 98.7%). Western blots (immunoblots) of four of the seven serum samples that produced false-negative results in the ELISA showed that three of them reacted weakly with gp41 and strongly with gp120, p55, and/or p24. Potential diagnostic difficulties raised by the reported C1q binding capacity of this peptide were also evaluated: few and weak false-positive results were found among sera from patients with rheumatoid arthritis (1 of 31) and neurocysticercosis (2 of 111). In fact, strong reactivity with the peptide spotted an undetected HIV infection underlying clinical neurocysticercosis.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.     

Immunogenic and antigenic dominance of a nonneutralizing epitope over a highly conserved neutralizing epitope in the gp41 envelope glycoprotein of human immunodeficiency virus type 1: its deletion leads to a strong neutralizing response

The Kennedy peptide, (731)PRGPDRPEGIEEEGGERDRDRS(752), from the cytoplasmic domain of the gp41 transmembrane envelope glycoprotein of HIV-1 contains a conformationally dependent neutralizing epitope (ERDRD) and a linear nonneutralizing epitope (IEEE). No recognized murine T cell epitope is present. The peptide usually stimulates virus-specific antibody, but this is not always neutralizing. Here we show that IEEE (or possibly IEEE plus adjacent sequence) is immunogenically and antigenically dominant over the ERDRD neutralizing epitope. Thus rabbits immunized in a variety of routes, doses, and adjuvants with a chimeric cowpea mosaic virus (CPMV) expressing the Kennedy peptide on its surface (CPMV-HIV/1) synthesized IEEE-specific serum antibody but no ERDRD-specific or HIV-1-neutralizing antibody. To test if this resulted from immunodominance or from a hole in the antibody repertoire, we immunized rabbits with chimera CPMV-HIV/29, which expresses the GERDRDR part of the Kennedy sequence. This chimera readily stimulated ERDRD-specific, neutralizing antibody. In mice the situation was less extreme, but individual animals with low neutralizing titers had a high ratio of IEEE-specific:ERDRD-specific antibody. Data are consistent with immunodominance of IEEE over ERDRD in the Kennedy peptide. IEEE-specific antibody was also antigenically dominant and prevented ERDRD-specific antibody from binding to its epitope and from neutralizing HIV-1. It may be that HIV-1 has evolved a nonneutralizing immunodominant epitope that allows it to possess a neutralizing epitope without suffering the consequences, and this idea is supported by the covariance of both epitope sequences. To our knowledge this is the first example of a defined sequence that controls the activity of an adjacent epitope.     

Monoclonal antibody identifies a highly conserved and immunodominant epitope of the human immunodeficiency virus transmembrane protein

A murine monoclonal antibody (MAb), 10E9, has been generated which identifies a conserved and immunodominant epitope of the human immunodeficiency virus (HIV) transmembrane protein, gp41. The MAb reacts with the protein backbone of the mature env gene product and also with polyprotein precursor, gp160. Human sera were tested for their ability to competitively inhibit the immunoreactivity of MAb 10E9. Of 100 serum samples obtained from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC), all showed strong inhibition to the reaction. In contrast, sera obtained from normal donors or those with other viral infections failed to perturb the binding activity of MAb 10E9. The geographic diversity of the AIDS/ARC patients studied provides evidence that the 10E9 epitope of gp41 is highly conserved.     

Characterization of neutralizing sites in the second variable and fourth variable region in gp125 and a conserved region in gp36 of human immunodeficiency virus type 2.

Several determinants of human immunodeficiency virus (HIV) have been suggested to harbor sites important for neutralization. The third variable region (V3) of the envelope glycoprotein (gp) is an important neutralizing determinant for both serotypes of HIV. The localization of additional neutralizing regions is an urgent task because the virus appears to mutate to phenotypes that escape neutralizing antibodies. Therefore, we have focused on the possibility of finding other immunodominant regions in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2). By immunization of guinea pigs with peptides corresponding to different selected regions of gp125 and gp36, we have found three antigenic determinants located in the V2 and V4 regions of the envelope protein gp125, and one region in the glycoprotein gp36, which are important for human antibody binding and also as targets for neutralization. The peptide representing the V2 region had the most pronounced capacity to induce neutralizing anti-HIV-2 antibodies in guinea pigs. Neutralizing activity was also detected in an antipeptide guinea pig sera representing a linear site in gp36, amino acids 644-658. A substitution set of peptides representing the conserved antigenic site in the central part of gp36 was used to identify the role of individual amino acids important for human antibody binding.     

Mutations within the human immunodeficiency virus type 1 gp160 envelope glycoprotein alter its intracellular transport and processing

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor polyprotein, gp160, proceeds via the endoplasmic reticulum (ER) and Golgi complex. We examined gp160 processing during the production of HIV-1 virions in transfected HeLa cells using wild-type and env mutant proviral molecular clones. Results from pulse-chase analyses indicated that a single amino acid substitution within a highly conserved domain of the env gene impaired gp160 export from the ER, leading to an increase in oligomeric forms of gp160 and a decrease in gp120 production. In contrast, gp160 which contained a mutated cleavage site was able to traverse the ER/Golgi complex, even in the absence of proteolytic processing, and become incorporated into budding virions. These findings indicate that export from the ER is a point in the intracellular trafficking of gp160 that is crucial to the production of the mature envelope components.     

Performance of human immunodeficiency virus type 1 gp41 assays for detecting enfuvirtide (T-20) resistance mutations

The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared to a "home brew" assay and clonal sequence analysis.     

Definition of an immunodominant T cell epitope contained in the envelope gp41 sequence of HIV-1.

The majority of the immunodominant amino acid sequences of HIV-1 that have been characterized to date are coded for by hypervariable gene sequences. These variable sequences are however interspersed with sequences that are highly conserved between HIV strains. Immunogenic viral products with amino acid sequences that vary minimally between strains, and that consistently elicit both humoral and cellular immune responses, may be ideal for inclusion in a subunit vaccine. We studied HIV-seronegative and HIV-infected persons, classified as asymptomatic (AS), ARC or AIDS. Initially, we assessed the cellular immune status of each subject from results of T cell phenotype analyses, assays for serum levels of surrogate markers of disease progression, and responses to mitogens and recall antigen. In addition, we tested whether three short synthetic peptides derived from the conserved sequences of the envelope gp120 (aa 262-284) and gp41 (aa 579-601), and core p17 (aa 106-125) regions of the HTLV-IIIB isolate, could elicit B cell as well as T cell responses in HIV-infected subjects. Only the gp41-derived sequence was immunogenic at both B and T cell levels. To further characterize the gp41 epitope, we used a series of overlapping synthetic peptides derived from a conserved region of the envelope gp41 (aa 572-613). We thus identified an immunodominant 12-mer peptide sequence, gp41(8)(aa 593-604), which consistently elicited both T cell blastogenic and B cell (antibody) responses in AS HIV-seropositive individuals but not in ARC and AIDS patients. Linear regression analysis showed that in AS persons there was a strong positive correlation (P less than 0.0005) between the absolute CD8+ T cell numbers and the magnitude of blastogenic responses to the gp41(8)(aa 593-604). Furthermore, those AS subjects with T cells that proliferated in response to this gp41 analogue also had significantly greater serum levels of antibody to the same short peptide sequence than symptomatic ARC and AIDS patients. These results suggest that cellular responses to the immunodominant and highly conserved envelope sequences of HIV-1, associated with increased CD8+ T cells, may be important in the pathogenesis of HIV disease.     

Human herpesvirus 8 reactivation and human immunodeficiency virus type 1 gp120

CONTEXT: Human herpesvirus 8 (HHV-8) is the presumed etiologic agent of Kaposi sarcoma (KS), the most common neoplasm in patients with acquired immunodeficiency syndrome. Current evidence indicates HHV-8 is necessary, but not sufficient, for KS development without the involvement of other cofactors. One potentially important cofactor is human immunodeficiency virus type 1 (HIV-1). Although HIV-1 is not essential for development of KS, studies have shown factors released from HIV-1-infected cells, including HIV-1 proteins and cytokines, promote the growth of KS cells in vitro. Recently, studies have shown that coculture of HIV-1-infected T cells with HHV-8-infected primary effusion lymphoma cell lines results in HHV-8 reactivation. This response was due, in part, to cytokines. However, only a portion of induced HHV-8 replication could be accounted for by cytokine stimulation, indicating that other factors, including HIV-1-associated proteins, may also be involved. OBJECTIVE: To investigate a possible role for HIV-1 gp120 in HHV-8 reactivation. DESIGN: Using an in vitro model system, we examined the effect of recombinant HIV-1 gp120 protein on HHV-8 replication in latently infected primary effusion lymphoma cell lines. MAIN OUTCOME MEASURES: Reactivation of HHV-8 was analyzed using Northern blot analysis and quantitative polymerase chain reaction for ORF26 messenger RNA expression, a gene encoding for the HHV-8 minor capsid protein produced only during reactivation. The results were extended and confirmed using a luciferase reporter construct driven by the HHV-8 ORF50 promoter, the first promoter activated during HHV-8 replication. RESULTS: No evidence of enhanced HHV-8 replication was found following treatment with HIV-1 gp120. In addition, HIV-1 gp120 was unable to act synergistically with interferon-gamma or hepatocyte growth factor/scatter factor to enhance reactivation of the virus in infected primary effusion lymphoma cell lines. CONCLUSIONS: HIV-1 gp120 does not appear to be responsible for the reactivation of HHV-8 demonstrated in our previous studies. Further studies are necessary to determine if other HIV-associated proteins, particularly Tat, gp160, and/or gp41, which are also released from infected cells, may be important in inducing HHV-8 reactivation.     

Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.     

Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41

Two α-helical heptad repeats, N-HR and C-HR, located in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, play an important role in membrane fusion by forming a 6-helix bundle. C34, a peptide mimicking C-HR, inhibits the formation of the 6-helix bundle; thus, it has potential as a novel antiretroviral compound. In order to improve the inhibitory effect of C34 on HIV-1 replication, we designed new C34-derived peptides based on computational analysis of the stable conformation of the 6-helix bundle. Newly designed peptides showed a stronger inhibitory effect on the replication of recombinant viruses containing CRF01_AE, subtype B or subtype C Env than C34 or a fusion inhibitor, T-20. In addition, these peptides inhibited the replication of a T-20-resistant virus. We propose that these peptides could be applied to develop novel antiretroviral compounds to inhibit the replication of various subtypes of HIV-1 as well as of T-20-resistant variants.     

Synthetic env gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection

An enzyme immunoassay (EIA) for serum antibodies to human immunodeficiency virus type 1 (HIV-1), based on the synthetic pentadecapeptide SGKLICT-TAVPWNAS, a segment of the transmembrane glycoprotein (gp41) of the virus, was developed and tested for sensitivity and specificity. Sera of 152 individuals at various stages of HIV-1 infection, including two prospectively and six retrospectively studied patients exposed to HIV-1 but seronegative on initial testing in whole-virus EIA and immunoblotting, were screened with the gp41 peptide antibody EIA. The reference population consisted of 1,000 healthy HIV-1 antibody-negative blood donors. In addition, five individuals with antibodies to HIV-2 were studied. Antibodies to the synthetic peptide were detected in 100% of those with asymptomatic infection. Only one patient with LAS failed to react in the peptide EIA. Patients with HIV-2 infection did not react in this test. The peptide antibodies appeared rapidly after infection, were detectable at the time when seroconversion was observed by immunoblotting, and preceded reactivity in whole-virus EIA. Sera of seven patients with verified HIV-1 infection did not react with gp41 in immunoblotting, although antibodies were readily detectable in the gp41 peptide EIA.