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1.
Antifusion activity in sera from persons infected with human immunodeficiency virus type 1.
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B S Graham J M Rowland A Modliszewski D C Montefiori 《Journal of clinical microbiology》1990,28(12):2608-2611
Cell-to-cell fusion plays an important role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infections. An assay to measure the antifusion activity of serum has been developed by using the fusion event that occurs between H9 cells chronically infected with HIV-1 (H9IIIB) and fusion-susceptible MT-2 cells. The endpoint is determined by measuring neutral red uptake in cells after syncytium formation is allowed to occur in the presence of various serum dilutions. The assessment of antifusion activity in serum by neutral red uptake has been shown to correlate with syncytium reduction as determined by direct counting. The optimal number and ratio of cells in the suspension for efficiency and speed of the assay have been determined. With this assay it was shown that 50% of 36 serum specimens capable of neutralizing cell-free virions failed to inhibit syncytium formation. The assay can thus measure a distinct activity in HIV-1-immune human sera which is a subset of neutralization activity. Because of the potential role of this activity in the rate of disease progression and protective immune responses, the antifusion assay will be an important tool for the investigation of disease pathogenesis and for acquired immunodeficiency syndrome vaccine development. The assay can also be applied to the investigation of the pathogenesis of the fusion event at the cellular level. The ability to use absorbance measurements rather than syncytium counts as the endpoint facilitates direct computer-assisted data analysis, which expedites the performance of the assay. 相似文献
2.
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. 相似文献
3.
Immunoprecipitation of human immunodeficiency virus type 2 glycoproteins by sera positive for human immunodeficiency virus type 1.
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Analysis by radioimmunoprecipitation of serum samples from 27 different human immunodeficiency virus type 1 (HIV-1)-infected individuals residing in Chile showed that the sera of 26% of these individuals also react with glycoprotein gp125 of HIV type 2 (HIV-2). This cross-reaction seems to reflect a qualitative difference among infected individuals, because the titer of antibodies against gp120 of HIV-1 in the cross-reacting samples did not differ significantly from that in the non-cross-reacting samples. Most of the HIV-1-seropositive sera, including many that did not react with gp125 of HIV-2, reacted with gp140, the precursor of HIV-2 glycoproteins. The observed cross-reactions allowed us to distinguish three groups of HIV-1-infected individuals: (i) those whose sera react with both gp140 and gp125, (ii) those whose sera react with gp140, and (iii) those whose sera react with neither of these glycoproteins. The possible cause and significance of these differences is under study. 相似文献
4.
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. 相似文献
5.
Synthetic env gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection 总被引:10,自引:0,他引:10
Ale Nrvnen Mirja Korkolainen Jukka Suni Jukka Korpela Sari Kontio Paul Partanen Antti Vaheri Marja-Liisa Huhtala 《Journal of medical virology》1988,26(2):111-118
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. 相似文献
6.
L H Gosting J McClure E S Dickinson S M Watanabe K Shriver L C Goldstein 《Journal of clinical microbiology》1987,25(5):845-848
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. 相似文献
7.
Viveros M Dickey C Cotropia JP Gevorkian G Larralde C Broliden K Levi M Burgess A Cao C Weiner DB Agadjanyan MG Ugen KE 《Virology》2000,270(1):135-145
Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600-614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits. 相似文献
8.
Comparison of antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in sera from HIV-1-infected individuals from Tanzania and from the United States.
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R Q Warren W M Nkya J F Shao S A Anderson H Wolf C W Hendrix P Kanda M Wabuke R N Boswell R R Redfield et al. 《Journal of clinical microbiology》1992,30(1):126-131
In this study, we compared sera from 159 human immunodeficiency virus type 1 (HIV-1)-infected individuals from Tanzania and 103 infected individuals from the United States for antibodies reactive with 10 HIV-1 gp160 epitopes defined by synthetic peptides. Our data indicate that the anti-gp160 antibody fine specificity differs between infected individuals from these two geographically diverse populations. For example, 50% of the Tanzanian sera contained antibodies reactive with an immunodominant HIV-1 gp41 epitope defined by peptide 600-611, whereas 91% of the sera from the United States were reactive. Differences in serologic reactivity between HIV-1-infected individuals from Tanzania and the United States were also observed with gp160 epitopes defined by peptides 503-528 and 846-860. Included among the peptides examined were four which corresponded to the V3 region of gp120. The majority of sera from either country contained antibodies reactive with peptide RP142, whose V3 sequence is based upon that of HIV-1 isolate MN. Further characterization of serologic reactivity suggested that sera from Tanzania were more likely to neutralize HIV-1 isolate IIIB or MN in vitro than were sera from the United States. These differences in antibody fine specificity between HIV-1-infected individuals from Tanzania and the United States suggest that regional isolates of HIV-1 may exist. 相似文献
9.
Jianhui Nie Aijing Song Sihong Xu Xiuhua Li Youchun Wang 《Archives of virology》2010,155(11):1813-1822
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. 相似文献
10.
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 相似文献
11.
Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping 总被引:3,自引:0,他引:3
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Agwale SM Robbins KE Odama L Saekhou A Zeh C Edubio A Njoku OM Sani-Gwarzo N Gboun MF Gao F Reitz M Hone D Folks TM Pieniazek D Wambebe C Kalish ML 《Journal of clinical microbiology》2001,39(6):2110-2114
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. 相似文献
12.
West African populations are infected with divergent strains of human immunodeficiency virus type 2 (HIV21, some of which are closely related to simian immunodeficiency virus (SIV) and it has been postulated that the HIV2 epidemic might have arisen by cross-species spread of SIV into the human population in West Africa. To gain some insight into the possible basis for cross protection between these two closely related viruses, the T-helper responses to 15 synthetic peptides from SIV gag synthetic peptides were investigated in seven HIV2-infected subjects and in seven healthy controls. Significant reactivity to at least one of the synthetic peptides tested was found in all patients and a statistically significant correlation between CD4+ lymphocyte absolute numbers and the number of reacting peptides was observed. A marginal lymphocyte reactivity was found in two of the healthy controls studied. In conclusion, this preliminary evidence that HIV2-infected patients exhibit T-cell responses to SIV gag peptides suggests that both viruses share t-helper epitopes in the gag viral region and raises the possibility of cross protection between SIV and HIV2 which may be relevant for HIV2 vaccine research based on closely related retroviruses. © 1995 WiIey-Liss, Inc. 相似文献
13.
Monitoring human immunodeficiency virus type 1-infected patients by ratio of antibodies to gp41 and p24 总被引:6,自引:1,他引:5
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G Schmidt K Amiraian H Frey J Wethers R W Stevens D S Berns 《Journal of clinical microbiology》1989,27(5):843-848
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. 相似文献
14.
15.
Performance of human immunodeficiency virus type 1 gp41 assays for detecting enfuvirtide (T-20) resistance mutations
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Marcial M Lu J Deeks SG Ziermann R Kuritzkes DR 《Journal of clinical microbiology》2006,44(9):3384-3387
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. 相似文献
16.
Lu C Gordon GM Chandran B Nickoloff BJ Foreman KE 《Archives of pathology & laboratory medicine》2002,126(8):941-946
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. 相似文献
17.
目的提高人免疫缺陷病毒Ⅰ型(HIV-1)包膜糖蛋白gp120基因在原核系统中的表达量。方法采用聚合酶链反应(PCR)技术扩增出560bp的HIV-1LAV株gp120N端基因片段,经EcoRⅠ及SalⅠ酶切后插入高效表达载体pET28a,得到重组质粒pET120,并转化表达宿主菌BL21(DE3),经诱导高效表达出HIV-1gp120基因片段。结果间接酶联免疫吸附试验(ELISA)及Westernblot实验表明,表达产物具有良好的抗原性及特异性。SDS-PAGE电泳分析结果表明,gp120表达量占总菌体蛋白的50%。结论在原核系统中高效表达了HIV-1gp120基因 相似文献
18.
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. 相似文献
19.
人类免疫缺陷病毒(human immunodeficiency virus type,HIV-1)包膜蛋白gp41序列相对保守,其近膜区(MPER)具有一些已知广谱中和抗体(bNAbs)的表位.在HIV疫苗研究中,尽管利用gp41的MPER中和表位进行疫苗设计,但其免疫效果仍不理想.近年发现,一些靶向于gp120和gp41交界面的bNAbs为HIV疫苗的研制提供了新的靶标.如何更好的模拟天然状态的gp41结构以诱导bNAbs的产生仍是学者们的主要研究方向,有大量研究关于靶向于gp41的bNAbs的表位、gp41免疫原的修饰、免疫策略及免疫效果等,因而探讨gp41作为免疫原候选的可行性及提高其免疫效果的方法具有重要意义. 相似文献