A new capture test using conjugated peptides for the detection of HIV antibodies.

A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591-611) YLKDQQLLGIWGCSGKLICTT, the gp120 sequence (314-329) IRIQRGPGRAFVTIGK and the p27 sequence (182-198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N-hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indirect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies.     

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.     

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.     

重组HIV-1抗原的应用及评价

将纯化的基因重组HIV-1P24和融合蛋白P24-gp41包被微孔板,用ELISA分别检测正常人血清和HIV-Ab国家参比品（Panel),rP24对20份HIV-Ab阳性Panel的检出率为95%（19/20）,对20份HIV-Ab阳性Panel通过率为90%（18/20）,P24-gp41对20份HIV-Ab阳性Panel检出率为90%（18/20）,对20份HIV-Ab阴性Panel的通过率为60%（12/20）;rP24和P24-gP41对正常人血清的检测结果均为阴性,结果表明研制的P24具有较高的敏感性和特异性,可作为组份抗原用于HIV抗体诊断试剂盒的生产。     

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

HIV蛋白质优势B细胞抗原表位的筛选、重组表达与抗原性鉴定

设计了一种新的病原体蛋白质B细胞抗原表位的筛选和重 组表达方法。不须使用抗原,而通过交替用病人血清IgG抗体“淘洗”(biopanning)随机肽库和用正常人血清IgG反向吸附,来获得特异抗原表位资料。用HIV病人血清的IgG抗体淘洗噬菌体递呈随机十二肽库,再以正常人IgG抗体吸附,筛选到了能和HIV病人血清发生特异反应的噬菌体克隆,经ELISA、DNA测序等,成功地筛选出了位于HIV gp41外膜蛋白、高亲和力、构型特异的优势B细胞抗原表位(602GCSGKLICTTNV613)。用大肠杆菌硫氧还蛋白作为骨架,在其活性部位以“内融合”形式重组表达了该抗原表位,纯化的重组蛋白具有良好的抗原性,能与HIV(+)IgG抗体及艾滋病人血清呈特异反应,表明本技术路线可以有 效地进行HIV蛋白质的B细胞抗原表位筛选和重组表达。此方法也可移植于其它病原微生物抗原或自身抗原的表位研究,继而为基于抗原表位水平的特异诊断试剂的研制、疫苗的设计提供依据。     

Construction of Peptides Mimicking a HIV-1 gp41 Epitope Recognized by Virus-Neutralizing Antibodies 2F5

A phage peptide library was screened with virus-neutralizing monoclonal antibodies (MAb) 2F5 which recognize a conserved epitope of HIV-1 gp41. Phages that expose peptides specifically binding with MAb 2F5 were selected by ELISA. Amino acid sequence analysis revealed homology to region 662–671 of HIV-1 HB10 gp160 for most peptides. The major role in recognition was ascribed to Asp-664, Lys-665, and Trp-666. The epitope-mimicking peptides were tested for immunogenicity. Antibodies to gp41 were detected in the serum of immunized rabbits.     

Construction of peptide mimetics of an epitope of the human immunodeficiency virus (HIV-1) gp41 protein, recognized by virus-neutralizing antibodies 2F5

A phase peptide library was screened with virus-neutralizing monoclonal antibodies (MCA) 2F5 which recognize a conserved epitope of HIV-1 gp41. Phages that expose peptides specifically binding with MCA 2F5 were selected by ELISA. Amino acid sequence analysis revealed a homology to region 662-671 of HIV-1 HB10 gp160 for most peptides. The major role in recognition was ascribed to Asp-664, Lys-665, and Trp-666. The epitope-mimicking peptides were tested for immunogenicity. Antibodies to gp41 were detected in serum of immunized rabbits.     

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。     

Characterization of a novel human anti-HIV-1 gp41 IgM monoclonal antibody designated clone 37

Human monoclonal antibodies (HuMAbs) demonstrate great potential for passive immunotherapy against HIV-1. The gp41 transmembrane envelope glycoprotein of HIV has an important role in the pathogenicity of AIDS and importantly displays considerably less hypervariability than the gp120 surface envelope HIV glycoprotein, which makes it particularly a better candidate for the development of passive and active immunotherapies. The general aim of this study was to develop HuMAbs to HIV surface glycoproteins and particularly gp41. Peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-seropositive long-term nondisease progressing patient. B-cells from this individual were then immortalized by Epstein-Barr virus (EBV) transformation, and antibody production was stabilized by fusion of transformed cells with a heteromyeloma. Subsets of the human heterohybridomas so generated were analyzed by ELISA. The hybridoma with the highest binding by immunoassay against gp160 was further analyzed. This hybridoma, designated as clone 37 (C37), was determined to be an IgM Kappa antibody and overlapping peptides of HIV envelope proteins (derived from the MN tissue culture line adapted HIV isolate) were used to map the specific binding domain of this HuMAb. Overlapping peptides designated 2026 (SWSNKSLDDIWNN, AA614-626), and 2027 (DDIWNNMTWMQWEREIDNYT, AA621-640) within the HIV-1 gp41 transmembrane glycoprotein were demonstrated to bind to C37 indicating that the specific binding domain for the antibody was DDIWNN. High affinity binding of C37 by ELISA to recombinant gp41 was demonstrated as well. Few IgM HuMAbs against HIV have been generated and characterized. Theoretically, because of the pentameric binding nature of IgM antibodies as well as their very efficient ability to activate complement, such reagents could have potential as anti-HIV agents.     

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.     

Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.     

Evaluation of ELISA for the Detection of Toxoplasma Antibodies in Swine Sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from > 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving < 10 EIU were negative in the other tests, and all those with > 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10–70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Antigenic activity of three chimeric synthetic peptides of the transmembrane (gp41) and the envelope (gp120) glycoproteins of HIV-1 virus

The antigenicity of three chimeric synthetic peptides (Qm, Qm-16, and Qm-17) incorporating an immunodominant epitope of the gp41 transmembrane protein (587-617) and the different epitopes of the gp120 envelope protein (495-516), (301-335), (502-516) of human immunodeficiency virus (HIV-1), separated by two glycine residues, was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HIV-1 positive sera (n = 47). The specificity was evaluated with samples from healthy blood donors (n = 20) and anti-HIV-2 positive samples (n = 10). The results indicate that the chimeric peptide, Qm, was the most reactive one because it detected antibodies to virus efficiently. This may be related to peptide adsorption onto the solid surface, the C-terminal region of HIV-1 gp120 (495-516) combined with gp41 (587-617) in the chimera, and the epitope accessibility to the antibodies. This study showed the usefulness of the chimeric peptides as antigen to detect antibodies to HIV-1 virus.     

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.     

Immunogenetic properties of peptides mimicking a human immunodeficiency virus gp41 (HIV-1) epitope recognized by virus-neutralizing antibody 2F5

Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (MCA) 2F5. Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages. Antibodies to gp41 were detected in the sera of immunized animals. The virus-neutralizing activity of the sera was examined.     

Immunogenic Properties of Peptides Mimicking a HIV-1 gp41 Epitope Recognized by Virus-Neutralizing Antibodies 2F5

Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (mAb) 2F5. Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages. Antibodies to gp41 were detected in the sera of immunized animals. The virus-neutralizing activity of the sera was examined.     

Exploring optical spectroscopic techniques and nanomaterials for virus detection

Viral infections pose significant health challenges globally by affecting millions of people worldwide and consequently resulting in a negative impact on both socioeconomic development and health. Corona virus disease 2019 (COVID-19) is a clear example of how a virus can have a global impact in the society and has demonstrated the limitations of detection and diagnostic capabilities globally. Another virus which has posed serious threats to world health is the human immunodeficiency virus (HIV) which is a lentivirus of the retroviridae family responsible for causing acquired immunodeficiency syndrome (AIDS). Even though there has been a significant progress in the HIV biosensing over the past years, there is still a great need for the development of point of care (POC) biosensors that are affordable, robust, portable, easy to use and sensitive enough to provide accurate results to enable clinical decision making. The aim of this study was to present a proof of concept for detecting HIV-1 pseudoviruses by using anti-HIV1 gp41 antibodies as capturing antibodies. In our study, glass substrates were treated with a uniform layer of silane in order to immobilize HIV gp41 antibodies on their surfaces. Thereafter, the HIV pseudovirus was added to the treated substrates followed by addition of anti-HIV gp41 antibodies conjugated to selenium nanoparticle (SeNPs) and gold nanoclusters (AuNCs). The conjugation of SeNPs and AuNCs to anti-HIV gp41 antibodies was characterized using UV–vis spectroscopy, transmission electron microscopy (TEM) and zeta potential while the surface morphology was characterized by fluorescence microscopy, atomic force microscopy (AFM) and Raman spectroscopy. The UV–vis and zeta potential results showed that there was successful conjugation of SeNPs and AuNCs to anti-HIV gp41 antibodies and fluorescence microscopy showed that antibodies immobilized on glass substrates were able to capture intact HIV pseudoviruses. Furthermore, AFM also confirmed the capturing HIV pseudoviruses and we were able to differentiate between substrates with and without the HIV pseudoviruses. Raman spectroscopy confirmed the presence of biomolecules related to HIV and therefore this system has potential in HIV biosensing applications.