利用gp41蛋白的合成肽抗原检测HIV—1抗体

采用固相多肽合成技术对人免疫缺损病毒-1型(HIV-1)gp41蛋白的一段代表优势抗原表位的22肽进行了化学合成,经反相高效液相层析(HPLC)和多肽序列测定表明,合成肽成品均质性良好,其氨基酸序列与设计相符。利用该合成肽作为包被抗原,以间接 ELISA 法检测 HIV-1血清抗体,其特异性、灵敏性和重复性均达到与国外生产的 gp41合成肽抗原相同的水平.     

Neutralization of HIV-1 primary isolate by ELDKWA-specific murine monoclonal antibodies

ELDKWA on HIV-1 gp41 is a conserved epitope recognized by one broadly neutralizing monoclonal antibody 2F5, which is a promising candidate target for vaccine design. Here we report two ELDKWA-specific monoclonal antibodies (mAbs), 18F11 and 7E10, that were screened from the splenocytes of mice immunized by recombinant GST-(ELDKWA)4 protein. In further evaluation, these mAbs exhibited appreciable neutralizing activities against HIV-1 primary isolate 92US675 (clade B) with IC50 (50% inhibition concentration) of 6.84 +/- 0.36 microg/ml and 10.66 +/- 1.69 microg/ml, respectively. Unexpectedly, neither of these two murine mAbs could neutralize laboratory-adapted strain HIV-1 IIIB (clade B). As a control, human mAb 2F5 neutralized both primary and laboratory-adapted strains. These data strongly suggest that ELDKWA-specific antibodies induced by different antigenic formats show different neutralizing activities against HIV-1, which implies another complication in the development of effective vaccines.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4- binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.      

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-β in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-β (aa128–134) and HIV-1 gp41 (aa586–595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-β antibody levels by ELISA. The levels of anti-IFN-β antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-β in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-β recognized rsgp41 (recombinant soluble gp41, Env amino acid 539–684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-β and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128–134 of human IFN-β and the immunosuppressive domain (aa583–599) of HIV-1 gp41. The increased levels of antibodies against interferon-β in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-β and HIV-1 gp41.     

HIV-1 gp41 NHR结合性环肽的研究

目的 筛选可与HIV-1 gp41 NHR结合的环肽，为研制抗HIV-1早期感染的小分子药物奠定基础。方法 采用P＋LS方法，用源于gp41 NHR的合成肽N36肽筛选噬菌体环七肽库，ELISA鉴定噬菌体克隆，根据阳性克隆的DNA序列，合成环肽并鉴定其与N36肽结合。结果 经3轮筛选、鉴定，得到11个和N36肽结合的噬菌体克隆。DNA测序并推导氨基酸序列，表明这11个克隆展示同一序列CDRHQHKRC。根据此序列合成的环肽NA（Biotin—SACDRHQHKRCGG）经与载体蛋白BSA偶联后，EIJSA鉴定表明交联物可特异地与N36肽结合，这种结合可被游离N36肽、以及源于gp41 CHR的肽C34抑制。结论 sACDRHQHKRCGG环肽为HIV-1 gp41 NHR结合肽。     

抗HIV-1 gp41 合成多肽C34单抗的制备及生物学活性

目的 制备针对C34和C46单抗，并以此为工具研究gp41表位，进一步了解HIV－1包膜蛋白的作用机理和病毒的感染机制，为寻找新的治疗靶点提供抗体工具。方法 常规动物免疫、细胞融合、克隆化制备抗C34和C46的单克隆抗体，并鉴定其特异性位点，还用ELISA法、MTTI法及荧光分子探针技术对单抗的生物学活性进行研究。结果 获得了3株抗C34单克隆抗体、2株抗C46单抗。此5个单抗均与C34结合，并抑制N36肽与C34肽复合物的形成；其中效价最高的1G1对H9／HIVⅢB细胞的生长有刺激作用，对H9／HIVⅢB细胞和MT－2细胞的融合无明显影响。结论 得到5株可与C34反应，并可抑制C34与N36多肽结合的单抗，其中1G1所识别的表位可能为增强性或刺激性表位。     

Enhanced HIV-1 envelope-tumor protection by a recombinant vaccinia virus expressing anchored HIV-1 gp120 lacking gp41

A recombinant vaccinia virus (rvv) expressing, human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein, gp120, fused to a non-cleavable transmembrane protein, vvE13, elicited protection against a tumor cell line expressing HIV-1 full length envelope glycoprotein, gp160, in mice. Mice vaccinated with vvE13 exhibited a decreased incidence of tumor development and significantly smaller tumors in comparison to mice vaccinated with rvv gp160, vvE1, or a thymidine kinase minus (TK-) rvv, vSC11, or phosphate-buffered saline (PBS) injected controls. vvE13 and vvE1 also delayed tumor development, compared to vSC11 and PBS-injected controls; however, a statistical correlation could not be demonstrated due to the development of tumors in so few animals. Specificity toward HIV-1 envelope glycoprotein, was shown, since HIV-1 envelope-tumor prevention (incidence for vvE13 and size for vvE1 and vvE13 and delay for vvE1 and vvE13) was statistically superior with HIV-1 envelope expressing tumors compared to parenteral tumors. The vvE13 recombinant vaccinia virus expressing the HIV-1 envelope glycoprotein gp120 fused to a non-cleavable transmembrane protein elicits superior protection against tumors expressing the gp160 envelope glycoprotein, as compared to vvE1 expressing gp160.     

Epitope mapping of apamin by means of monoclonal antibodies raised against free or carrier-coupled peptide.

A panel of 20 monoclonal antibodies raised against the bee-venom peptide apamin (18 residues, 2 disulfide bridges) was prepared. Nine monoclonal antibodies (mAb) were obtained from a mouse immunized with free apamin and 11 from a mouse immunized with a mixture of free and carrier-coupled peptide. Using a panel of 11 synthetic apamin analogs, we examined the fine antigenic specificity of each antibody. The mAb generated against free apamin preferentially bound to the central part of the peptide and less frequently recognized the N- and C-terminal regions. However, monoclonal antibodies obtained by immunization with carrier-bound apamin showed a broader range of specificities, consistent with the possibility of the entire surface of this small antigen becoming immunogenic upon coupling to the carrier.     

模拟HIV-1 gp41 CHR表位短肽的筛选及研究

目的 利用针对HIV-1跨膜蛋白gp41CHR序列合成肽C34的单克隆抗体1G1筛选噬菌体12肽库，旨在找寻模拟C34肽表位的序列，同时探索该短肽成为HIV-1gp41NHR与CHR结合抑制物的可能性。方法 以1G1为钓饵蛋白对噬菌体12肽库进行亲和筛选，以双夹心ELISA鉴定阳性克隆。结果 经3轮筛选后，随机挑选17个噬菌体克隆，其中6个克隆与1G1显示出较强的结合活性，上述6个阳性克隆经DNA测序，氨基酸序列相同；HYEFWAWNWEAN，其明显的疏水性质类似于G34N末端，特异性鉴定显示这些克隆均能够与HIV-1gp41N多肽结合，结论 该噬菌体克隆展示肽可模拟HIV-1gp41CHR多肽表位，并可与N多肽结合。     

HIV—1gp41基因的分段克隆和表达

目的 在大肠杆菌中表达HIV－1gp41N肽和C肽基因。方法 用PCR方法从含HIV－1gp160基因的质粒中扩增HIV－1gp41N肽和C肽基因,重组人pGEX-4T-1载体,并亚克隆人pGEM7zf( )中测定核苷酸序列,限制性酶切鉴定后进行原核表达。结果 成功地扩增到HIV－1gp41N肽和C肽基因,酶切鉴定及测序结果与已知HIV－1亚型的gp41N肽区和C肽区基因序列一致,SDS－PAGE结果显示,表达出与预期分子量大小相同的蛋白。结论 N肽和C肽基因的成功表达,为进一步研究其结构和功能奠定了基础。     

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.     

Antibodies to HIV-1 gp41 recognize synthetic peptides of human IFN-alpha and IFN-beta

Based on our finding that a common epitope exists between HIV-1 gp41 and human type I interferons (IFN-alpha and IFN-beta), and increased levels of antibodies against human IFN-alpha and IFN-beta were observed in HIV-1-infected individuals, we tried to explain the mechanism of increased levels of antibodies. Mouse antisera recognizing HIV-1 recombinant soluble (rs) gp41 (aa 539-684) interacted with two synthetic peptides sequence-corresponding to the IFN-alpha/beta receptor binding site on human IFN-alpha and IFN-beta, while normal mouse serum (pooled normal sera) did not. The anti-rspg41 antisera after adsorption by IFN-beta sepharose column lost the activity of interaction with both synthetic peptides. In another experiment, rsgp41 could bind to sepharose column conjugated with anti-IFN-beta polyclonal antibodies (IgG). These results indicate that the common epitope on gp41 and type I interferons could induce antibodies recognizing the receptor binding site on IFN-alpha and IFN-beta, suggesting that increased levels of antibodies against IFN-alpha and IFN-beta in HIV-1-infected individuals could be induced by gp41.     

The effect of sCD4 on the binding and accessibility of HIV-1 gp41 MPER epitopes to human monoclonal antibodies

Two human monoclonal anti-HIV-1 antibodies, 2F5 and 4E10, were utilized to investigate the accessibility and conservation of gp41 MPER epitopes on five different clades of HIV-1 in the absence and presence of sCD4. The binding of human monoclonal antibodies (mAbs) to HIV-1 was dependent upon the virus clade. Soluble CD4 significantly increased the accessibility of gp41 MPER-binding epitopes on several isolates that previously showed little or no binding with 2F5 and 4E10 mAbs as determined by a modified ELISA-based virus capture assay and surface plasmon resonance. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus. However, neither binding per se nor the total envelope content per virion was a predictor of neutralization. The hidden or conformational gp41 MPER epitopes unmasked by sCD4 may provide additional targets for vaccine design.     

Analysis of a subclass-restricted HIV-1 gp41 epitope by omission peptides.

To define the amino acids involved in IgG subclass reactivity to two overlapping HIV-1 gp41 (E34/32; amino acid positions 582-613) peptides, sera from 18 HIV-infected individuals were studied. Peptides mimicking E34 but with single amino acid deletions or glycine substitutions were used to define the amino acid residues necessary for antibody binding. Two dominating immunogenic epitopes, containing highly hydrophilic amino acids, were found on the original peptide. Further analysis was undertaken with two corresponding omission sets of dodecapeptides representing halves of the complete E34 plus a terminal cystein peptide. The subclass reactivities usually differed between the patients with regard to the epitopes with which the different IgG subclasses reacted and also to the importance of different amino acids in antibody binding. The 600 glycine and the 601 lysine were involved in the binding of all IgG1, 2 and 4 and most IgG3. The development of E34/32-reactive IgM and IgG subclasses showed different patterns in four patients with primary HIV infections, contradicting the existence of a general pattern for the development of IgG subclasses to this peptide. The findings suggest that different progenitor clones are selected for synthesis of the different subclasses.     

HIV-1 gp41 and type I interferon

HIV-1 gp41-like human type I interferon (IFN) could inhibit lymphocyte proliferation and up-modulate MHC class I and II and ICAM-1 molecule expression. Sequence comparison indicates that a similar epitope RILAV-YLKD exists between N-domain of gp41 and two regions in IFN-alpha(aa29-35 and 113-129), IFN-beta (aa31-37 and 125-138) and IFN-omega (aa29-35 and 123-136), which was shown to form IFN-alpha/beta-receptor binding site. Weak sequence similarity was also found to exist in both regions on gp41 and type I IFN of murine and bovine. Experimental studies indicated that a common immunological epitope exists between gp41 and IFN-alpha and -beta. Antibodies against human IFN-alpha and -beta recognized the common immunological epitope and inhibited gp41-binding to the potential cellular receptor protein p45. Moreover, the polyclonal antibody to IFN-beta completely inhibited gp41-binding to human T, B cells and monocytic cells, while IFN-alpha could only inhibit this binding incompletely. It was interestingly observed that human IFN-beta after preincubating with cells could incompletely inhibit the binding of gp41 to human B cells and monocytic cells, and very weakly inhibit the binding to human T cells, indicating that the receptor for IFN-beta-binding may be involved in gp41 binding. This potential relationship may be based on the amino acid sequence homology in the receptor binding region between gp41 and IFN-beta. It was observed that the increased levels of antibodies against human IFN-alpha and -beta exist in HIV-1-infected individuals and are associated with the common epitope on gp41. Besides, several studies provided experimental evidence that the common immunological epitope could induce protective activity against HIV-1. The IFN-alpha-based vaccine has showed a significant reduction of disease progression in IFN-alpha-vaccine-treated HIV-infected patients. Recent experimental evidence indicates that gp41 and IFN-beta were involved in downregulation of CCR5 expression and induction of cell activation or signal transduction. Whether it may be performed by a similar mechanism is still to be investigated.     

Isolation and characterization of monoclonal antibodies elicited by trimeric HIV-1 Env gp140 protein immunogens

Eleven anti-HIV Env monoclonal antibodies (MAbs) were isolated from mice immunized with soluble Env proteins derived from the clade B Env, SF162, or DeltaV2 (a derivative of SF162 lacking the V2 loop). All six anti-gp120 MAbs studied, neutralized SF162 and their activities were dependent by the glycosylation patterns of the V1, V2 or V3 loops. Only one anti-gp120 MAb (an anti-V3 MAb) displayed cross-neutralizing activity, which was influenced by the type of V1 loop present on the target heterologous viruses. None of the five anti-gp41 MAbs studied displayed anti-SF162 neutralizing activity. Our studies indicate that the current limitation of soluble HIV Env gp140 immunogens to elicit robust cross-reactive neutralizing antibody responses is not only due to the elicitation of high titers of homologous antibodies but also due to the elicitation of antibodies whose epitopes are naturally occluded, or not present, on the virion-associated Env.     

Characterization of a trimeric MPER containing HIV-1 gp41 antigen

The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.     

Development of monoclonal antibodies reactive with methamphetamine raised against a new antigen

Monoclonal antibodies (McAbs) specific to methamphetamine (MA) were produced using p-amino MA coupled to bovine serum albumin (BSA) with glutaraldehyde (GA) as an immunogen and with conventional hybridoma techniques. Hybridoma clones secreting the McAbs were selected by an enzyme-linked immunosorbent assay (ELISA) system using both the above conjugate and BSA modified with GA as screening antigens. In the ELISA system were used avidin and biotinyl-alkaline phosphatase which converts nicotinamide adenine dinucleotide phosphate (NADP) into NAD. The final enzyme activity was determined using diformazan of nitroblue tetrazolium formed together with the NAD produced, alcohol dehydrogenase and phenazine methosulfate. The McAbs from 9 clones were characterized by a crossreactivity test using the ELISA. The McAbs recognized MA (100%), methoxyphenamine (8.0%), ephedrine (2.3%), but did not react with metylephedrine, amphetamine, OH-amphetamine, dimethylamphetamine, beta-phenylethylamine, norephedrine, phentermine and ranitidine. An inhibition curve for MA was obtained in the range of 0.75 to 50 ng.     

人源抗HIV-1 gp120趋化蛋白受体结合位点Fab单克隆抗体基因的获得

目的：筛选人源抗HIV－1 gp120趋化蛋白受体结合位点噬菌体Fab单克隆抗体基因。方法：根据HIV－1 gp120趋化蛋白受体结合位点的氨基酸序列合成23肽，并以此为固相抗原从HIV－1噬菌体Fab抗体库筛选阳性克隆，并进行鉴定及序列测定。结果：获得了1株抗HIV－1 gp120趋化蛋白受体结合位点的人源Fab抗体克隆，具有较高的亲和力、特异性和抑制率，序列测定及分析显示该抗体属IgG I亚类，κ型，重、轻链可变区分别属VhⅢ和VκⅢ基因家族。结论：成功地获得了人源抗HIV－1 gp120趋化蛋白受体结合位点噬菌体Fab单克隆抗体基因。