HIV-1和HIV-2混合感染的发现

人免疫缺陷病毒(ＨＩＶ)分两个型,即ＨＩＶ1和ＨＩＶ2。继ＨＩＶ1于1983年从淋巴腺病病人分离到后,1986年又从西非的ＨＩＶ感染人群分离到与ＨＩＶ1在基因序列相差达55%以上的另一种ＨＩＶ,后者被分类为ＨＩＶ2[1,2]。ＨＩＶ1和ＨＩＶ2一样都能引起免疫缺陷和艾滋病。但两...     

HIV-1感染引起组蛋白乙酰化和增加p21WAF1表达

目的：DNA甲基化和组蛋白乙酰化是基因表达调控的主要形式。人类免疫缺陷病毒（HIV-1）可引起T淋巴细胞DNA甲基化酶上调。本文旨在明确HIV-1对细胞周期依赖刺激酶抑制剂p21^WAF1表达的影响。方法：建立HIV-1感染的Hut78细胞系；以RT-PCR和Westem blotting 分析p21^WAF1表达情况；以亚硫酸氢钠修饰DNA和基因测序，研究p21^WAF1基因启动子甲基化，以Western blot-ting 和染色体免疫测定探究总组蛋白和与p21^WAF1基因启动子相关的组蛋白乙酰化水平。并以GST pull-down和免疫沉淀分析HIV-1导致乙酰化及乙酰化引起p21^WAF1过表达的可能机理。结果：HIV-1感染后，其反式激活蛋白Tat与辅助转录因子P/CAF、hGCN5结合，共同刺激组蛋白H3乙酰化。尽管p21^WAF1启动子部分区域有甲基化发生，但p21^WAF1表达仍上调。这可能与E2A对p21^WAF1的作用有关。结论：HIV-1感染可引起T淋巴细胞p21^WAF1基因的甲基化和乙酰化紊乱，导致p21^WAF1表达增强。     

胆红素衍生物体外抗HIV-1的初步研究

目的 初步研究胆红素衍生物（DTB）体外抑制HIV-1病毒复制的作用。为进一步研究其作用机理奠定基础。方法 将DTB加入到感染HIV-1的细胞中，在适当条件下培养，最终以其培养上清液中的HIV-1p24抗原滴度（ELISA法）及MTT染色细胞毒法（MTT法）判断DTB抑制病毒的效果。结果 DTB在对细胞无毒浓度作用下能够有效抑制HIV-1的复制，当DTB浓度为160，80和40mg/ml时，抑制率分别为93.0%,56.2%和18.1%。结论 DTB可以有效抑制体外培养HIV-1病毒的复制。     

小鼠作为HIV—1实验动物模型的研究进展

本文就转基因小鼠，严重联合免疫限制病鼠和正常的Ｓｗｉｓｓ小鼠用于ＨＩＶ－１感染实验动物模型进行了简单综述，小鼠模型对ＨＩＶ－１感染机制，抗逆转录病毒的治疗学和抗病毒药物的筛选、效果，ＡＩＤＳ疫苗的研究及效果观察都有实用价值。     

血友病患者中丙肝病毒的负荷与人类免疫缺陷病毒—1疾病发展相关联

目的　炎性细胞因子在胰腺缺血再灌注损伤中的作用尚不清楚 ,FR16 76 5 3是近年来新研制的能有效抑制白介素 -1β和肿瘤坏死因子 -α产生的复合制剂 ,本实验的目的是观察 FR在狗胰尾局部缺血再灌注损伤中的作用。方法　胰尾缺血 90 min。在阻断血流期间 ,胰头被切除。观察对照组 (n=14 )和 FR治疗组 (n=11)的动物成活率 ,组织血流 ,动脉氧分压 ,血清淀粉酶和脂肪酶水平 ,血糖和胰岛素 ,肝脏酶学 ,肌酐 ,白介素 - 1βm RNA在外周血中的变化和组织病理改变。结果　对照组狗死亡 6只 ,治疗组死亡 2只。治疗组血清淀粉酶、脂肪酶水平 (P=0 .…     

HIV—1耐药性研究的新进展

随着多种抗逆转录病毒治疗（ART）药物的广泛使用，以及高效抗逆转录病毒疗法（HAART，highly active antiretroviral therapy)的应用和推广，HIV－1感染者的并发症和AIDS病人的死亡率明显下降，抗HIV／AIDS的治疗效果是极其明显的，但是，HAART应用的局限性也是显而易见的。其中，HIV－1的耐药性是维持HAART长期疗效的障碍，如何加强HIV－1耐药性的临床和基础研究对治疗有十分重要的意义。本文旨在对产生HIV－1耐药性的分子机制，HIV－1治疗药物及其耐药性与HIV－1基因突变的相关性，HIV－1耐药性检测方法及其临床应用的可靠性和指导意义等方面的新进展作一综述报道。     

HIV—1及其nef蛋白存在有与正常人组织呈交叉反应的表位

本文报导了从大量抗HIV-1结构蛋白单抗和抗nef肽、nef蛋白单抗中发现的HIV-1及其nef蛋白存在有与正常人组织呈交叉反应的表位。这些单抗的特异性为抗HIV-1 p55+p24;抗gp160+gp120+gp41;抗p18+p27;抗nef蛋白(表位为由LHGM组成的四个氨基酸顺序)及抗nef蛋白肽(表位为由HPE或HPEY组成的氨基酸顺序)。它们与正常人扁桃体中的基质、血管内皮或平滑肌起反应。文中对AIDS病人自身免疫反应性进行了讨论。     

表达HIV-1 gag-pol△和gp 140 TM蛋白复制缺陷型重组腺病毒的构建及免疫效果研究

目的构建表达人免疫缺陷病毒Ⅰ型gag-pol△和gp140TM基因的非复制型重组腺病毒.方法首先将HIV-1的gag-pol△和gp140TM基因插入穿梭载体,再与腺病毒骨架质粒pAdEasy-1共转化E.coli BJ5183,获得重组子.转化293细胞后获得重组病毒.重组腺病毒与DNA疫苗联合免疫小鼠,ELISA检测小鼠血清中的抗体.结果获得两株重组腺病毒vAd-gag-pol△和vAd-gp140TM,能正确表达Gp140TM、Gag蛋白以及经剪切加工的P24蛋白,与DNA疫苗联合免疫小鼠后能产生高滴度的HIV-1特异性的抗体.结论成功构建了表达HIV-1结构基因的重组腺病毒,能有效诱导HIV-1特异性抗体.     

Comparison of the biological activities of human immunodeficiency virus 1 P24 and GP41 expressed in Spodoptera frugiperda cells by use of bac-to-bac system

Recombinant transposing plasmids pFH24 and pFH41 were constructed by cloning the human immunodeficiency virus 1 (HIV-1) p24 and gp41 genes, respectively, into the transposing vector pFastBacHTa. Recombinant bacmids rBH24 and rBH41 were obtained by transposing pPolh/p24 and pPolh/gp41 expression cassettes from recombinant plasmids pFH24 and pFH41, respectively. Recombinant viruses rAcH24 and rAcH41 were generated by transfection of the Spodoptera frugiperda (Sf9) cells with the DNAs of plasmids rBH24 and rBH41, respectively. Analysis of the expressed p24 or gp41 proteins with an antiserum to HIV-1 (HIV-1 antiserum) by an enzyme-linked immunosorbent assay (ELISA) and dot blot assay showed high biological activity of these proteins; p24 was more active than gp41. Also a Western blot analysis showed stronger bands for p24 than for gp41. The high reactivities of p24 and gp41 with the HIV-1 antiserum suggest that these proteins could also be used as specific standard antigens in HIV-1 diagnostics.     

Comparison of cervicovaginal humoral immunity in clinically asymptomatic (CDC Al and A2 category) patients with HIV-1 and HIV-2 infection

Paired sera and cervicovaginal secretions (CVS) from 11 HIV-1- and 11 HIV-2-infected women, all clinically asymptomatic (CDC A1 and A2 categories), were analyzed for total IgG, IgA, albumin (HSA), IgG, and IgA antibodies toenvencoded surface glycoproteins of HIV-1 (gp160) and of HIV-2 (gp105), by comparison to 15 age-matched healthy controls. Secretion rates of IgG and IgA into CVS were evaluated by calculation of their relative coefficients of excretion (RCE) by reference to HSA. Cervicovaginal production of anti-HIV antibodies was evaluated by comparison between specific antibody activities of IgG and of IgA to HIV in CVS and in sera. In HIV-1-infected women, total IgG and IgA in CVS were, respectively, 6- and 4-fold increased, whereas the secretion rate of total IgG was 2.1-fold increased and that of total IgA was 2.5-fold reduced. In contrast, total IgG and IgA as well as their secretion rates were normal in HIV-2-infected women. In HIV-1- but not in HIV-2-infected women, HSA levels in cervicovaginal washings were twofold increased, demonstrating alteration of the mucosal barrier in HIV-1 infection. In HIV-1-infected patients, IgG and IgA to gpl60 were detected in all sera and CVS. In HIV-2-infected patients, IgG to gp105 was detected in all sera and CVS, whereas IgA to gp105 could be detected in only half of sera and one-third of CVS. Cross-reactivity by IgG and/or IgA to HIV-1 or HIV-2 against the surface glycoprotein of the other HIV type was observed in sera as well as in CVS, and more frequently in HIV-2- than in HIV-1-infected women. Finally, the mean specific activities of IgG and of IgA to gp160 or gp105 were higher in CVS than in sera, evidencing a possible local synthesis of both isotypes in HIV-1 as well as in HIV-2 infections. As early as the asymptomatic stages, HIV-1 affects the cervicovaginal mucosa more than HIV-2 does, suggesting higher viral replication within the female genital tract in HIV-1 infection than in HIV-2 infection.     

In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection.

We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection.     

5株不同亚型HIV-1毒株gp120序列特征分析及其蛋白的表达

目的 分析我国HIV-1主要亚型毒株的gp120区域的序列特征,并表达出gp120糖基化蛋白,为研究gp120的致病机制和设计疫苗奠定基础.方法 利用巢式PCR从来自于不同省份的5名HIV-1感染者外周血单个核细胞DNA中扩增全长gp120基因并测序,对序列特征进行分析,并将获得的gp120基因克隆入真核表达载体,体外表达获得gp120糖基化蛋白.结果 序列分析显示,此5条gp120序列分属HIV-1 Thai-B、BC重组和AE重组哑型,虽然亚型不同,但这些gp120序列在相同的位置都存在着一些保守的糖基化位点,且都具备相同的Furin蛋白酶第一切割位点,与参考序列比较发现,不同的HIV亚型序列中,除V3序列长度较为保守外,V1、V2、V4、V5各区域都有不同程度的缺失现象,与BC重组和AE重组亚型相比,Thai-B亚型V3的顶端环呈现出多种组合.最终将这5条gpl20序列都克隆人真核表达载体并表达出糖基化的gp120蛋白.结论 在设计疫苗和检测试剂时应考虑到膜区的高变性,gp120糖蛋白的体外表达有利于进一步开展针对我国主要HIV流行毒株膜蛋白致病机制和疫苗学的研究.     

HIV antibodies in whole saliva detected by ELISA and western blot assays

Paired serum and saliva samples were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) for the presence of human immunodeficiency virus (HIV) antibodies. The study group included 36 individuals known to be HIV seropositive and 14 healthy, seronegative controls. HIV antibodies were detected in all but one of the saliva samples of the seropositive subjects. In this particular patient, seroconversion was documented 1 week earlier by sequential testings. A further saliva sample obtained 2 months later was ELISA positive for salivary HIV antibodies. Antibodies against HIV proteins gp120 and gp160 were detected by Western blot assay in all saliva specimens taken from HIV seropositive subjects (including the ELISA-negative patient who seroconverted. Antibodies against other viral proteins (p65, p55, p51, gp41, p35, p24 p18) were found in saliva haphazardly without any clear-cut correlation with the clinical stage of the disease. Pretreatment of the saliva with protease inhibitor was essential for the diagnostic use of saliva for the detection of HIV antibodies by Western blot assay. Calculation of the ratio of titres in serum to those in saliva showed the highest ratios in symptomless subjects (mean +/- SD; 1844 +/- 1412) and the lowest in patients with acquired immune deficiency syndrome (AIDS) (mean +/- SD; 811 +/- 445). The ratio of serum to saliva by ELISA showed a positive correlation with salivary flow rate, indicating a dilution of salivary HIV antibodies with increasing salivary flow rate. The gingival bleeding index was negatively correlated with the ratio, supporting the concept that salivary HIV antibodies transudate from blood to saliva via gingival fluid.     

Das erregerspezifische Antikörperprofil in den verschiedenen Stadien der HIV-1-Infektion

Summary The westernblot analysis of 170 patients with HIV-1-infection demonstrated that 47% of the patients in latent stage, 58% of the patients with lymphadenopathy-syndrome and only 25% of the patients with the full-blown picture of AIDS showed the complete pattern of HIV-specific antibody response. This antibody response is mainly directed against the env-encoded envelope proteins gp160, gp120 and gp41, against the gag-encoded core proteins p55, p24 and p17 as well as against the pol-encoded enzymatic proteins p66, p51 and p31.Antibodies against gp160 and gp120 were present in nearly all patients, whereas the prevalence of the other antibodies decreased with the stage of the disease. Statistical significant differences were found particularly between patients with LAS or AIDS respectively. Antibodies against p17 were detected in 74% of the patients with LAS but only in 25% of the patients with AIDS. The lack of antibodies against p17, p24 or p51 was significantly associated with lower mean CD4/CD8-ratios (p<0,007) and higher mean serum levels of IgA (p<0,001) and beta-2-microglobulin (p<0.001). One third of the patients with LAS and this reduced pattern of antibody response developed AIDS within six months. These results demonstrate that the detection of antibodies against p17, p24 or p51 is of prognostic importance. A serological profile which lacks the antibody response against at least two of those three viral antigens indicates a progression of the disease activity.

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Abkürzungsverzeichnis LAS Lymphadenopathie-Syndrom - ELISA Enzyme-linked-Immunosorbent-Assay Herrn Prof. Dr. N. Zöllner zum 65. Geburtstag gewidmet     

Comparison of antibody responses to different forms of HIV-1 core antigens by epitope mapping

The specificity of antibodies to HIV-1 capsid (p24CA) and matrix (p17MA) proteins, produced in mice against unprocessed immature assembled polyprotein (wild-type p55 virus-like particles or chimeric p55 virus-like particles) or against the monomeric mature form (rp24CA/rp17MA), was analyzed by a microplate epitope mapping assay using a panel of synthetic peptides covering the entire p24CA plus p17MA sequences of HIV-1_LAI. All immunized mice developed anti-p24CA and anti-p17MA antibodies, although the spectrum of specificity of these antibodies was different. Four p24 CA epitopes (residues 176–192, 201–218, 233–253, 285–304) were recognized by anti-rp24CA/rp17MA antibodies, whereas one p17MA epitope (residues 11–25) and one p24CA epitope (residues 176–192) were constantly recognized by anti-p55 virus-like particle antibodies. These results suggest a different specificity pattern of anti-p24CA and anti-p17MA antibodies depending on whether they are produced against the soluble mature form or the immature assembled form of the gag proteins. J. Med. Virol. 51:145–151, 1997. © 1997 Wiley-Liss, Inc.     

Investigation of atypical western blot (immunoblot) reactivity involving core proteins of human immunodeficiency virus type.

Serum specimens which originally exhibited a narrow (indeterminate) 24-kilodalton core protein (p24) or p24/p55 pattern of reactivity with human immunodeficiency virus (HIV) in the Western blot (immunoblot) test were studied to gather information on antibody specificity. A total of 12 specimens were initially reevaluated with an indirect immunofluorescence assay (IFA), three enzyme-linked immunosorbent assays (ELISAs), and Western blot analyses. Five of the specimens were IFA positive and contained anti-gp160/gp120 antibodies which were observed only when an HIV Western blot antigen rich in gp160 and gp120 was used. The remaining seven serum specimens were nonreactive by IFA and showed variable reactivity in HIV antibody ELISAs. The specimens did not cross-react with core antigens for human T-cell leukemia virus types 1 and 2 or contain detectable levels of HIV p24 antigen. The p24/p55 reactivity of six of the seven indeterminate specimens could be reduced or eliminated by preincubating the specimens with disrupted, HIV-infected H9 cells but not with uninfected H9 cells. The six specimens also exhibited discernible reactivity with recombinant HIV p24 antigen. When an additional 23 indeterminate specimens were assayed, all of the serum specimens were nonreactive by IFA while 65% (15 of 23) showed various degrees of reactivity with the recombinant p24 protein. There was no indication that any of the HIV core antibody reactivity was caused by HIV infection. Indeterminate results for five patients with specific p24 reactivity, who were retested after a period of weeks or months, remained indeterminate for HIV antibody with no significant change in ELISA or Western blot reactivity.     

HIV-1 gp41 binding to human T- and B-lymphocytes and monocytes is modulated by phorbol myristate acetate (PMA)

HIV-1 gp41 independently of CD4 binds to human T cells, B cells and monocytic cells. Since PMA downmodulates CD4 (HIV receptor) expression and inhibits HIV-1 dependent syncytia formation, we wanted to examine whether PMA could affect gp41 binding protein expression on human cells. The strong binding of HIV-1 recombinant soluble gp41 (rsgp41; Env aa539–684) to monocytes (CD14⁺) and B-lymphocytes (CD19⁺) and B lymphoblastoid cells (Raji) could be clearly decreased by treating the cells with PMA for 48 h, while the weak binding to T lymphocytes was slightly increased by this treatment. The PMA inhibitory- and enhancing-effects could be avoided by pretreatment with staurosporine (protein kinase C inhibitor). The PMA treatment of Raji and U937 (monocytic) cells resulted in a 50–60% decrease of gp41 binding proteins (gp41bps) detectable in cell lysates of these cells in comparison with lysates of buffer-treated cells, while in the case of H9-cells PMA treatment resulted in an increase of available gp41bps by about 35% in comparison with buffer-treated H9. Staurosporine pre-treatment could prevent these effects of PMA. Further studies of rsgp41-eluates from these buffer-treated or PMA-treated cells demonstrated that PMA modulated mainly expression of rsgp41bps of 37, 45, 50 and 62 kDa. These results indicate that PMA exerts different effects on human T, B and monocytic cells. Production by and expression on cells of HIV-1 gp41bps appear to depend on protein kinase C, supporting that the four proteins on human cells may act as receptor proteins for HIV-1 gp41.