HIV-1病毒分子的生物学研究进展

自1981年美国报告首例艾滋病(AIDS)以来,艾滋病正以惊人的速度在全球传播和扩散。全球累计有5000多万HI V/AIDS,投入大量的人力、物力研究HI V的生物学特征、基因变异及分子进化,为艾滋病的预防、治疗寻找科学依据,本文拟从HI V的分子生物学角度,介绍目前人类对于艾滋病研究所达到的最新分子水平,为艾滋病预防、治疗提供一个全面了解和认识。1HI V的基本结构及其分型HI V是一种RNA逆转录病毒,根据血清学反应和病毒核酸序列测定,HI V可分为Ⅰ型(HI V-1)[1],Ⅱ型(HI V-2)[2],二者之间存在一定的免疫交叉反应。艾滋病绝大多数由…     

艾滋病疫苗研究进展

艾滋病的广泛流行对全球公共卫生和社会稳定造成严重的冲击,目前全球艾滋病的传播仍在进一步扩大.虽然宣教和行为干预对控制艾滋病的传播起到了一定作用,但仅靠这些预防措施不足以完全控制艾滋病的传播.     

血清中高浓度的HIV-1易造成病毒传播

美国马利兰州巴尔的摩的约翰．霍普金斯大学学Thomas Quinn及其同事报告说，血清中有较高浓度HlV一1的个体较那些有较低浓度的个体更易使其配偶感染。HIV-1 RNA浓度上升10倍较上升2倍更易造成传播。Quinn指出，“结果提示静脉血中较低的病毒负荷可以降低其传播力。”     

HIV-1获得性耐药研究进展

HIV-1耐药性影响艾滋病的传播、治疗和预后,随着发现即治疗政策的全面推广,艾滋病治疗人数不断增加,HIV-1耐药已经成为越来越重要的公共卫生问题。HIV-1耐药包括传播性耐药和获得性耐药,传播性耐药指HIV感染者未经抗病毒治疗就发生的耐药,而获得性耐药指艾滋病病人接受抗病毒治疗后产生的耐药。本文拟从HIV-1获得性耐药现状,耐药位点分布、耐药检测方法、耐药结果解释系统和耐药产生的原因等方面进行文献整理,对我国的艾滋病抗病毒治疗和耐药监测工作有一定的参考作用。     

全自动分析系统定量检测HIV-1病毒载量分析

目的:研究全自动分析系统定量在检测HIV-1病毒载量中的作用.方法:取笔者所在医院2017年4月～2018年4月收治的艾滋病患者68例为研究对象,随机均分成对照组和观察组各34例.对照组给予罗氏自动检测仪检查,观察组给予雅培m2000全自动分析仪进行检查,对比两组检查正确率和符合度.结果:两种方法检测后,观察组HIV病...     

HIV-1感染相关基因的多态性研究进展

近年来,通过对人免疫缺陷病毒I型(HIV-1)协同受体(如CCR5、CXCR4、CCR2、CCR3和CXCR4的天然配基SDF1等)的基因多态性分析,发现CCR5-△32等位基因型的个体对M-tropic病毒株感染有天然的抵抗力;CCR5-△32和CCR2-64I等位基因型分别对艾滋病(AIDS)的发病进程有显著的延缓作用,SDF1-3′A等位基因型也直接影响到AIDS的预后。因此,协同受体的发现使HIV-1感染和AIDS发病机制的研究有了重大的突破。尤其是通过揭示CCR5、CCR2和SDF1等基因多态性与HIV-1感染的相互关系及特点,对AIDS的预防、诊断和治疗都具有重要的意义。     

人乳头瘤病毒治疗性活载体疫苗的研究进展

高危型人乳头瘤病毒(HPV)-16和HPV-18在宫颈癌的致病过程中起着重要作用.近年来,HPV预防性疫苗已成功上市,但其费用较高,且不能治疗已感染的患者及相关的损伤.多种靶向E6/E7抗原的HPV治疗性疫苗已进入临床前模型和临床试验,包括活载体疫苗,多肽、蛋白疫苗,核酸疫苗及细胞疫苗.此文就HPV治疗性活载体疫苗进行...     

HIV-1病毒储存库的清除策略研究进展

HAART可有效控制HIV复制,使HIV/AIDS患者生存期延长和死亡率降低,但由于HIV-1病毒储存库的存在,无法根除HIV。此文将介绍HIV-1病毒库的定义、病毒库检测方法及可能的清除机制,包括IFN联合HAART治疗、重新激活静息的HIV感染细胞、采用免疫疗法杀灭潜伏感染的细胞、编辑基因诱导目标细胞产生抗性等,为实现治愈HIV的目标提供参考依据。     

广州市发现首例HIV-1/HIV-2混合感染

目的探讨首例HIV-1/HIV-2混合感染发现的经过以及对本地艾滋病预防控制的意义。方法对广州市某医院一名门诊患者血样2份(包括初筛试验前后各1份)进行HIV抗体检测。初筛试验采用酶联免疫吸附试验(ELISA)和明胶颗粒凝集试验(PA),确认试验采用蛋白印迹法(WB)。结果患者为男性,非洲马里人,血清经初筛试验为HIV阳性。初筛阳性的血清,以HIV-1/HIV-2 HIVBLOT2.2试剂做蛋白印迹试验(WB),确认为HIV-1阳性,同时HIV-2的特异条带gp36阳性;样本再用HIV-2型的BLOT1.2膜条确认为HIV-2阳性。结论被检者为HIV-1/HIV-2混合感染,这是广州市、广东省首次发现HIV-1/HIV-2混合感染,提示HIV-2可能会以对外交流等方式传入广东。     

一种CpG佐剂增强HIV-1DNA疫苗免疫原性的研究

目的以一种CpG寡聚核苷酸为HIV-1DNA疫苗候选佐剂,研究该CpG佐剂增强DNA疫苗免疫原性,体外促进DC细胞成熟等特点。方法在Balb/c小鼠模型上连续3次联合免疫HIV-1DNA疫苗及CpG佐剂,通过IFN-γ、IL-2ELISPOT及ELISA检测HIV特异性细胞免疫反应及体液免疫应答强度;体外制备小鼠骨髓来源的树突状细胞,通过FACS技术、高通量细胞因子检测等方法评价CpG佐剂刺激活化DC的能力。结果 CpG能够增强HIV-1DNA疫苗诱导的特异性细胞免疫反应水平,降低DNA疫苗使用剂量;CpG体外刺激原代小鼠骨髓来源的树突状细胞(BMDC),能显著上调CD40、CD80、CD86等BMDC表面共刺激分子的表达,活化BMDC并分泌各型细胞因子IL-5、IL-12p70,促炎症因子IL-1α、IL-1β、IL-6、IL-10、MIP-2、KC、MIG、Eotaxin、GM-CSF等以发挥佐剂效应。结论综合体内体外实验数据,证实该型CpG能够充分活化BMDC,显著提高HIV-1DNA疫苗免疫原性,降低疫苗使用剂量,可成为HIV-1DNA疫苗临床试验用候选佐剂。     

Characterization of recombinant influenza A virus as a vector for HIV-1 p17

We have generated a recombinant influenza A virus with the HIV-1 p17^Gag (rFlu-p17) gene inserted into the influenza virus neuraminidase (NA) gene. Expression of HIV-1 p17 protein was detected by conventional Western blot analysis and also by liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis of rFlu-p17 infected cells. The latter method does not depend on protein-specific antibody preparations and proved to be a powerful tool to detect proteins of interest. Next, antigen presentation of p17 expressed after infection of antigen-presenting cells was determined. Cloned p17-specific CD8+ T-cells were co-cultured with rFlu-p17 infected B-cells and produced IFN-γ upon stimulation. Furthermore, we showed that immunization with rFlu-p17 elicited a humoral immune response in mice. This study shows that replication-deficient rFlu-p17 is antigenic in vitro and immunogenic in vivo and warrants further development as a candidate vaccine vector.     

Packaging limits and stability of HIV-1 sequences in a coxsackievirus B vector

Enteroviruses elicit protective mucosal immune responses that could be harnessed as part of a strategy to prevent sexual transmission of the human immunodeficiency virus-1 (HIV-1). We report the construction of replication-competent recombinant vectors of coxsackievirus B3 (CVB3) that express one or more portions of the HIV-1 Gag protein. Vectors containing the capsid domain of Gag were initially genetically unstable with protein expression lost after brief passage in tissue culture. Codon modification to increase the G/C content of the HIV-1 capsid sequence resulted in enhanced genetic stability of CVB3 vectors during in vitro passage. Cells infected with a vector expressing the matrix (MA) subunit of the HIV-1 Gag protein were susceptible to lysis by CD8 T cell clones specific for the SL9 epitope found within MA. These studies suggest that CVB3 vectors may be useful as vaccine vector candidates, if hurdles in class I antigen presentation and stability can be overcome.     

Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy

A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.     

转座介导的HIV-1整合位点分析

目的 建立转座酶反应介导的HIV-1整合位点鉴定方法,以鉴定HIV-1潜伏或复制性感染有关的整合特征.方法 采用转座酶反应介导整合序列的捕获与扩增方法,对照人基因组进行序列分析.结果 50株细胞克隆中,有45株可检测到HIV-1整合片段,共获得9个整合位点.HIV-1潜伏或复制型细胞内整合位点的分布无显著差异.HIV-1的整合位点分布于所有基因目录内,包括转录活跃基因、非编码区或重复元件,并与相邻的表观分布相关.结论 转座酶反应介导的整合位点鉴定方法简便快捷,能以最大随机方式还原整合位点.HIV-1感染的整合与基因组特征存在联系.     

Induction of HIV-1 subtype B and AE-specific neutralizing antibodies in mice and macaques with DNA prime and recombinant gp140 protein boost regimens

We developed highly expressing clade B and AE DNA and envelope protein (Env) vaccines for evaluation in mice and macaques as DNA prime/protein boost regimens. High levels of Env-specific antibodies were induced in mice, albeit with limited neutralizing activity in vitro. A combined clade B and AE regimen induced high titer Env-specific antibody in two pigtail macaques that neutralized several strains of HIV-1. However, upon mucosal challenge with SHIV_SF162P3 no protection from infection was observed. Although the vaccines tested provide a platform for inducing robust humoral immunity, further refinements to broaden coverage against divergent strains and induce mucosal immunity are needed.     

2013-2015年全国HIV-1病毒载量检测能力验证方法的比较

目的 通过分析2013-2015年全国HIV-1病毒载量检测能力验证（VQA-PT）结果,了解法国bioMerieux公司的NucliSens EasyQ HIV-1 v2.0（EasyQ）、美国Siemens Healthcare Diagnostics公司VERSANT HIV-1 RNA 3.0 assay（bDNA）、美国罗氏分子诊断公司COBAS AmpliPrep/COBAS TaqMan HIV-1 test（Taqman）、美国Abbott Molecular公司Abbott Real Time HIV-1 Kit（M2000）、中山大学达安基因股份有限公司和东北制药集团辽宁生物医药有限公司的HIV-1核酸定量检测试剂盒（国产试剂）5种方法检测结果之间的定量换算关系,为不同方法检测HIV-1病毒载量结果的横向比较提供参考。方法 参照《HIV-1病毒载量测定及质量保证指南》（2013版）的要求,由中国CDC性病艾滋病预防控制中心参比实验室统一组织全国HIV-1的VQA-PT。采用多中心回溯性研究方法,2013-2015年共计考核6次,考核实验室155家,涉及阳性样本22个,相应结果2 954个。针对每个阳性样本的所有结果,首先转换为对数值,而后按照检测方法分类,计算对数值的均值,获得不同方法检测相同样本的一组数据。对最终获得的22组数据进行Bland-Altman分析和线性回归分析。结果 Bland-Altman分析显示,EasyQ及bDNA与Taqman的一致性为100%,M2000及国产试剂与Taqman的一致性≥ 90%。回归分析结果显示,EasyQ、bDNA、M2000及国产试剂检测结果在数值上与Taqman之间存在相应的换算关系（P<0.01）。结论 对于我国HIV-1感染者,使用不同病毒载量检测方法获得的结果一致性良好,不同方法的结果之间可以转换,进而对治疗效果进行分析评价。     

Cell-associated infectious HIV-1 viral load as a predictor of clinical progression and survival among HIV-1 infected injection drug users and homosexual men

Cell-associated infectious HIV-1 viral load was measured using semi-quantitative microculture techniques to determine its predictive capability for progression to AIDS or survival among HIV-1 infected injecting drug users (IDU) and homosexual men (HM). The authors followed 296 IDU and 240 HM from February 1992 through September 1995 for: (i) death, (ii) AIDS, and (iii) AIDS or bacterial infection. At baseline, viral load was quantified using microculture techniques to determine infectious units per million peripheral blood mononuclear cells (IUPM). Data were analyzed using standard statistical methods for survival analysis. Of the 536 total participants, 106 died (20%), and 98 of the 481 AIDS-free participants developed AIDS (20%). The relative hazard of AIDS for a viral load of 100 IUPM, relative to a negative culture (0 IUPM), was 6.73 (95% CI: 2.23–20.3) after adjusting for risk group, initial CD4+ count, and other covariates. The adjusted relative hazard of death for a viral load of 100 IUPM vs. 0 IUPM was 2.57 (95% CI: 0.97–6.80). Viral load predicted time to death within the <200 cells/l CD4+ stratum. The predictive value of viral load on HIV-1 progression did not vary by risk group. These data show that cell associated infectious HIV-1 viral load was significantly predictive of progression across risk groups for AIDS and death among those severely immune compromised.     

162例HIV-1抗体阳性血清WB带型分析

通过对本实验室１９９６－１９９８年间以ＷＢ法确认的１６２例ＨＩＶ－１抗体阳性血清ＷＢ带型分析，表明ＨＩＶ早期感染带型占确认阳性数的比例呈增长趋势，提示相关的传播途径并未得到完全有效控制；随着ＡＩＤＳ期带型所占比例的增加，预示着我省ＨＩＶ感染者逐步进入临床发病期，必须引起重视。     

Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development

The failure to develop an effective vaccine against HIV-1 infection has led the research community to seek new ways of raising qualitatively different antibody and cellular immune responses. Towards this goal, we investigated the yellow fever 17D vaccine strain (YF17D), one of the most effective vaccines ever made, as a platform for HIV-1 vaccine development. A test antigen, HIV-1 p24 (clade B consensus), was inserted near the 5′ end of YF17D, in frame and upstream of the polyprotein (YF-5′/p24), or between the envelope and the first non-structural protein (YF-E/p24/NS1). In vitro characterization of these recombinants indicated that the gene insert was more stable in the context of YF-E/p24/NS1. This was confirmed in immunogenicity studies in mice. CD8⁺ IFN-γ T-cell responses against p24 were elicited by the YF17D recombinants, as were specific CD4⁺ T cells expressing IFN-γ and IL-2. A balanced CD4⁺ and CD8⁺ T-cell response was notable, as was the polyfunctionality of the responding cells. Finally, the protective efficacy of the YF17D recombinants, particularly YF-E/p24/NS1, in mice challenged with a vaccinia expressing HIV-1 Gag was demonstrated. These results suggest that YF17D warrants serious consideration as a live-attenuated vector for HIV-1 vaccine development.     

The distribution of HIV-1 recombination breakpoints

We find that recombination breakpoints are non-randomly distributed across the genomes of HIV-1 intersubtype recombinants. In particular we find two recombination prone regions, "hot spots", located approximately either side of the envelope gene. To investigate this, we test whether there is a correlation between the distribution of the recombinant breakpoints with (1) genetic similarity, (2) predicted locations of secondary RNA structure, (3) regions identified as recombinant hot spots from experimental studies and (4) the predicted locations of positively selected sites. No detectable relationship with RNA secondary structure was found. A weak relationship with genetic similarity exists but it does not account for the recombination hot spots. The comparison with the published experimental studies indicated that the identified recombination hot spots differ in their locations, indicating that selection is having an impact on HIV-1 recombinant structures in infected individuals. We observe an association between recombination prone regions and strong positive selection across the envelope gene in support of this hypothesis.