【作者】 李悦；

【导师】 陈启民；

【作者基本信息】 南开大学 ， 微生物学， 2009， 博士

【摘要】 艾滋病已成为影响人类健康最严重的传染性疾病之一,各国迫切需要研制安全有效的艾滋病疫苗,然而至今还未有成功的范例。疫苗的安全性、有效性以及免疫策略需要在合适的动物模型中进行评价,SHIV/恒河猴模型是应用最为广泛的动物模型之一。SHIV,即SIV/HIV嵌合病毒,是利用基因重组技术,将SIV和HIV的相应基因进行置换而构建出来的重组嵌合病毒,然而尚未有代表中国HIV流行株高致病SHIV和动物模型。构建中国HIV-1 CRF07 BC主要流行株基因CCR5嗜性的致病性SHIV/恒河猴艾滋病模型,对于设计艾滋病疫苗和免疫保护效果评价可以发挥重要作用。本研究以SHIV-89.6P的克隆株SHIV-KB9为主要骨架,将从AIDS患者临床样本中制备gp120和部分gp41（从N末端的50到780个氨基酸）的KpnI-BamHI基因片段替换SHIV-KB9的相应区域,得到76个携带中国主要流行亚型CRF07 BC不同毒株env基因的SHIV cDNA嵌合克隆。将这些重组病毒cDNA转染293T细胞,收集病毒上清,分别感染TZM细胞系、人和猴的PBMC细胞,对所有克隆的生物学活性进行细胞水平鉴定。通过SIV p27衣壳蛋白滴定,筛选到4株具有感染活性的SHIV cDNA克隆,分别为SHIV-XJN0363A4,SHIV-XJN036388,SHIV-XJN0421A17和SHIV-XJDC6431。通过对这4株SHIV病毒在恒河猴淋巴细胞中感染能力的比较,筛选活性最高的SHIV-XJDC6431进行一系列细胞水平生物活性的比较。透射电镜观察到SHIV-XJDC6431 cDNA克隆在293T转染细胞系中包装出病毒颗粒;利用表达不同辅助受体的细胞系确定SHIV-XJDC6431具有CCR5嗜性;SIVp27衣壳蛋白滴定描绘出其在人、恒河猴PBMC中的复制动力学曲线,复制能力低于SHW-KB9,但高于本室构建的SHIV-XJ01270和SHIV-CN97001;被感染过的恒河猴PBMC出现明显的细胞病变;DNA PCR检测到病毒已成功整合到人、恒河猴PBMC的基因组中;RT-PCR检测到病毒在人、恒河猴PBMC培养上清中gag-pol RNA转录本和（或）RNA基因组的形成。随后,将1000TCID₅₀的SHIV-XJDC6431病毒静脉注射两只中国恒河猴443号和444号,两只恒河猴感染特征基本一致。感染后20天左右出现病毒载量,峰值出现时间较晚,仅达到10^5.12pg/ml和10^5.47pg/ml。检测结果表明SHIV-XJDC6431可在第一代恒河猴443、444中有效复制,两只动物均表现出潜伏期稳定的低水平病毒血症过程。在444感染后第65天进行了一次猴体传代实验,发现经过体内适应的病毒在第二代恒河猴体内（445号）表现出较高的感染活性和复制能力,感染445后第15天可检测到病毒载量,且峰值出现在19天,可达到10_6.98pg/ml。通过病毒载量和外周血CD4⁺T淋巴细胞计数分析,发现第一代动物表现出的感染活性较弱,与SHIV-KB9相比病毒载量峰值较低并且检测到载量的时间点滞后。总之,本研究成功构建了四株在细胞水平具有感染活性的携带中国流行亚型HIV-1 env基因片断的SHIV病毒株,其中一株SHIV-XJDC6431在动物体内已验证具有生物学活性。同时,经过猴体传代试验能够提高其感染、复制能力,并期望经过后期多次动物体内传代实验,能够筛选到高致病性的携带中国流行亚型env基因的SHIV病毒,为构建评价AIDS/HIV疫苗的动物模型提供物质基础。更多还原

【Abstract】 Human immunodeficiency virus(HIV) infection and AIDS continue to be a growing problem for the world’s population.The need to develop a safe and efficacious vaccine against HIV is more pressing than ever.Human immunodeficiency virus(HIV) and simian immunodeficiency virus(SIV) are classified to Lentivirus of Retroviridae.The genome of HIV and SIV is highly homologous and most closely in Lentivirus group.SHIV,SIV/HIV chimeric virus,was constructed by replacing the corresponding region of SIV and HIV.A pathogenic R5 simian-human immunodeficiency virus(SHIV) encoding gene of the predominant prevalent HIV-1 B’/C Recombinant(CRF07_BC) strain in China was highly desirable to study the role of HIV-1 envelopes in transmission and pathogenesis as well as to evaluate candidate AIDS vaccines in nonhuman primates. SHIV-KB9,the clone of SHIV-89.6P was used as starting backbone for the construction of Chinese recombinant B’/C SHIV.The 2.1kb KpnI-BamHI fragment encoding the Chinese HIV envelope gene(covering N-terminal 50 to 780 amino acids) amplified from different HIV infections genomic DNA was replaced with the corresponding region of SHIV-KB9 individually.76 full-length SHIV clones were constructed and their infectious activity was tested in TZM,macaque and human PBMC.Among all full-length proviral clones derived from 7 different HIV-1 samples,only four clones were found to be infectious,they are SHIV-XJN0363A4,SHIV-XJN0363B8,SHIV-XJN0421A17 and SHIV-XJDC6431.One clone SHIV-XJDC6431 was chosed to be the most infectious activity due to dected infectivity in vitro,and was used to check the infectious charactization in vitro/in vivo.The virus particle of SHIV-XJDC6431 was packed in transfected 293T cells and could be observed under transmitted electron micrograph.The coreeeptor usage of SHIV-XJDC6431 was tested in GHOST cell lines,which showed that was CCR5-specific SHIVs.The infectivity of SHIV-XJDC6431 in human and rhesus monkey’s PBMC were verified by DNA polymerase chain reaction(PCR),reverse transcriptase PCR(RT-PCR) and Simian immunodeficiency virus(SIV) p27 gag antigen titration assay.Also the distinct cytopathogenicity of rhesus monkey PBMC infected could be observed under optical microscope.To this end,SHIV-XJDC6431 was adapted by two passages in three Chinese-origin Rhesus Macaques,separately.Up to now,SHIV-XJDC6431 could replicate effectively in first passage 443,444 and second passage 445.It was revealed that the infectivity property of SHIV-XJDC6431 in first passage 443 and 444 was low replicated by measuring viral load and CD4/CD8 ratio analysis. Compared with SHIV-KB9 and SHIV-CN97001,the peak of viral load was low and the time point of deteced plasma viremia was delayed in two macaques post SHIV-XJDC6431 infection.But,the two animals infected had inclined setpoint viral load.After that,444 was passaged in vivo at day 65 post SHW-XJDC6431 infection. It was founded that the infectivity property of SHW-XJDC6431 enhanced during in vivo passage,the viral RNA load of second passage was higher.In conclusion,we constructed four R5-tropic and CRF07_BC SHIV strains which could establish persistent infection in human and macaque lymphocyte cells in vitro,and one of them SHIV-XJDC6431 could establish infection also in Chinese rhesus macaque in vivo.In addition,we observed that SHIV-XJDC6431 was capable of replicating more efficiently in macaque through animal passage,and hope for gaining more replication capacity and pathogenicity through continuous passaging in the rhesus macaque passage,which will entitle this strain as a useful tool in AIDS/HIV pathogenic and vaccine research.更多还原