【作者】 刘强；

【导师】 邵一鸣； 杨贵波；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2008， 博士

【摘要】 目前,艾滋病已成为影响人类健康的最严重的传染性疾病之一。世界各国严峻的流行形式迫切需要研制出安全有效的预防性艾滋病疫苗。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。其中,SHW/印度恒河猴艾滋病模型是应用最为广泛的动物模型之一。包含中国HIV-1 CRF07 BC主要流行株基因CCR5嗜性的致病性SHIV/中国恒河猴艾滋病模型在基于中国HIV-1主要流行株设计的艾滋病疫苗免疫保护效果评价中可以发挥非常重要的作用。SHIV-XJ02170感染性分子克隆是以B亚型强毒株SHIV-KB9基因为框架重组入中国CRF07 BC亚型HIV-1主要流行株env基因构建而成,本研究将SHIV-XJ02170连同Ruth M Ruprecht教授惠赠的另一株包含CRF07 BC亚型HIV-1 env的SHIV-CN97001分别在中国恒河猴体内快速传代。经过近4年的连续猴体传代和长期适应后,通过病毒载量和外周血CD4⁺T淋巴细胞计数分析未发现致病性强毒株的出现。不过,两株SHIV病毒在猴体传代过程中表现出病毒毒力增强的特点。其中,SHIV-CN97001在前3代中病毒复制能力持续升高。更为重要的是,SHIV-XJ02170传代中的两只动物表现出潜伏期稳定的低水平病毒血症过程。为了分析SHIV-CN97001和SHIV-XJ02170传代过程中序列变异特点,扩增各代动物急性期病毒载量峰值时间点病毒RNA Gp120克隆序列并分析病毒准种之间的基因离散率和遗传多样性。SHIV-CN97001传代过程中基因距离表现出先升后降的趋势,其中第三代动物中基因离散率和遗传多样性最显著。SHIV-XJ02170传代中的408动物体内病毒相对于原始序列基因离散率在适应过程中持续上升。分析发现各代动物基因离散率和病毒复制能力之间存在相关性。基因多态性分析发现两株SHIV病毒Gp120 V3区在传代过程中均最为保守。基于V3区关键氨基酸位点分析发现所有RNA克隆序列均利用CCR5作为辅助受体,传代过程中未发生改变。总体而言,SHIV-CN97001和SHIV-XJ02170传代过程中Gp120基因距离未出现明显上升,并且每代动物传代早期出现典型的“瓶颈效应”。按照Mullins教授提出的HIV-1感染后致病过程不同阶段基因距离变化理论分析,SHIV-XJ02170在第二代动物408体内目前已经处于感染的后期阶段。病毒准种基因变异分析可以部分解释两株SHIV病毒在中国恒河猴体内传代过程中未出现致病性强毒株的原因。为建立致病性SHIV-KB9/中国恒河猴艾滋病疫苗评价模型并比较SHIV-KB9在中国恒河猴和印度恒河猴体内致病模式的差异。7只Mamu-A^*01阴性的中国恒河猴静脉途径接种1-1000 MID₅₀剂量的SHIV-KB9并监测病毒载量、CD4/CD8比值、SHIV特异性抗体滴度以及病毒序列变异。在中国恒河猴中,SHIV-KB9表现出和文献报道的印度恒河猴中相同的三种致病模式。然而,SHIV-KB9在两亚种动物中的主要致病模式是不同的。另外,SHIV-KB9感染中国恒河猴后三种致病模式中病毒血症的特点与印度恒河猴是不同的。对比发现,病毒载量和CD4⁺T细胞的维持能力与体液免疫反应相关。值得注意的是,在CD4⁺T细胞显著下降的动物体内SHIV-KB9病毒准种的基因离散率和遗传多样性明显升高。SHIV-KB9在中国恒河猴体内表现出相对印度恒河猴致病过程进展更加缓慢的特点,这一特点更加接近HIV-1在人体的致病特点并更适合做为艾滋病疫苗的评价模型。另外,SHIV-CN97001和SHIV-KB9感染中国恒河猴后可以诱导针对SHIV-KB9的异源性和同源性免疫保护反应。因此,SHIV病毒成功感染中国恒河猴后继续进行SHIV-KB9二次攻毒试验在中国恒河猴艾滋病疫苗免疫保护效果评价中是不适合的。为了控制艾滋病在中国的流行,本研究团队研制了包含中国CRF07_BC亚型主要流行毒株HIV-1 CN54 gag,pol,nef以及gp140基因的DNA疫苗和复制性天坛株重组痘苗病毒载体疫苗（rTV）。为了评价本疫苗的免疫源性和攻毒保护效果并分析DNA疫苗的初免效果,采用rTV单免和DNA/rTV联合免疫两种策略免疫12只中国恒河猴,并选用同源性SHIV-CN97001和异源性SHIV-KB9二次攻毒的方式分析疫苗的免疫保护效果。免疫和攻毒后,监测细胞和体液免疫反应以及病毒载量的变化。HIV特异性结合抗体滴度和IFN-γ分泌性T细胞免疫反应分别采用ELISA和ELISPOT方法分析,中和抗体滴度通过测定SHIV-KB9在TZM-bl细胞的感染能力进行分析。相比rTV单免组,DNA/rTV联合免疫组动物疫苗免疫后可以诱导10倍以上水平的IFN-γ分泌性T细胞免疫反应,但诱导的结合抗体和中和抗体滴度水平没有明显差异。两种免疫策略均可以在最后一次痘苗免疫9个月后诱导出很强的记忆性免疫反应。同源性SHIV-CN97001病毒攻击后,rTV单免组全部4只动物均获得完全性保护的效果。然而,DNA/rTV联合免疫组3只动物中仅有1只动物获得完全性保护效果,其它2只动物获得部分性保护效果。10个月之后,两组中各挑选1只针对SHIV-CN97001完全性保护的动物进行异源性SHIV-KB9的二次病毒攻击。联合免疫组中M7动物通过病毒载量和血液传代分析依然保持完全性保护的效果,而rTV单免组M2动物仅获得部分性保护的效果,表现为急性期病毒载量峰值水平下降以及潜伏期病毒载量下降至低于最低检测限水平。分析发现在同源性SHIV-CN97001免疫保护方面中和抗体可能发挥了关键性作用,而在异源性SHIV-KB9免疫保护方面IFN-γ分泌性T细胞免疫反应和免疫保护密切相关。DNA-复制性重组痘苗病毒艾滋病疫苗免疫中国恒河猴后提供有效且持续性的免疫反应,可以有效的保护动物抵抗同源性和异源性SHIV病毒的攻击。本研究可以对艾滋病疫苗的临床试验提供有效的参考数据。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.A pathogenic R5 simian-human immunodeficiency virus（SHIV） encoding genes 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. To this end,SHIV-XJ02170 and SHIV-CN97001 which also carrying CRF07_BC env, were adapted by serial passage in thirteen Chinese-origin Rhesus Macaques,separately. Up to now,there were no pathogenic viral strain obtained during in vivo passage by viral load and CD4/CD8 ratio analysis.But,it was revealed that the infectivity property of the two SHIVs enhanced during in vivo passage.The viral RNA load of third passage was highest during SHIV-CN97001 passage and two animals infected SHIV-XJ02170 had inclined setpoint viral load.To identify variation in the gpl20 region of SHIV-CN97001 and SHIV-XJ02170 during passage,the fragments of gpl20 gene were amplified by RT-PCR from the plasma of infected animals at the peak viral load time point and the gene distances （divergence,diversity） were calculated using DISTANCE.The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest and the gene divergence from founder strain in M408 serially inclined during SHIV-XJ02170 in vivo adaption.It had a relationship between viral divergence from the founder strain and viral replication ability.In the two SHIV constructs,the nucleic acid sequence of the V3 region was highly conservative and all the RNA clones were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop.These results showed that there was no significant increase in the genetic distance during SHIV-CN97001 and SHIV-XJ02170 passage,and the two SHIV gp120 gene evolved toward ancestral states upon transmission to a new host.But,the SHIV-XJ02170 virus in one of second passage animals（408） maybe has involved the late infection phase according to gene distance analysis.This could partly explain why there has no pathogenic viral strain obtained during in vivo passage.To develop a SHIV-KB9/Chinese-origin rhesus macaques（Ch Rh） model for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh with published reports from Indian-origin rhesus macaques（Ind Rh）.Seven mamu-A*01 negative Ch Rh were inoculated intravenously with 1-1000 MID₅₀ of SHIV-KB9.The monkeys were monitored by measured viral load,CD4,CD8,SHIV-specific antibody and virus genetic variation.In Ch Rh,SHIV-KB9 displayed three identical disease progression patterns,compared to what observed in Ind Rh.However,the primary pattern was not same for the two subspecies.The level of plasma viremia differed in SHIV-KB9-infected Ch Rh exhibiting different outcomes in contrast to in Ind Rh. Generally,the viral load values and the maintenance of CD4⁺ T cells were associated with the humoral immune responses.Otherwise,the viral genetic distances （divergence,diversity） were larger in animals（M419,M425） in which the CD4⁺ T cells were profound depleted.Ch Rh model employing SHIV-KB9 displayed a relative slower progression to AIDS compared with Ind Rh,which may more accurately reflect the potential of candidate vaccines in humans.Interesting, SHIV-CN97001 and SHIV-KB9 infection in Chinese-origin Rhesus Macaques could induce heterologous and homologous immune protective response to SHIV-KB9 superinfection.So it is not suitable for SHIV-KB9 superinfection in Chinese monkeys systemicly infected with SHIVs.To contain the pandemic of HIV/AIDS in china,plasmids containing nef,gag, pol,gp140 and replication competent Tiantan vaccinia（rTV） containing gag,pol and gp140 from B’/C recombinant HIV-1 CN54,the predominant prevalent strain in China,were constructed as candidate AIDS vaccines.To evaluate the immunogenicity and protective effect against homologous SHIV-CN97001 and heterologous SHIV-KB9 challenge,12 Chinese-origin Rhesus Macaques were immunized and challenged,rTV-only vaccination was compared with a DNA prime/rTV boost strategy.Animals were followed prospectively for immune parameters and viral RNA loads.HIV-specific antibody titers and CTLs responses were determined with ELISA and ELISPOT based assay respectively.Neutralizing antibody titers were measured using a viral infectivity assay of TZM-bl cells.Compared to the DNA/rTV vaccine, the rTV-only vaccine raised less than 1/10 the number of vaccine-specific T cells,but comparative titers of binding antibody and neutralizing antibody.Otherwise,the candidate vaccines were able to induce strong memory responses in all the vaccinated animals nearly 9 months after the last immunization.After homologous SHIV-CN97001 challenge,the rTV-only-vaccinated animals had achieved better control of the infection.4 of 4 animals in rTV group were completely protected. However,only 1 of 3 animals in DNA prime/rTV boost group（M7） was complete protected,the other two animals were partially protected.After 10 months,we selected two animals which completely protected against SHIV-CN97001 to be rechallenged with heterologous SHIV-KB9,only one for each group.One monkey （M7） in DNA prime/ rTV boost group was also completely protected against SHIV-KB9 rechallenge,which was comfirmed by blood passage analysis,another one （M2） in rTV-only group was partially protected with declined peak and setpoint viral load.It was indicated that the rapid responses having neutralizing activity might contributed to the clearance of the homologous SHIV-CN97001 challenge virus and the IFN-γsecreting T cell immune response had correlation to protection from heterologous SHIV-KB9 rechallenge.DNA-replication competent Tiantan vaccinia combined vaccine could provide the potent,persistent immunity necessary to protect against homologous and heterologous SHIV.This study provides a foundation for evaluation of these candidate vaccines in human body.更多还原