New SHIV R5 env's (based on all subtypes) for effective microbicide testing in ma

描述(申请人提供)：含有HIV-1 env基因的SIV毒株(SHIVenv)已成功地通过静脉和粘膜途径感染猕猴。这些猕猴模型对艾滋病毒发病机制、疫苗和杀微生物剂测试的研究至关重要。然而，很少有SHIVenv毒株能保持稳定和长期的感染。在抗HIV-1杀微生物剂的测试中有几个明显的挑战，其中许多挑战来自于测试疗效的糟糕的动物模型。在R21方案中，我们概述了一种基于急性/早期感染的A、B、C和D亚型的env和pol基因构建和检测SHV感染性的系统。在目标1中，我们将利用快速酵母重组克隆方法，将大约400个HIV-1 env基因穿梭到HIV-1NL4-3或SIV骨架mac239和KB-9中。HIV-1 A、C和D亚型env基因将在感染三个月内或感染后三年内从乌干达和津巴布韦妇女的子宫内膜或血液中进行聚合酶链式反应扩增。已经构建了20多种HIV-1包膜嵌合病毒，并使用这些患者样本的包膜基因进行了测试。如果克隆能够在分别表达人或恒河猴CD4/CCR5的细胞系上以及分别在人或恒河猴外周血单核细胞中复制，则HIV和SIV环境嵌合病毒将被包括在亚型特异池中。在目标2中，这些池的致病性将通过(1)使用阴道外植体和(2)通过猕猴的阴道暴露来获得。然后，在外植体组织和猕猴中建立感染的克隆可以被重组到A、B、C和D亚型的“致病”亚型池中，用于本提案R33部分(目标3)中描述的杀微生物剂研究。首先，我们将确定是否需要更高浓度的cmpd167或PSC-RANTES来抑制人类或恒河猴阴道外植体组织中的A、B、C和D亚型HIV或SHIV的“致病”池(与标准SHIVSF162-P3相比)。我们确定任何即使在药物存在的情况下也能感染的艾滋病毒或希沃克病毒克隆(S)的身份。然后，可以测试这些特定的HIV-1克隆(由原始DNA克隆产生)对CMPD167和PSC-RANTES的敏感性，并确定感染是否与耐药性有关。最后，也是最重要的一点是，杀菌剂CMPD167和PSC-RANTES将在接触A、B、C和D亚型池以及标准SHIVSF162-P3之前，经阴道应用于恒河猴。我们怀疑，大多数接受治疗的猕猴将免受SHIVSF162-P3感染。相反，杀菌剂的保护作用可能会减弱，在一些动物中，病毒血症的轻微延迟(与未经治疗的动物相比)可能是由于SHIV池中对CMPD167和PSC-RANTES敏感性降低的特定克隆感染的结果。阴道杀微生物剂提供了一种很好的方法来保护妇女免受艾滋病毒-1感染，但在人类使用这些产品之前对其进行测试仍然是一个挑战。一种猴子物种(例如恒河猴)和HIV-1的病毒表亲(SHIV)被用来测试这些化合物的保护水平。在这项提案中，我们设计了与HIV-1更密切相关的新SHIV，并为未来的人类使用提供了更严格的杀微生物剂测试。

DESCRIPTION (provided by applicant): SIV strains containing HIV-1 env genes (SHIVenv) have been successfully employed to infect macaques through intravenous and mucosal routes. These macaque models have been crucial for studies on HIV pathogenesis, vaccine, and microbicide testing. However, few SHIVenv strains can maintain stable and prolonged infections. Several challenges are apparent in the testing of anti-HIV-1 microbicides and many of these stem from poor animal models to test efficacy. In the R21 proposal, we have outlined a system to construct and test the infectivity of SHIV based on the env and pol genes of subtype A, B, C, and D from acute/early infections. In aim 1, we will utilize a rapid yeast recombination cloning approach to shuttle approximately 400 HIV-1 env genes into an HIV-1NL4-3 or SIV backbones of mac239 and KB-9. The HIV-1 subtype A, C, and D env genes will be PCR amplified from the endocervix or blood of Ugandan and Zimbabwean women within three months or after three years of infection. Over 20 HIV-1 env chimeric viruses have already been constructed and tested using env genes from these patient samples. HIV and SIV env chimeric viruses will be included in subtype-specific pools if the clone is capable of replication on cell lines expressing human or rhesus CD4/CCR5 (respectively) and in human or rhesus PBMCs (respectively). In aim 2, the pathogenicity of these pools will then be accessed (1) using vaginal explants and (2) through vaginal exposure in macaques. The clones that establish infection in both the explant tissue and macaques can then be reconstituted into the "pathogenic" subtype A, B, C, and D pools for the microbicide studies described in the R33 section of this proposal (aim 3). First, we will determine if higher concentrations of cmpd167 or PSC- RANTES are required to inhibit the "pathogenic" subtype A, B, C, and D pools of HIV or SHIVs (as compared to the standard SHIVSF162-P3) in human or rhesus vaginal explant tissues. We determine the identity of any HIV or SHIV clone(s) that are capable of infection even in the presence of the drug. These specific HIV-1 clones (produced from original DNA clones) can then be tested for sensitivity to CMPD167 and PSC-RANTES and to determine if infection was related to drug resistance. Finally and most importantly, microbicides CMPD167 and PSC-RANTES will be vaginally applied to rhesus macaques prior to exposure with the "infectious" subtype A, B, C, and D pools as well as the standard SHIVSF162-P3. We suspect that the majority of the treated macaques will be protected from SHIVSF162-P3 infection. In contrast, the protective effects of the microbicides may be reduced and that in some animals, a slight delay in viremia (as compared to untreated animals) may be the result of infection by specific clone in the SHIV pool with reduced sensitivity to CMPD167 and PSC-RANTES. Vaginal microbicides provide an excellent method to protect women from HIV-1 infection but testing these products prior to human use remains a challenge. A monkey species (e.g. Rhesus macaques) and virus cousin of HIV-1 (SHIV) are used to test the level of protection by these compounds. In this proposal, we have designed new SHIVs that are more closely related to HIV-1 and provide more stringent testing of microbicides for future human use.