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

描述（由申请人提供）：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HIV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毒株能够维持稳定和长时间的感染。在抗hiv -1杀菌剂的测试中存在一些明显的挑战，其中许多挑战源于测试功效的动物模型不佳。在R21提案中，我们概述了一个基于急性/早期感染的a、B、C和D亚型的env和pol基因构建和测试SHIV传染性的系统。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环境基因转染到HIV-1NL4-3或mac239和KB-9的SIV主干中。HIV-1亚型A、C和D env基因将在感染3个月或3年后从乌干达和津巴布韦妇女的宫颈内或血液中进行PCR扩增。利用来自这些患者样本的env基因，已经构建和测试了20多种HIV-1环境嵌合病毒。如果克隆能够在表达人类或恒河猴CD4/CCR5的细胞系（分别）和人类或恒河猴PBMCs（分别）上复制，HIV和SIV环境嵌合病毒将被包括在亚型特异性池中。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重组为“致病性”亚型A、B、C和D，用于本提案R33部分中描述的杀微生物剂研究（目标3）。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组织中HIV或shiv的“致病性”亚型A、B、C和D（与标准SHIVSF162-P3相比）。我们确定即使在药物存在的情况下也能感染的任何HIV或SHIV克隆的身份。然后可以测试这些特异性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，也是最重要的是，在接触“传染性”亚型A、B、C和D池以及标准SHIVSF162-P3之前，将杀微生物剂CMPD167和PSC-RANTES阴道施用于恒河猴。我们怀疑大多数接受治疗的猕猴将免受SHIVSF162-P3感染。相反，杀微生物剂的保护作用可能会降低，并且在一些动物中，病毒血症的轻微延迟（与未治疗的动物相比）可能是由于SHIV池中的特定克隆感染了对CMPD167和PSC-RANTES敏感性降低的结果。阴道杀微生物剂是保护妇女免受HIV-1感染的一种极好的方法，但在人类使用之前对这些产品进行检测仍然是一项挑战。一种猴子物种（如恒河猴）和HIV-1病毒的表亲（SHIV）被用来测试这些化合物的保护水平。在本提案中，我们设计了与HIV-1更密切相关的新型shiv，并为未来人类使用的杀微生物剂提供了更严格的测试。

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.