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

新的 SHIV R5 env（基于所有亚型）可在马进行有效的杀菌剂测试

• 批准号: 7690343
• 负责人: ERIC J ARTS
• 金额: $ 21.1万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-25 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7690343
• 关键词: Acute; Animal Model; Animals; Blood; CCR5 gene; CXCR4 gene; Cell Line; Cercopithecine Herpesvirus 1; Cervical; Chronic Myeloproliferative Disorder; Cloning; Comparative Study; DNA; Drug resistance; Endocervix; Exposure to; Frequencies; Future; Genetic Recombination; Genetic Variation; HIV; HIV-1; Homologous Gene; Human; Infection; Intravenous; Macaca; Macaca mulatta; Methods; Modeling; Monkeys; Pathogenesis; Pathogenicity; Patients; Pharmaceutical Preparations; Population Heterogeneity; RANTES; RANTES, N(alpha)-(n-nonanoyl)-desSer(1)-(thioproline(2),cyclohexylglycine(3))-; Recombinants; Resistance; Route; SIV; Sampling; System; Testing; Tissues; Topical application; Vaccines; Vagina; Vertebral column; Viral; Viremia; Virus; Woman; Yeasts; anti-HIV-1 microbicide; base; design; efficacy testing; env Genes; human tissue; inhibitor/antagonist; microbicide; pol genes; prevent; protective effect; reconstitution; rectal; simian human immunodeficiency virus; stem; transmission process; vaginal microbicide

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.

描述（由申请方提供）：含有HIV-1 env基因（SHIVenv）的SIV毒株已成功用于通过静脉内和粘膜途径感染猕猴。这些猕猴模型对于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菌株可以维持稳定和长期的感染。在抗HIV-1杀微生物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，我们已经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基因构建和测试SHIV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-1 NL 4 -3或SIV mac 239和KB-9的骨架中。HIV-1亚型A、C和D env基因将在感染后三个月内或三年后从乌干达和津巴布韦妇女的宫颈内膜或血液中进行PCR扩增。已经使用来自这些患者样本的env基因构建并测试了超过20种HIV-1 env嵌合病毒。如果克隆能够在表达人或恒河猴CD 4/CCR 5的细胞系（分别）和人或恒河猴PBMC（分别）中复制，则HIV和SIV env嵌合病毒将被纳入亚型特异性合并液中。在目标2中，然后将（1）使用阴道外植体和（2）通过猕猴的阴道暴露评估这些样本池的致病性。然后，可将在外植体组织和猕猴中建立感染的克隆重组为“致病性”亚型A、B、C和D库，用于本提案R33部分所述的杀微生物剂研究（目的3）。首先，我们将确定是否需要更高浓度的化合物167或PSC-RANTES来抑制人或恒河猴阴道外植体组织中HIV或SHIV的“致病”亚型A、B、C和D库（与标准SHIVSF 162-P3相比）.我们确定即使在药物存在下也能够感染的任何HIV或SHIV克隆的身份。然后可以测试这些特定的HIV-1克隆（由原始DNA克隆产生）对CMPD 167和PSC-RANTES的敏感性，并确定感染是否与耐药性有关。最后且最重要的是，杀微生物剂CMPD 167和PSC-RANTES将在暴露于“感染性”亚型A、B、C和D合并物以及标准SHIVSF 162-P3之前经阴道应用于恒河猴。我们怀疑大多数经治疗的猕猴将免受SHIVSF 162-P3感染。相反，杀微生物剂的保护作用可能降低，并且在一些动物中，病毒血症的轻微延迟（与未处理的动物相比）可能是SHIV库中对CMPD 167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保护妇女免受HIV-1感染的极好方法，但在人类使用之前测试这些产品仍然是一个挑战。使用猴种（例如恒河猴）和HIV-1（SHIV）的病毒近亲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关系更密切的新型SHIV，并对未来人类使用的杀微生物剂进行了更严格的测试。