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（由申请人提供）：已成功使用了含有HIV-1 Env基因（SHIVENV）的SIV菌株通过intectect和粘膜途径感染猕猴。这些猕猴模型对于研究HIV发病机理，疫苗和杀生型测试至关重要。但是，很少有Shivenv菌株能够保持稳定和长时间的感染。在测试抗HIV-1杀菌剂时，显而易见的挑战是显而易见的，其中许多源于较差的动物模型来测试功效。在R21提案中，我们概述了一个系统，以基于急性/早期感染的亚型A，B，C和D的ENV和POL基因来构建和测试SHIV的感染性。在AIM 1中，我们将利用快速的酵母重组克隆方法将大约400 HIV-1 ENV基因穿梭到MAC239和KB-9的HIV-1NL4-3或SIV骨架中。 HIV-1亚型A，C和D ENV基因将在三个月内或感染三年后从乌干达和津巴布韦妇女的内部或血液中扩增。已经使用这些患者样本的ENV基因构建和测试了20多种HIV-1 ENV嵌合病毒。如果克隆分别能够在表达人或恒河猴CD4/CCR5的细胞系以及人类或恒河类PBMC上复制，则HIV和SIV ENV嵌合病毒将包括在亚型特异性池中。在AIM 2中，将使用阴道外植体访问这些池的致病性（1）通过猕猴中的阴道暴露。然后，可以将在本植物组织和猕猴中建立感染的克隆重组为“致病”亚型A，B，C和D池，以在此提案的R33部分中描述的微生物剂研究（AIM 3）。首先，我们将确定是否需要较高浓度的CMPD167或PSC-rantes来抑制HIV或SHIV的“致病性”亚型A，B，C和D池（与人类或恒河类阴道的标准SHIVSF162-P3相比，与标准的SHIVSF162-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），以确定感染是否与耐药性有关。最终，最重要的是，在与“感染性”亚型A，B，C和D池以及标准的Shivsf162-P3接触之前，将在恒河猕猴中阴道应用微生物CMPD167和PSC-RANTES。我们怀疑大多数处理过的猕猴将受到SHIVSF162-P3感染的保护。相比之下，菌皮的保护作用可能会降低，并且在某些动物中，病毒血症的略有延迟（与未经治疗的动物相比）可能是SHIV池中特定克隆感染的结果，对CMPD167和PSC-Rantes的敏感性降低。阴道微生物提供了一种保护女性免受HIV-1感染的极好方法，但是在人使用之前对这些产品进行测试仍然是一个挑战。 HIV-1（SHIV）的猴子（例如恒河猕猴）和病毒表弟用于测试这些化合物的保护水平。在此提案中，我们设计了与HIV-1密切相关的新SHIV，并为未来的人类使用提供了更严格的生体测试。