Impact of Non - B HIV - 1 Subtype on second line Protease Inhibitor Regimens in Africa (INSPIRE)

研究摘要 发现大多数病毒学失败的患者(HIV-1RNA和gt；=1000cp/mL)在二线PI-1上- 含药方案缺乏耐PI蛋白(PR)基因突变是主要原因之一 抗逆转录病毒耐药性研究的谜团。我们假设env和gag序列 来自这些患者的基因含有代偿性突变，特别是在环境中，这使得PI 抵抗。Gp41CT在PI抗性中的作用可能与病毒成熟有关， 在激活Env融合活性时，由PR触发。近年来，越来越多的临床病例 有报道称，在缺乏整合酶(IN)的情况下，含DTG的治疗失败 突变，这表明IN外的突变可能会在这些患者中产生耐药性。我们的 研究表明，第一次对环境病毒突变进行从头选择可以带来 体外对多洛替格列韦(DTG)的耐药性(未发表的数据)。我们将这种表型归因于 环境病毒突变体介导高效的细胞间传递的能力，导致 感染的多样性增加。此外，最多将 在没有PR突变的情况下，分别观察到对ATV、DRV和LPV的易感性 在HIV-1、CRF_02AG和G亚型感染者中。这些发现具有广泛的意义 对我们理解Env和Gag功能以及HIV-1药物进化的启示 抵抗。我们的目标是确定在ATV/r或LPV/r上预测病毒学失败(VF)的因素 接受PI方案治疗的室性心动过速患者未来的治疗效果不佳，可能处于 在三线方案中开发整合酶耐药抑制剂的风险增加。协作型 由尼日利亚人类病毒学研究所领导的调查小组将利用Robust 2L方案的纵向队列患者(喀麦隆、尼日利亚和乌干达)，再加上良好的 严谨的嵌套病例对照研究的特征和存档的临床标本 设计。 为了检验这些相互关联的假设，我们建议：(I)使用我们新开发的HIV-xGen Next 世代测序方法扩大对病毒进化和艾滋病毒耐药性的了解 并确定是否存在非B型HIV-1的有效修饰 亚型，(Ii)鉴定携带目标1中确定的突变的重组病毒，使用 建立表型分析以确定它们对药物敏感性和定点的影响 诱变，最后(Iii)确定复制适合性在药物敏感性上的差异 基于酵母重组的克隆系统的使用。我们计划研究这些患者和他们的 病毒(A、C、D、G亚型和CRF02_AG、CRF43_02G、HIV-1O和HIV-1N)提供 独一无二的机会来确定基因改变的表型和临床意义 HIV-1 Gag、Gag-Poll和env基因。总而言之，这项提议将允许令人兴奋的流行病学， 临床和公共卫生研究机会。

Abstract of research The finding that most patients with virologic failure (HIV-1 RNA >=1,000cp/mL) on second-line PI- containing regimens lack PI-resistance mutations in the protease (PR) gene is one of the main enigmas of antiretroviral drug resistance research. We hypothesized that env and gag sequences from these patients contain compensatory mutations, specifically in the Env, that confer PI resistance. A role of the gp41 CT in PI resistance may be linked to the role of virus maturation, triggered by PR, in activating Env fusion activity. In recent years, increasing numbers of clinical reports have observed failure of DTG-containing therapy in the absence of integrase (IN) mutations, suggesting that mutations outside IN may confer resistance in these patients. Our studies demonstrated the first instance of de novo selection of Env mutations that confer resistance to Dolutegravir (DTG) in vitro (unpublished data). We attribute this phenotype to the ability of the Env mutants to mediate highly efficient cell-to-cell transmission, resulting in an increase in the multiplicity of infection. In addition, up to 20-, 6- and 24-fold reduction in susceptibility to ATV, DRV and LPV, respectively, was observed in absence of PR mutations among HIV-1 CRF_02AG and subtype G infected patients. These findings have broad implications for our understanding of Env and Gag functions and the evolution of HIV-1 drug resistance. Our goal is to identify factors that predict virological failure (VF) on ATV/r or LPV/r as patients with VF on a PI-containing regimens have suboptimal future treatment and may be at an increased risk for developing integrase resistance inhibitor on a third-line regimen. A collaborative team involving investigators led by the Institute of Human Virology Nigeria will exploit robust longitudinal cohorts (Cameroon, Nigeria and Uganda) of patients on 2L regimens, coupled to well- characterized and archived clinical specimens that underpin a rigorous nested case-control study design. To test these interlocking hypotheses, we propose to: (i) use our newly developed HIV-xGen next generation sequencing method to extend understanding of viral evolution and HIV drug resistance across the viral genome and to determine whether there is effect modification by non-B HIV-1 subtypes, (ii) characterize recombinant viruses carrying mutations identified in Aim 1, using established phenotypic assays to determine their effect on drug susceptibility and site-directed mutagenesis, and lastly (iii) determine differences in replicative fitness on drug susceptibility with use of a yeast recombination-based cloning system. Our plans to study these patients and their viruses (subtype A, C, D, G, and CRF02_AG, CRF43_02G, HIV-1 O and HIV-1 N) provides a unique opportunity to determine the phenotypic and clinical significance of genotypic changes in HIV-1 gag, gag-pol and env genes. All in all, this proposal will permit exciting epidemiological, clinical, and public health research opportunities.