An Ultrasensitive In Vivo Latent HIV Viral Outgrowth Assay Using Humanized Mice

 描述(由申请人提供)： 摘要：完全治愈的“柏林病人”的例子给艾滋病毒领域注入了许多乐观情绪，目前的主旨是推进新的艾滋病毒-1根除战略。在此背景下，必须开发可靠的测试，以验证可诱导的病毒库中的显著减少，并预测抗逆转录病毒治疗(ART)中断(ATI)后的治疗策略的反应。除ATI外，通过共培养方法筛选潜伏感染细胞的病毒外生长分析(VOA)对于量化可诱导或具有复制能力的残留病毒至关重要，这些残留病毒可能会在治疗策略后持续存在。然而，在一些长期无法检测到血浆和细胞相关艾滋病毒-1的患者中，例如两名接受异基因造血干细胞移植的“波士顿患者”和出生后立即接受早期抗逆转录病毒治疗的“密西西比州儿童”，传统的VOAS无法在外周CD4T细胞中检测到残留病毒，尽管病毒在治疗中断后有所反弹。因此，需要开发和验证使用新的创新系统的更敏感的VOAs，例如对HIV敏感的人源化小鼠(HU-MICE)。在最近的一项研究中，通过将SIV感染的猕猴的CD4T细胞过继转移到幼稚的动物身上，成功地从完全抑制病毒的SIV感染的猕猴(由所有标准测试确定)中恢复了潜伏病毒。这表明，使用体内系统可以捕获和诱导超低水平的原本无法检测到的潜伏感染细胞。我们目前的建议是基于这些最新的发现和假设，即体内人源化小鼠病毒生长试验(HmVOA)在检测HIV患者低水平持续感染方面将比传统的体外VOAS更敏感。我们的具体目标是：1.建立和优化利用患者来源的静息CD4T细胞检测潜伏HIV的体内人源化小鼠病毒生长试验(HmVOA)；2.使用患者来源的静息CD4T细胞将hmVOA与传统的VOA进行比较，并确定hmVOA是否更敏感；3.确定hmVOA是否可以检测到病毒在包括VOA在内的任何其他方法都无法检测到的患者中的持续感染；4.评估hmVOA是否可以比标准VOA更容易地从长期完全抑制的患者的组织储藏室/避难所检测到病毒潜伏细胞，5.确定体内注射HIV潜伏期反转剂如罗米地辛和潘诺比妥能否进一步提高hmVOA的敏感性。

 DESCRIPTION (provided by applicant): ABSTRACT: The fully cured "Berlin patient" example instilled a lot of optimism in the HIV field and the current thrust is to forge ahead with developing novel HIV-1 eradication strategies. In this context, it is essential that reliable tests are developed that can verify significant reductins in inducible viral reservoirs and predict responses to curative strategies after antiretroviral therapy (ART) interruptions (ATI). Other than ATI, viral out growth assays (VOA) that screen for latently infected cells by co-culturing methods have been critical to quantifying inducible or replication competent residual virus that may persist after a curative strategy. However, in some patients with prolonged undetectable plasma and cell-associated HIV-1 such as the two "Boston patients" who underwent allogeneic HSCT and the "Mississippi child" who had undergone early ART right after birth, traditional VOAs could not detect residual virus in peripheral CD4 T cells although virus rebounded after treatment interruption. Thus, it has become important that more sensitive VOAs that employ novel and innovative systems such as HIV susceptible humanized mice (hu-mice) need to be developed and validated. In a recent development, latent virus was successfully recovered from fully virus suppressed SIV infected macaques (as determined by all standard tests) undergoing intensive ART by adoptive transfer of their CD4 T cells to naive animals. This showed that ultralow levels of otherwise undetectable latently infected cells could be captured and induced using an in vivo system. Our current proposal is based on these recent findings and the hypothesis that an in vivo humanized mouse viral outgrowth assay (hmVOA) will be more sensitive than traditional in vitro VOAs at detecting low levels of persistent infectin in HIV patients. Our specific aims are to: 1. Develop and optimize an in vivo humanized-mouse viral outgrowth assay (hmVOA) for latent HIV using patient derived resting CD4 T cells, 2. Compare hmVOA with traditional VOA using patient derived resting CD4 T cells and determine if hmVOA is more sensitive, 3. Determine whether hmVOA can detect persistent HIV infection in patients in which virus is undetectable by any other method, including VOA, 4. Evaluate if hmVOA can more readily detect virus-latent cells from tissue reservoirs/ sanctuaries of long-term fully suppressed patients' compared to standard VOA, 5. Determine if sensitivity of hmVOA can be further improved by in vivo treatment of cell infused mice with HIV latency-reversing agents such as romidepsin and panobinostat.