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

使用人源化小鼠进行超灵敏体内潜伏 HIV 病毒生长测定

• 批准号: 8966520
• 负责人: Ramesh Akkina
• 金额: $ 66.29万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-30 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966520
• 关键词: Adoptive Transfer; Aftercare; Allogenic; Animals; Anti-HIV Therapy; Area; Berlin; Biological Assay; Birth; Boston; CD4 Positive T Lymphocytes; Cells; Child; Coculture Techniques; Colorado; Development; Goals; Growth; HIV; HIV Infections; HIV-1; In Vitro; Infection; Interruption; Latent Virus; Macaca; Methods; Mississippi; Mus; Names; Patients; Peripheral; Pharmaceutical Preparations; Plasma Cells; Residual state; Rest; SIV; Sampling; System; Testing; Tissues; Universities; Viral; Virus; Work; antiretroviral therapy; base; experience; forging; improved; in vivo; innovation; novel; optimism; public health relevance; research study; response

 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.

 描述（由申请人提供）： 摘要：“柏林患者”完全治愈的例子给艾滋病领域注入了很多乐观情绪，当前的重点是继续开发新的 HIV-1 根除策略。在这种情况下，必须开发可靠的测试来验证诱导型病毒库中的显着还原素，并预测抗逆转录病毒治疗（ART）中断（ATI）后对治疗策略的反应。除了 ATI 之外，通过共培养方法筛选潜伏感染细胞的病毒生长测定 (VOA) 对于量化在治疗策略后可能持续存在的可诱导或有复制能力的残留病毒至关重要。然而，在一些血浆和细胞相关HIV-1长期检测不到的患者中，例如两名接受同种异体HSCT的“波士顿患者”和出生后立即接受早期ART的“密西西比儿童”，传统的VOA无法检测到外周CD4 T细胞中的残留病毒，尽管治疗中断后病毒反弹。因此，需要开发和验证采用新颖和创新系统（例如 HIV 易感人源化小鼠（hu-mice））的更灵敏的 VOA 变得非常重要。在最近的一项研究中，通过将 CD4 T 细胞过继转移给幼稚动物，成功地从完全病毒抑制的 SIV 感染猕猴（根据所有标准测试确定）进行强化 ART 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否则无法检测到的潜伏感染细胞。我们目前的提议是基于这些最新发现和假设，即体内人源化小鼠病毒生长测定 (hmVOA) 在检测 HIV 患者中低水平的持续感染方面比传统的体外 VOA 更敏感。我们的具体目标是： 1. 使用患者来源的静息 CD4 T 细胞开发和优化针对潜伏 HIV 的体内人源化小鼠病毒生长测定 (hmVOA)，2. 使用患者来源的静息 CD4 T 细胞将 hmVOA 与传统 VOA 进行比较，并确定 hmVOA 是否更敏感，3. 确定 hmVOA 是否可以检测病毒存在的患者中的持续性 HIV 感染。 无法通过任何其他方法（包括 VOA）检测到，4. 评估与标准 VOA 相比，hmVOA 是否可以更容易地检测来自长期完全抑制患者的组织储存库/避难所的病毒潜伏细胞，5. 确定通过用 HIV 潜伏期逆转剂（如罗米地辛和帕比司他）对细胞输注小鼠进行体内治疗，是否可以进一步提高 hmVOA 的敏感性。