Retargeting cytotoxic T lymphocytes in HIV/SIV infection to kill infected cells

项目摘要 艾滋病毒感染的绝育治疗仍然是难以捉摸的，并且仍然是世界上最大的挑战之一。 领域病毒的快速突变率及其随后逃避适应性免疫的识别 这一系统不仅阻碍了预防性疫苗接种策略，而且也阻碍了从体内根除病毒 因此，需要终身抗逆转录病毒治疗。这种病毒式的“逃离”， 免疫识别继续对旨在诱导抗病毒的研究工作构成巨大挑战， 通过细胞毒性T淋巴细胞（CTL）和抗体（Ab），在免疫学上和治疗上都具有免疫性。在 在这项研究中，我们将开发和评估一种新的免疫治疗策略的体外功效， HIV感染的SIVmac 239模型我们试图诱导组成型CTL介导的SIVmac 239 - 1杀伤。 通过含有单克隆抗体（mAb）和负载肽的MHC I的双功能融合蛋白感染细胞 （pMHCI）结构域。mAb结构域将负责融合蛋白定位于感染的细胞表面， 而pMHCI结构域将募集并激活CTL以杀死感染的细胞。在拟议研究的目标1中， 我们将评估SIVmac 239包膜（Env）结合mAb用于靶向和标记SIVmac 239- 被感染的细胞将筛选15种SIVmac 239 Env结合mAb结合感染细胞的能力， 因为它们对病毒逃逸的敏感性。这些mAb包括已知的感染细胞结合物eCD 4-IG和5L 7 IgG， 沿着的还有Watkins实验室从感染动物中分离的13种新型Env结合mAb。在目标2中， 将产生由SIVmac 239 Env-结合mAb和负载有 免疫优势CTL表位，然后测试这些融合蛋白诱导CTL介导的杀伤细胞的能力。 体外感染细胞。我们将开始使用eCD 4-IG和5L 7 IgG的融合蛋白研究，这两种蛋白都结合 感染的细胞，并没有出现选择逃逸突变体的基础上，以前的研究。我们的目标是招募高- 频率的CTL群体（>整个宿主CTL库的10%），因此将包括免疫显性的 表位SIVmac 239达特SL 8和恒河猴巨细胞病毒（RhCMV）IE 1 VY 9。通过招募 大量和普遍存在的CTL群体杀死感染的细胞，我们假设这些融合蛋白可以 提供对病毒复制的强力和长期抑制，如果不是灭菌的话。重要的是，这一战略将 允许外源性递送负载有不依赖于免疫原性的恒定肽抗原的MHC I分子， 病毒基因型因此，这些融合蛋白将诱导SIVmac 239感染的细胞的组成性杀伤， 而不管病毒是否携带CTL逃逸突变。这项研究将提供对mAb介导的深入了解 靶向HIV/SIV感染细胞，抗逆转录病毒CTL应答的效力和特异性，以及 CTL逃逸的治疗挑战，所有这些都将促进抗逆转录病毒药物的未来发展。 免疫疗法最重要的是，本研究的结果可以为pMHCI-mAb提供概念验证 融合蛋白作为一种新的策略，用于治疗和潜在的消毒艾滋病毒/SIV感染。

PROJECT SUMMARY A sterilizing cure for HIV infection has remained elusive and persists as one of the greatest challenges in the field. The rapid mutation rate of the virus and its subsequent escape from recognition by the adaptive immune system has hindered not only prophylactic vaccination strategies, but also eradication of the virus from the bodies of infected individuals, thus necessitating lifelong antiretroviral therapy. This viral “escape” from immunorecognition continues to pose a tremendous challenge for research efforts aimed at inducing antiviral immunity via cytotoxic T lymphocytes (CTLs) and antibodies (Abs), both prophylactically and therapeutically. In this study, we will develop and evaluate the in vitro efficacy of a novel immunotherapeutic cure strategy using the SIVmac239 model of HIV infection. We seek to induce constitutive CTL-mediated killing of SIVmac239- infected cells via a bifunctional fusion protein containing monoclonal Ab (mAb) and peptide-loaded MHC I (pMHCI) domains. The mAb domain will be responsible for fusion protein localization to the infected cell surface, while the pMHCI domain will recruit and activate CTLs to kill the infected cell. In Aim 1 of the proposed study, we will evaluate the use of SIVmac239 envelope (Env)-binding mAbs for targeting and marking SIVmac239- infected cells. Fifteen SIVmac239 Env-binding mAbs will be screened for their ability to bind infected cells and for their susceptibility to viral escape. These mAbs include the known infected cell-binders eCD4-Ig and 5L7 IgG, along with 13 novel Env-binding mAbs isolated from infected animals by the Watkins laboratory. In Aim 2, we will produce fusion proteins composed of a SIVmac239 Env-binding mAb and a MHC I molecule loaded with an immunodominant CTL epitope, then test the ability of these fusion proteins to induce CTL-mediated killing of infected cells in vitro. We will begin our fusion protein studies with eCD4-Ig and 5L7 IgG, both of which bind infected cells and do not appear to select for escape mutants based on previous studies. We aim to recruit high- frequency CTL populations ( > 10% of entire host CTL repertoire), and will therefore include the immunodominant epitopes SIVmac239 Tat SL8 and rhesus cytomegalovirus (RhCMV) IE1 VY9 in our fusion proteins. By recruiting abundant and ubiquitous CTL populations to kill infected cells, we hypothesize that these fusion proteins could provide robust and long-term suppression of viral replication, if not sterilization. Importantly, this strategy would allow exogenous delivery of MHC I molecules loaded with invariant peptide antigens that are independent of viral genotype. Thus, these fusion proteins would induce constitutive killing of SIVmac239-infected cells, regardless of whether the virus harbors CTL escape mutations. This study will provide insight into mAb-mediated targeting of HIV/SIV-infected cells, the potency and specificity of antiretroviral CTL responses, and the therapeutic challenge of CTL escape, all of which will facilitate the future development of antiretroviral immunotherapies. Most importantly, the results of this study could provide proof-of-concept for pMHCI-mAb fusion proteins as a novel strategy for treating and potentially sterilizing HIV/SIV infection.