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

重新定位 HIV/SIV 感染中的细胞毒性 T 淋巴细胞以杀死受感染的细胞

基本信息

批准号：
10001322
负责人：
Brandon C Rosen
金额：
$ 5.05万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10001322
关键词：
AIDS/HIV problem Acquired Immunodeficiency Syndrome Adaptive Immune System Affinity Animals Anti-Retroviral Agents Antibodies Antigens Autologous Binding Biological Assay Biotechnology CCR5 gene CD4 Positive T Lymphocytes CXCR4 gene Cell surface Cells Chimeric Proteins Coculture Techniques Codon Nucleotides Cytomegalovirus Cytotoxic T-Lymphocytes Data Development Enzyme-Linked Immunosorbent Assay Escape Mutant Exhibits Fab Immunoglobulins Flow Cytometry Frequencies Future Genotype HIV HIV Envelope Protein gp120 HIV Infections HIV-2 Harvest Human Immunodominant Epitopes Immunoglobulin G Immunotherapeutic agent Immunotherapy In Vitro Individual Infection Laboratories Lead Lymphocyte Suppression Mediating Modeling Monitor Monoclonal Antibodies Mutation Peptides Plasmids Population Predisposition Production Proteins RNA Research Resistance Rhesus SIV Serial Passage Specificity Stains Structure Surface T-Lymphocyte Epitopes Testing Therapeutic Transfection Viral Virus Virus Replication acute infection adaptive immunity anti-IgG antiretroviral therapy antiviral immunity arm base chronic infection cytotoxic CD8 T cells design experimental study fluorophore in silico in vivo in vivo evaluation insight neoplastic cell novel novel strategies prophylactic recruit response single molecule treatment strategy vaccination strategy viral fitness

项目摘要

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.

项目概要 HIV 感染的绝育疗法仍然难以实现，并且仍然是人类面临的最大挑战之一。病毒的快速突变率及其随后逃避适应性免疫识别的领域。系统不仅阻碍了预防性疫苗接种策略，而且阻碍了从体内消灭病毒感染者，因此需要终身抗逆转录病毒治疗。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) 产生预防性和治疗性免疫。在这项研究中，我们将开发并评估一种新型免疫治疗策略的体外疗效，使用 HIV感染的SIVmac239模型我们试图诱导CTL介导的SIVmac239-介导的组成型杀伤。通过含有单克隆抗体 (mAb) 和肽负载 MHC I 的双功能融合蛋白感染细胞 (pMHCI) 结构域将负责融合蛋白定位到受感染的细胞表面，而 pMHCI 结构域将招募并激活 CTL 来杀死受感染的细胞。我们将评估 SIVmac239 包膜 (Env) 结合单克隆抗体用于靶向和标记 SIVmac239 的用途将筛选 15 种 SIVmac239 包膜结合单克隆抗体，以确定它们与感染细胞结合的能力。这些单克隆抗体包括已知的感染细胞结合剂 eCD4-Ig 和 5L7 IgG，在目标 2 中，我们与沃特金斯实验室从受感染动物中分离出 13 种新型环境结合单克隆抗体。将产生由 SIVmac239 Env 结合 mAb 和负载 MHC I 分子组成的融合蛋白免疫显性 CTL 表位，然后测试这些融合蛋白诱导 CTL 介导的杀伤的能力我们将开始使用 eCD4-Ig 和 5L7 IgG 进行融合蛋白研究，两者都结合。根据之前的研究，感染细胞似乎不会选择逃逸突变体。频率 CTL 群体（> 整个宿主 CTL 库的 10%），因此将包括免疫显性通过招募我们的融合蛋白中的 SIVmac239 Tat SL8 和恒河猴巨细胞病毒 (RhCMV) IE1 VY9 表位。丰富且无处不在的 CTL 群体来杀死受感染的细胞，我们发现这些融合蛋白可以重要的是，这种策略即使不是绝育，也将提供对病毒复制的强有力和长期的抑制。允许外源递送负载有独立于的不变肽抗原的 MHC I 分子因此，这些融合蛋白会诱导 SIVmac239 感染细胞的组成型杀伤，无论病毒是否含有 CTL 逃逸突变，这项研究都将提供对 mAb 介导的深入了解。 HIV/SIV 感染细胞的靶向、抗逆转录病毒 CTL 反应的效力和特异性，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最重要的是，这项研究的结果可以为 pMHCI-mAb 提供概念验证。融合蛋白作为治疗和潜在消灭 HIV/SIV 感染的新策略。