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

• 批准号: 10227032
• 负责人: Brandon C Rosen
• 金额: $ 5.1万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 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/10227032
• 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; Preventative vaccination; 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

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.

项目总结 艾滋病毒感染的消毒疗法仍然难以捉摸，而且仍然是世界上最大的挑战之一 菲尔德。病毒的快速变异率及其随后逃脱适应性免疫的识别 该系统不仅阻碍了预防性疫苗接种战略，而且还阻碍了病毒从体内的根除。 因此，需要终生抗逆转录病毒治疗。这种病毒式的“逃脱” 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(Abs)进行免疫，预防和治疗。在……里面 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疗效 HIV感染的SIVmac239模型。我们试图诱导结构性CTL介导的SIVmac239杀伤- 含有单抗和多肽MHC I的双功能融合蛋白感染细胞 (PMHCI)域。MAb域将负责将融合蛋白定位到受感染的细胞表面， 而pMHCI结构域会招募和激活CTL来杀死受感染的细胞。在拟议研究的目标1中， 我们将评估SIVmac239包膜(Env)结合单抗用于靶向和标记SIVmac239- 被感染的细胞。将对15株SIVmac239包膜结合单抗进行筛选，以了解其结合感染细胞和 因为他们对病毒逃逸很敏感。这些单抗包括已知的感染细胞结合子eCD4-Ig和5L7 Ig G， 以及沃特金斯实验室从感染动物中分离到的13株新的Env结合单抗。在目标2中，我们 将产生由SIVmac239囊膜结合单抗和MHC I分子组成的融合蛋白 免疫优势CTL表位，检测这些融合蛋白诱导CTL介导的杀伤作用 体外感染的细胞。我们将从eCD4-Ig和5L7免疫球蛋白开始我们的融合蛋白研究，这两种抗体都可以结合 受感染的细胞，根据以前的研究，似乎没有选择逃逸突变体。我们的目标是招募高- 频率CTL群体(占整个宿主CTL谱系的10%)，因此将包括免疫显性 融合蛋白中含有SIVmac239Tat SL8和恒河猴巨细胞病毒(RhCMV)IE1 VY9表位。通过招聘 大量且普遍存在的CTL群体来杀死感染的细胞，我们假设这些融合蛋白可以 对病毒复制提供强有力的长期抑制，如果不是消毒的话。重要的是，这一战略将 允许外源性递送MHC I分子，这些分子装载有独立于 病毒基因。因此，这些融合蛋白将诱导对感染SIVmac239的细胞的结构性杀伤， 无论病毒是否含有CTL逃逸突变。这项研究将对mAb介导的 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的未来发展 免疫疗法。最重要的是，这项研究的结果可以为pMHCI-mAb提供概念验证 融合蛋白作为一种治疗艾滋病毒/SIV感染的新策略，并可能使其绝育。