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: 10450682
• 负责人: Brandon C Rosen
• 金额: $ 4.74万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10450682
• 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; 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.

项目摘要 对艾滋病毒感染的消毒治疗仍然难以捉摸，并且仍然是最大的挑战之一 场地。病毒的快速突变及其随后从适应性免疫识别的逃脱 系统不仅阻碍了预防性疫苗接种策略，而且还阻碍了体内的病毒 受感染的个体，因此必要的终身抗逆转录病毒疗法。这种病毒“逃脱” 免疫识别继续对旨在诱导抗病毒物的研究工作构成巨大挑战 通过预防性和治疗性通过细胞毒性T淋巴细胞（CTL）和抗体（ABS）免疫。 这项研究，我们将使用新型免疫治疗策略的体外效率开发和评估 HIV感染的SIVMAC239模型。我们试图诱导组成型CTL介导的SIVMAC239- 通过含有单克隆AB（MAB）和加载的MHC I的双功能融合蛋白感染细胞 （PMHCI）域。 MAB结构域将负责融合蛋白定位到感染细胞表面， 而PMHCI域将招募并激活CTL以杀死受感染的细胞。在拟议的研究的目标1中， 我们将评估使用SIVMAC239 INVELOPE（ENV）的使用mAb来靶向和标记SIVMAC239- 感染细胞。将筛选15个SIVMAC239 ENV结合MAB，以结合感染细胞的能力和 因为他们对病毒逃生的敏感性。这些mAb包括已知的感染细胞限制者ECD4-Ig和5l7 IgG， 目标2，我们 将产生由SIVMAC239 ENV结合MAB和带有A的MHC I分子组成的融合蛋白 免疫主导CTL发作，然后测试这些融合蛋白诱导CTL介导的杀戮的能力 体外感染细胞。我们将使用ECD4-Ig和5L7 IgG开始我们的融合蛋白研究，这两者都结合 受感染的细胞，并且似乎没有根据先前的研究选择逃生突变体。我们旨在招募高级 频率CTL种群（>整个主机CTL曲目的10％），因此将包括免疫主导者 融合蛋白中的Sivmac239 sivmac239 tat Sl8和恒河猴（RHCMV）IE1 VY9。通过招聘 富含和无处不在的CTL种群杀死受感染的细胞，我们假设这些融合蛋白可以 如果不灭菌，则提供对病毒复制的强大而长期的抑制。重要的是，这种策略将 允许外源输送MHC I分子，该分子装有不变的肽抗原，独立于 病毒基因型。那就是这些融合蛋白会诱导SIVMAC239感染细胞的构型杀死， 不管病毒是否具有CTL逃脱突变。这项研究将提供有关mab介导的洞察力 靶向HIV/SIV感染的细胞，抗逆转录病毒CTL反应的效力和特异性以及 CTL逃生的治疗挑战，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 融合蛋白是一种治疗和潜在消毒HIV/SIV感染的新型策略。