HIV-Receptor Interactions and Related Anti-HIV Strategies

HIV 受体相互作用和相关抗 HIV 策略

• 批准号: 9161460
• 负责人: Edward Berger
• 金额: $ 79.07万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9161460
• 关键词: Acute; Animals; Anti-Retroviral Agents; Antibodies; Antigen Receptors; Binding; Biological Assay; C-Type Lectins; CCR5 gene; CD209 gene; CXCR4 gene; Carbohydrates; Cell fusion; Cells; Chronic; Collaborations; Complement; Complex; Extracellular Domain; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Receptors; HIV-1; Human; Immunotoxins; In Vitro; Infection; Length; Link; Macaca mulatta; Mannose Binding Lectin; Mannose-Binding Lectins; Mediating; Membrane; Modeling; Molecular; Monoclonal Antibodies; N-terminal; National Institute of Allergy and Infectious Disease; Peripheral Blood Mononuclear Cell; Predisposition; Prevention; Proteins; Pseudomonas aeruginosa toxA protein; Recombinant Proteins; Refractory; Research Personnel; Resolution; Screening Result; Serum; Site; Surface Plasmon Resonance; Testing; Treatment Protocols; Tyrosine; Vaccine Research; Virus; X-Ray Crystallography; antiretroviral therapy; base; design; env Glycoproteins; in vivo; killings; langerin; mouse model; neutralizing antibody; nonhuman primate; novel; polypeptide; receptor; receptor binding; simian human immunodeficiency virus; sulfation

1. Design of soluble protein constructs displaying the extracellular domains of the HIV coreceptors CCR5 and CXCR4. The HIV-1 Env glycoprotein has been studied extensively at the atomic level (X-ray crystallography and high resolution cryo-EM), including constructs of the gp120 glycoprotein (the receptor-binding subunit) in unliganded form and bound to soluble CD4 and specific monoclonal antibodies. However the complex of gp120 bound to coreceptor (CCR5 or CXCR4) has thus far proven refractory to such analyses, in large part due to the intricate membrane associations of these molecules (they are GPCRs with 7 TM segments). In FY2015, we extended our design and analysis of soluble protein constructs containing extracellular domains of CCR5 known to be critical for coreceptor function, with particular emphasis on a recombinant protein containing sCD4 attached by a polypeptide linker to the CCR5 N-terminal region. By optimizing features of the construct (e.g. linker length) and its expression (e.g. to maximize tyrosine sulfation), we have shown in assays of Env-mediated cell fusion and pseudovirus neutralization that the CCR5 N-terminal moiety interacts specifically with its cognate site on gp120. Efforts are underway to further analyze these interactions using surface plasmon resonance, as a precursor to collaborative efforts to crystalize this protein construct in complex with gp120, hopefully leading to atomic level structural analysis. 2. Targeting killing of HIV-infected cells. We have proposed that complementing combination anti-retroviral therapy (cART) with an immunotoxin designed to selectively kill HIV-infected cells might offer a means to cure acute infection. In collaboration with Dr. Ira Pastan, NCI, we have conducted studies of a new immunotoxin based on Pseudomonas aeruginosa exotoxin A, employing a targeting domain from an optimized broadly neutralizing antibody isolated by the NIAID Vaccine Research Center. Our studies demonstrate excellent breadth and potency against genetically diverse HIV-1 isolates. To test the concept of curing acute infection in a nonhuman primate model, we have begun a collaborative study with investigators at the US MHRP and the VRC to test whether immunotoxin administration can complement cART to achieve a cure of acute SHIV infection of rhesus macaques, based on prevention of virus emergence after cessation of all treatment. At present, we are analyzing sera from animals intended for the study to test for pre-existing anti-PE antibodies; the SHIV challenge/treatment protocol will begin pending the results of this screen. As a potential functional cure of chronic HIV infection, we are extending our studies with chimeric antigen receptors (CARs) containing novel bispecific CD4-based targeting motifs. Our results with CARs containing CD4 linked to the carbohydrate recognition domains (CRDs) of various human C-type lectins indicate the greatly enhance potencies of the CRDs from human mannose-binding lectin 2 (MBL-2) and langerin, compared to those from DC-SIGN and L-SIGN. We have generated a CD4-MBL2 CAR construct in which all components are based entirely of sequences from rhesus macaques. Pending the expected confirmation of the in vitro efficacy of this CAR in virus suppression assays using SHIV-162P and rhesus PBMC, collaborative in vivo studies will be initiated with Dr. Mario Roederer of the VRC. Another collaborative study has been initiated with Dr. Victor Garcia-Martinez of UNC to test the human CD4-MBL2 CAR in the BLT humanized mouse model.

1。可溶性蛋白质构建体的设计，显示了HIV cocectors CCR5和CXCR4的细胞外域。 HIV-1 ENV糖蛋白已在原子水平（X射线晶体学和高分辨率冷冻EM）上进行了广泛的研究，包括无染色的GP120糖蛋白（受体结合亚基）的构建体，并以可溶性CD4和特定的Monoclonal抗体绑定。然而，与共肽（CCR5或CXCR4）结合的GP120的复合物已证明对这种分析的难治性很大程度上是由于这些分子的复杂膜关联（它们是具有7 TM段的GPCR）。在2015财年，我们扩展了对可溶性蛋白质构建体的设计和分析，这些蛋白质构建构建含有CCR5的细胞外域，已知对于coceptor功能至关重要，特别强调了由多肽链接到CCR5 N末端区域附着的重组蛋白，该蛋白包含重组蛋白。通过优化构建体的特征（例如接头长度）及其表达（例如，为了最大化酪氨酸硫酸化），我们已经在Env介导的细胞融合和中和中和的测定中表明，CCR5 N末端部分部分与GP120上的认知位点特异性相互作用。正在努力使用表面等离子体共振进一步分析这些相互作用，这是与GP120复杂化这种蛋白质构建体的合作努力的先驱，希望导致原子水平的结构分析。 2。靶向杀死HIV感染的细胞。我们已经提出，与旨在有选择性杀死HIV感染细胞的免疫毒素相互补充的抗逆转录病毒疗法（CART）可能会提供一种治愈急性感染的手段。通过与NCI的IRA Pastan博士合作，我们对基于铜绿假单胞菌Exotoxin A的新免疫毒素进行了研究，该毒素采用了通过NIAID疫苗研究中心分离的优化广泛中和抗体的靶向域。我们的研究表明，针对遗传多样的HIV-1分离株的广度和效力很高。为了在非人类灵长类动物模型中测试治愈急性感染的概念，我们已经开始与美国MHRP的研究人员和VRC进行协作研究，以测试免疫毒素给药是否可以补充CART，以治愈所有治疗后的病毒出现。目前，我们正在分析用于研究的动物的血清，以测试现有的抗PE抗体。 SHIV挑战/治疗方案将开始等待此屏幕的结果。作为慢性HIV感染的潜在功能治愈，我们正在使用含有新型双特异性CD4靶向基序的嵌合抗原受体（CAR）扩展研究。与DC-Sign and L-Sign和L-Sign和L-Sign和L-sign相比，我们的含有与各种人类C型凝集素的CD4相关的CD4的汽车与人类结合凝集素2（MBL-2）和Langerin相比，与DC-Sign and L-Sign和L-sign和L-sign符号相比，CRD的能力大大增强了CRD的能力。我们已经生成了一个CD4-MBL2 CAR构建体，其中所有组件完全基于恒河猕猴的序列。在使用SHIV-162P和PBMC抑制病毒抑制测定中的预期证实，将与VRC的Mario Roederer博士启动体内研究。另一项合作研究是与UNC的Victor Garcia-Martinez博士启动的，以测试BLT人源化小鼠模型中的人CD4-MBL2汽车。