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: 8555772
• 负责人: Edward Berger
• 金额: $ 53.94万
• 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/8555772
• 关键词: Adoptive Transfer; Animal Model; Antibodies; Antigen Receptors; Antiviral Agents; Area; Autologous; Bacterial Proteins; Binding; Biological Assay; Bone Marrow; CCR5 gene; CD28 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CXCR4 gene; Cell Therapy; Cells; Clinical; Clinical Research; Collaborations; Development; Engineering; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Receptors; HIV-1; Human; Immunotoxins; Infection; Liver; Longevity; Macaca mulatta; Malignant Neoplasms; Masks; Membrane; Memory; Methods; Modeling; Modification; Molecular; Molecular Conformation; Mus; Mutation; National Institute of Allergy and Infectious Disease; North Carolina; Pathogenesis; Phase; Predisposition; Prevention; Process; Property; Research Personnel; Signal Transduction; Site; Stem cells; Structure; Surface; Synapses; T-Lymphocyte; Testing; Thymus Gland; Time; Tropism; V3 Loop; Variant; Viral; Viral Load result; Virus; antiretroviral therapy; base; cell killing; cytotoxic; design; design and construction; extracellular; immunogenicity; improved; in vivo; in vivo Model; insight; interest; killings; molecular dynamics; neutralizing antibody; novel; prevent; receptor; synthetic peptide

1) Structure/function studies of the HIV Env trimer (collaboration with Dr. Paolo Lusso, NIAID). Extending our findings that V1V2 masking of the V3 loop of gp120 occurs by an intraprotomer (cis) rather than interprotomer (trans) mechanism, we are pursuing a proposal for a novel gp120 conformation that we believe represents the unliganded structure in the native trimer prior to CD4 binding. Our evidence comes from a combination of studies with monclonal antibodies and synthetic peptides based on newly predicted sites of intramolecular interactions, as well as gp120 and CD4 constructs (soluble and membrane anchored forms) with engineered mutations in the regions of interest. Molecular dynamics analyses provide support the new model. 2) Immunotoxiins for targeted killing of HIV-infected cells. We have initiated collaborations using in vivo models to test the ability of immunotoxins directed against HIV-1 Env expressed on the surface of productively infected cells to deplete infected cell reservoirs persisting during ART. In collaboration with Dr. Tom North (recently moved from U. C. Davis to Emory U.), we are examining the effects of sCD4-PE40 in SIVmac239-infected rhesus macaques whose viral loads have been suppressed below detectable levels by combination ART; the goal is to test whether the immunotoxin significantly delays (or, less likely, prevents) the virus rebound typically observed after ART cessation. Analysis of the first phase of this study is near completion. We have also initiated a collaboration with Drs. Victor Garcia-Martinez and Shailesh K Choudhary (U. North Carolina) to test the activity of CD4-PE40 and 3B3-PE38 in HIV-1-infected humanized mice (containing human bone marrow, liver, thymus, i.e. BLT mice); again, we wish to test whether the immunotoxins significantly delay (or prevent) viral rebound after cessation of ART. 3) Enhanced chimeric antigen receptors (CARs) for adoptive transfer of autologous CD8 T cells genetically modified to target HIV-1 Env (collaboration with Drs. Steven Rosenberg and Rick Morgan, NCI). The immunotoxin approach suffers from the inherent problem of immunogenicity due to the foreign moiety of such proteins (bacterial in the case of PE-based agents); thus the immunotoxin treatment is limited to short duration, a major limitation in view of the presence of reservoirs of latently infected cells that can become activated at a later time. Thus a more durable "targeted cell killing" method would seem preferable. Adoptive trasfer of engineered CD8 T cells represents an exciting option, particularly in view of the emerging successses of this strategy in the cancer filed. This has been tried for HIV infection more than a decade ago; while the transferred engineered T cells were found to populate many anatomical sites where HIV replication occurs, they persisted only at low levels, and no clinical benefits were obtained. We are introduciing two new features to the CAR constructs that designed to improve efficacy of this approach. The first includes enhancement of the intracellular signaling domains by including relevant motifs developed by cancer investigators in the CAR field namely regions of CD28 and 41BB; these are expected to enhance the functional activity and in vivo longevity of the adoptively transferred CD8 cells. Perhaps more importantly are modifications that we have introduced into the extracellular Env-targeting moiety. We have designed a modified variants of the 2-domain extracellualr region of CD4 (designated CD4-M) with two important features compared to the corresponding wild type motif: much higher binding efficiency to surface Env, and inability to support HIV entry into target cells expressing this molecule plus coreceptor CCR5 or CXCR4. Indeed we found that CAR constructs with wild type CD4 extracellular motifs used in previous clinical studies functioned very efficiently as entry receptors, suggesting the likely possibility that when transduced into T CD8 cells (which also express CCR5), such CAR constructs rendered the cells highly susceptible to HIV infection. We believe this is a likely scenario because CD8 T cells exert their cytotoxic activity against infected CD4 T cells by establishing intimate contacts with these targets; such conditions would be optimal for the undesired iinfection of the CD8 cells via cell-to-cell infection ("virologic synapse"). CARs containing the modified CD4-M extracellular motif were found to be completely devoid of of this undesired entry receptor activity. The second critical feature of CD4-M is that because of its greatly enhance binding to surface Env, it should significantly enhance the cytotoxic activity of the engineered CD8 T cells. Preliminary assays show only a modest improvement compared to the wild type CD4 moiety, but these assays were not performed under conditions expected to optimally reveal the improvements, i.e. they were performed with isolates whose Envs are inherently capable of efficiently binding WT CD4, and under conditions of high Env expression on the target cells (i.e. Env pseudotype viruses). More meaningful tests are underway using Envs from isolates known to bind CD4 much less efficiently (i.e. many clinical isolates), and under conditions of less abundant Env surface expression (as occurs during productive infection of primary CD4+ T cells). In our efforts to extend the CAR studies to a relevant animal model, we have begun studies to test the approach in rhesus macaques infected with SIVmac239 (collaboration with Dr. Mario Roederer, VRC). To this end, we have are designing CAR constructs with the corresponding rhesus-derived motifs, including the rhesus based CD4-M variant. We also plan to compare CAR efficacy with different subsets of rhesus CD8 cells, including the recently described memory CD8 T cells with stem cell-like properties.

1）HIV Env Trimer的结构/功能研究（与Niaid的Paolo Lusso博士合作）。扩展了我们的发现，即GP120的V3环的V1V2掩盖是由脑内（CIS）而不是Interlototomer（Trans）机制发生的，我们正在追求对新型GP120构型的建议，我们认为，我们认为这代表了在CD4结合之前本机内胶合体中无固定的结构。我们的证据来自基于新预测的分子内相互作用的部位以及GP120和CD4构建体（可溶性和膜锚定形式）以及在利益区域的工程突变的研究。分子动力学分析提供了支持新模型。 2）用于靶向杀死HIV感染细胞的免疫毒素。我们已经使用体内模型进行了合作，以测试针对在有效感染细胞表面表达的针对HIV-1 ENV的免疫毒素的能力，以消耗ART期间的感染细胞储层。与汤姆·诺斯（Tom North）博士（最近从美国戴维斯（U. C. Davis）迁至埃默里（Emory U.目的是测试免疫毒素是否会显着延迟（或较小的可能阻止）病毒反弹，通常在停止艺术后会观察到。对这项研究的第一阶段的分析即将完成。我们还与Drs启动了合作。 Victor Garcia-Martinez和Shailesh K Choudhary（美国北卡罗来纳州）在HIV-1感染的人源化小鼠中测试CD4-PE40和3B3-PE38的活性（含有人骨髓，肝，胸腺，胸腺，即BLT小鼠）；同样，我们希望测试免疫毒素在停止艺术后是否显着延迟（或预防）病毒反弹。 3）增强的嵌合抗原受体（CAR），用于对靶向HIV-1 Env的遗传修饰的自体CD8 T细胞（与Steven Rosenberg和NCI的Rick Morgan博士的合作）进行遗传修饰。免疫毒素方法由于这种蛋白质的外来部分（在基于PE的剂的情况下）而遭受了免疫原性的固有问题；因此，免疫毒素治疗仅限于持续时间短，这是鉴于有潜在感染细胞的储层的主要局限性，这些储层可以在以后被激活。因此，更耐用的“目标细胞杀死”方法似乎是可取的。工程CD8 T细胞的收养Trasfer代表了一个令人兴奋的选择，尤其是鉴于该策略在提交的癌症中的新兴成功方面。十多年前，已经尝试过用于艾滋病毒感染的艾滋病毒感染。虽然发现转移的工程T细胞填充了发生HIV复制的许多解剖部位，但它们仅在低水平上持续存在，并且没有获得临床益处。我们正在为汽车构造引入两个新功能，这些功能旨在提高这种方法的功效。首先包括通过包括由CD28和41BB区域的癌症研究者开发的相关基序来增强细胞内信号传导域；这些预计将增强收养CD8细胞的功能活性和体内寿命。也许更重要的是，我们已经引入了细胞外隔离部分的修改。与相应的野生型基序相比，我们设计了CD4（指定CD4-M）的两个重要特征的2域外胞外区域的修改变体：与表面Env的相应野生型基序相比，更高的结合效率，无法支持HIV进入表达该分子的靶细胞，表达该分子和CRECEPTOR CCR5或CXCR4。确实，我们发现，先前临床研究中使用的具有野生型CD4细胞外基序的汽车构建体作为入口受体非常有效地发挥作用，这表明当将转导为T CD8细胞（也表达CCR5）时，这种可能的可能性使这种汽车构建了这些细胞使该细胞对HIV感染极敏感。我们认为这很可能是一种情况，因为CD8 T细胞通过与这些靶标建立紧密接触而针对感染的CD4 T细胞发挥其细胞毒性活性。这种条件对于通过细胞对细胞感染（“病毒式突触”）的不期望的CD8细胞的I摄取将是最佳的。发现含有改良CD4-M细胞外基序的汽车完全没有这种不希望的入口受体活性。 CD4-M的第二个关键特征是，由于其大大增强了与表面Env的结合，因此应显着增强工程CD8 T细胞的细胞毒性活性。与野生型CD4部分相比，初步测定只有适度的改进，但是在预期的条件下，没有进行这些测定，即在最佳的情况下进行改进，即它们是用envs进行的分离株进行的，其ENV可以有效地结合WT CD4，并且在目标细胞上有效地具有高度结合的条件（即，在目标细胞上具有高度的条件（即env Pseudotype psseypepotype型）。使用来自已知结合CD4的分离株的ENV进行的更有意义的测试（即许多临床分离株），在较少丰富的ENV表面表达的条件下（如主CD4+ T细胞的生产性感染期间，发生的情况发生）。为了将汽车研究扩展到相关的动物模型，我们已经开始研究以Sivmac239感染的Rhesus Macaques（与Mario Roederer，VRC的合作）感染的方法。为此，我们正在设计具有相应的恒河源主题的汽车构建体，包括基于恒河猴的CD4-M变体。我们还计划将汽车疗效与不同子集CD8细胞（包括最近描述的具有干细胞样性能的记忆CD8 T细胞）进行比较。