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: 8946275
• 负责人: Edward Berger
• 金额: $ 80.96万
• 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/8946275
• 关键词: Acute; Adoptive Transfer; Animals; Antibodies; Antigen Receptors; Binding; Binding Sites; Biochemical; Biological Assay; C-Type Lectins; CCR5 gene; CD8B1 gene; CXCR4 gene; Carbohydrates; Cell fusion; Cells; Chronic; Clinical Research; Collaborations; Complement; Complex; Control Animal; Engineering; Extracellular Domain; Generations; Genes; Genetic; Goals; HIV; HIV Envelope Protein gp120; HIV Receptors; HIV-1; Immunotoxins; In Vitro; Individual; Infection; Interferometry; Lead; Ligands; Link; Macaca; Macaca mulatta; Mannose; Membrane; Methods; Modality; Modeling; Modification; Molecular; Molecular Conformation; National Institute of Allergy and Infectious Disease; Patients; Persons; Polysaccharides; Predisposition; Prevention; Proteins; Pseudomonas aeruginosa toxA protein; Recombinants; Resolution; Roentgen Rays; SIV; Scaffolding Protein; Signal Transduction; Structure; System; T-Lymphocyte; Testing; Time; Vaccinia; Vaccinium; Variant; Viral; Virus; X-Ray Crystallography; antiretroviral therapy; base; biophysical techniques; cell killing; design; design and construction; env Glycoproteins; extracellular; gp160; insight; killings; memory CD4 T lymphocyte; mouse model; neutralizing antibody; neutralizing monoclonal antibodies; novel; particle; pathogen; receptor; recombinant virus; simian human immunodeficiency virus

1) Design of soluble protein constructs displaying the extracellular domains of the HIV coreceptors CCR5 and CXCR4. In FY2014, we designed and characterized soluble constructs containing the extracellular domains of CCR5 previously identified as being critical to coreceptor function, i.e. the N-terminus and extracellular loop 2 (ECL2). While structural details of the HIV-1 Env glycoprotein (gp120 and gp41 subunits, as well as trimeric gp140) have been studied extensively at the atomic level (X-ray crystallography and high resolution cryo-EM), the coreceptor molecules have resisted such analyses, in large part due to their obligate complex membrane associations (they are GPCRs with 7 TM segments). Our soluble CCR5 constructs are designed to circumvent this problem. Two variant designs expressed in animal cells and tested, including: linking these domains to soluble CD4, or to a stable protein scaffold previously described as being useful to analyzing extracellular regions of other GPCRs. In assays of activation and neutralization of Env function using vaccinia-based cell fusion assays, we have found that some constructs appear to be specifically interacting with their corresponding binding sites on gp120. Further studies will employ biochemical and biophysical approaches to test this conclusion; supportive results will pave the way for collaborative X-ray crystallographic analyses of gp120-CCR5 interactions using these soluble coreceptor constructs. 2) Structure-function studies of the HIV-1 Env trimer. In FY2014, we have expanded our collaborative studies with Dr. Paolo Lusso, NIAID, on HIV-1 Env variants engineered to be 'locked' in a novel proposed conformation for gp120 in the unliganded trimeric state. in particular, we have extended previous studies on soluble monomeric gp120 to corresponding cell-associated Env trimers derived from gp160 constructs with the same modifications. The results provide further confirmation that the proposed structure indeed reflects the preferred gp120 conformation in the native Env trimer. These findings add additional structural insights beyond those recently described by the X-ray crystallographic structure of the engineered 'SOSIP' Env trimer construct. As another approach to study the native HIV-1 Env trimer, we are developing methods for real-time binding studies of antibodies and ligands to native Env expressed on pseudovirus particles, using biolayer interferometry (ForteBio Octet system). 3) Targeted killing of HIV-infected cells. In FY2014, we extended our targeting killing strategy using both novel immutoxins (for augmenting ART to cure acute infection) and chimeric antigen receptors (CARs) to achieve a 'functional cure' of chronic infection. Our previous studies (collaboration with Dr. Ira Pastan, NCI) have demonstrated highly potent and specific killing of HIV-infected cells with recombinant immunotoxins based on Pseudomonas aeruginosa exotoxin A (PE). Early collaborative studies in a humanized mouse model (Goldstein et al. J. Inf. Dis. 2000) indicated that these agents have dramatic effects when co-administered with ART during acute infection, resulting in (nearly) complete prevention of viral resurgence upon cessation of therapy. However in chronically infected BLT humanized mouse model (Denton et al. PLoS Pathogens 2014) as well as in SIV/macaque models (North et al., and Brenchley et al., unpublished), the immunotoxins significantly augmented ART activity but failed to eradicate the virus. These results suggest that immunotoxins may be of value as supplements to ART to achieve cure in HIV acute infection; to this end, collaborative studies (Pastan group) have been extended to generate new PE-based immunotoxins using recently identified broadly neutralizing mAbs. To achieve a 'functional cure' in chronically infected individuals, a more durable targeted cell killing strategy will be required. To this end, in collaboration with Dr. Steven Rosenberg, NCI, we have designed novel CD4-based chimeric antigen receptors (CARs) for genetic modification of patient T cells and adoptive transfer to the infected person. In addition to containing '2nd generation' intracellular signaling motifs, our CARs contain unique targeting domains that confer major benefits compared to 'standard' CD4-based CARs that proved ineffective in previous clinical studies: 1) enhanced potency, and 2) absence of CD4 entry receptor activity, which otherwise could lead to infection of the CAR-expressing CD8 T cells. We have designed two versions of such CARs, one containing CD4 linked to an scFv against the CD4-induced conserved bridging sheet involved in coreceptor binding, and another containing CD4 attached to the carbohydrate binding domain (CRD) of a C-type lectin, which binds to high mannose glycans universally expressed on all gp120 variants. Both CAR modalities display the indicated enhancements based on in vitro studies, and the latter is expected to be weakly (perhaps completely?) non-immunogenic. Preliminary collaborative studies (Dr. Tae-Wook Chun, NIAID) demonstrate that the CD4-scFv CARs effectively kill activated memory CD4+ T cells from ART-suppressed patients. Collaborative studies (Dr. Mario Roederer, VRC) were initiated with the CD4-scFv CAR in macaques infected with a SHIV; however the animals controlled this recombinant virus without the need for the genetically modified T cells. Studies are underway with SIV-infected macaques, using the CD4-CRD CAR (all sequences derived from corresponding rhesus macaque genes). Also planned are collaborative studies (Dr. Victor Garcia-Martinez, UNC) to test the CARs in the BLT humanized mouse model.

1）设计可溶性蛋白质构建体，显示出HIV CORECEPTORS CCR5和CXCR4的细胞外结构域。 在2014财年，我们设计并表征了包含先前鉴定为对共感受器功能至关重要的CCR5细胞外域的可溶性构建体，即N末端和细胞外环2（ECL2）。 While structural details of the HIV-1 Env glycoprotein (gp120 and gp41 subunits, as well as trimeric gp140) have been studied extensively at the atomic level (X-ray crystallography and high resolution cryo-EM), the coreceptor molecules have resisted such analyses, in large part due to their obligate complex membrane associations (they are GPCRs with 7 TM细分）。我们的可溶性CCR5构建体旨在解决此问题。在动物细胞中表达的两种变体设计，包括：将这些结构域与可溶性CD4联系起来，或与先前被描述为分析其他GPCR的细胞外区域有用的稳定蛋白支架。在使用基于疫苗的细胞融合分析的ENV函数激活和中和化的测定中，我们发现某些构建体似乎与GP120上的相应结合位点特别相互作用。进一步的研究将采用生化和生物物理方法来检验这一结论。支持性结果将为使用这些可溶性共素构建体的GP120-CCR5相互作用的协作X射线晶体学分析铺平道路。 2）HIV-1 ENV三聚体的结构功能研究。在2014财年，我们与Niaid的Paolo Lusso博士扩大了有关HIV-1 Env型的合作研究，该变体被设计为“锁定”，以“锁定”为在非配合三聚体状态的新颖的GP120拟议构型中。特别是，我们已经将有关可溶性单体GP120的先前研究扩展到了与具有相同修饰的GP160构建体衍生的相关细胞相关的ENK三聚体。结果提供了进一步的确认，即所提出的结构确实反映了天然ENV夹板中首选的GP120构象。这些发现增加了其他结构见解，而不是最近由工程“ SOSIP” ENV TRIMER构建体的X射线晶体结构所描述的见解。作为研究天然HIV-1 ENV三聚体的另一种方法，我们正在开发用于使用Biolayer干涉测定法（Fortebio Octet System）实时结合对在假病毒颗粒上表达的天然ENV的实时结合研究的方法。 3）靶向杀死HIV感染的细胞。 在2014财年，我们使用新型的不分毒蛋白（用于治愈急性感染）和嵌合抗原受体（CAR）扩展了靶向杀戮策略，以实现慢性感染的“功能治疗”。我们以前的研究（与IRA Pastan博士，NCI的合作）表明，基于铜绿假单胞菌Exotoxin A（PE），用重组免疫毒素对HIV感染的细胞高度有效且特异性杀死。早期的人源性小鼠模型（Goldstein etal。J.Inf。2000）中的早期协作研究表明，在急性感染期间与ART共同管理时，这些药物具有巨大的影响，从而（几乎）完全预防治疗后病毒疗法。然而，在慢性感染的BLT人源化小鼠模型（Denton等人PLOS病原体，2014年）以及SIV/猕猴模型（North等人和Brenchley等人，未发表）中，免疫毒素显着增强了艺术活性，但未能消除病毒。这些结果表明，免疫毒素可能是有价值的，可以作为ART的补充，以在HIV急性感染中治愈。为此，已经扩展了合作研究（Pastan组），以使用最近确定的广泛中和MAB生成新的基于PE的免疫毒素。为了在长期感染的个体中实现“功能治愈”，将需要更耐用的靶向细胞杀戮策略。为此，我们与NCI的Steven Rosenberg博士合作，我们设计了新型的基于CD4的嵌合抗原受体（CARS），以遗传对患者T细胞的遗传修饰并收养转移给受感染者。除了包含“第二代”细胞内信号传导基序外，我们的汽车还包含与“标准”基于CD4的CD4 CAR相比，在先前的临床研究中证明无效的靶向域：1）增强的效力，2）不存在CD4进入受体活性，否则可能会导致表达CD8 T细胞的CAR表达CD8 T细胞。我们设计了两个此类汽车的版本，其中一种包含与CD4诱导的涉及共纤维受体结合的保守的桥梁相关的CD4，另一种包含与C-type loctin的碳水化合物结合结构域（CRD）连接的CD4，该凝胶蛋白（C-type loctin）与高曼诺斯Glycans Glycans Glycans commented compersity consection conseption thorly gps consections contype loctin contype loctin（CRD）。两种CAR模式都基于体外研究表现出所指定的增强，后者预计将弱（也许是完全？）非免疫原性。初步协作研究（Niaid的Tae-Wook Chun博士）表明，CD4-SCFV汽车有效地杀死了被抑制ART抑制的患者的活化记忆CD4+ T细胞。合作研究（Mario Roederer，VRC博士）是在感染了SHIV的猕猴的CD4-SCFV汽车中启动的。然而，动物控制了这种重组病毒，而无需基因修饰的T细胞。使用CD4-CRD CAR（所有序列源自相应的恒河猴基因），使用SIV感染的猕猴进行了研究。还计划的是协作研究（Victor Garcia-Martinez，UNC博士），以测试BLT人源化小鼠模型中的汽车。