Interactions Of Human Immunodeficiency Virus With Receptors

人类免疫缺陷病毒与受体的相互作用

基本信息

批准号：
7732468
负责人：
Edward Berger
金额：
$ 68.15万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/7732468
关键词：
Antibodies Antiviral Agents B-Lymphocyte Epitopes Binding Sites Biological Assay Cell Line Cell fusion Cells Clinical Collaborations Complement Development Engineering Epitopes Goals HIV HIV Infections HIV-1 Helix (Snails)Highly Active Antiretroviral Therapy Immunoglobulin G Immunotoxins Infection Inhibitory Concentration 50 Laboratories Lactobacillus Left Life Masks Modification Molecular Mutation National Institute of Allergy and Infectious Disease Numbers Organism Pathogenesis Peptides Phase I Clinical Trials Plants Prevention Prevention strategy Process Proteins Range Recombinants Reporter Genes Residual state Resistance Sexual Transmission Site Surface Testing Tobacco Mosaic Virus Transmembrane Domain Tropism United States Food and Drug Administration V3 Loop Vaccines Variant Viral Load result Viremia Virus Replication Week Work base cell type design ear helix immunogenicity insight killings member microbicide neutralizing antibody neutralizing monoclonal antibodies novel prevent receptor receptor binding research study therapeutic protein vector

项目摘要

During FY2008, this laboratory has expanded studies on the molecules involved in HIV entry, and on the development of related HIV treatment and prevention strategies using recombinant bifunctional proteins. 1) sCD4-17b. We designed this recombinant bifunctional protein and showed previously that it had broad HIV-1 neutralizing activity, using the single cycle TZM-bl reporter gene assay. In FY2008 we greatly extended the number of isolates examined to include 12 clade B and 12 clade C isolates representing the standardized panels selected for vaccine analyses, plus 8 isolates from clades A and F. Strong potency was observed throughout, with the IC50 values generally ranging around 0.1 0.2 microgm/ml (2 4 nM) for laboratory-adapted strains and 0.5 4 microgm/ml (10 80 nM) for most primary isolates. Particularly dramatic was the efficacy of sCD4-17b on many isolates that are highly resistant (IC50 much greater that 50 microgm/ml) to one or more of the well characterized broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5 and 4E10. sCD4-17b also showed potent neutralization of spreading infection in PBMCs. The varying sCD4-17b sensitivities of different isolates was found to correlate with the reactivities of the functional Env trimers with CD4 rather than with the exposed 17b epitope. These results support the potential use of sCD4-17b for protection against HIV-1 infection. To this end, we have found modifications of the linker composition that give much better yields of functional sCD4-17b in Lactobacillus, supporting the use of this engineered commensal organism as a live microbicide to protect against sexual transmission. We have also initiated a collaboration with Dr. Kenneth Palmer (U. Louisville) who has developed recombinant tobacco mosaic virus as a vector to express large quantities of foreign proteins in Nicotinia plants (e.g. 60 grams of another candidate microbicide protein, 99% purity in 2 weeks). 2) 3B3-PE38 immunotoxin. We are continuing to develop this bifunctional protein for depletion of HIV-1 reservoirs that persist after HAART. Extending our collaboration with Dr. Ira Pastan (NCI), we have examined a variant of 3B3-PE38 with several mutations in known B cell epitopes, in the hope of reducing the immunogenicity of this protein for therapeutic application. The effect of the mutations on specific killing of HIV Env-expressing cells was found to be dependent on the cell type. Thus, the mutations had no effect when tested against a CHO-Env transfectant cell lines, but reduced the activity against an HIV-1 chronically infected cell line. These results suggest that only a subset of the mutations tested may be suitable for clinical use of this agent. Collaborative experiments with Dr. Tae-Wook Chun are being conducted to test whether 3B3-PE38 can deplete cells responsible for the residual viremia still detectable under highly suppressive HAART. With support from the NIAID Scientific Director, we are collaborating with Dr. Clifford Lane and Dr. Richard Davey (NIAID) to formulate a pre-IND with the goal of obtaining FDA approval for a phase 1 clinical trial of 3B3-PE38 in infected people with strong HAART suppression of viral load. 3) We are continuing studies of the functional features of the HIV-1 Env trimer. In an extension of previous work, we have observed functional complementation upon co-expression of a diversity of Envs containing mutations in distinct determinants involved in fusion. The results indicate that fusion does not require every member of the Env trimer to contain binding sites for CD4 or coreceptor; similar, every member need not contain an active fusion peptide or N-helix or normal transmembrane domain. These findings indicate functional cross-talk between different protomers within the Env trimer. In collaboration with Dr. Susan Zolla-Pazner, we are examining the molecular basis for masking of specific epitopes on the V3 loop. Our sCD4-activated cell fusion assay enables us to assess the potential neutralizing activities of antibodies whose epitopes become accessible only after receptor binding.

在2008财年期间，该实验室扩大了对参与HIV进入的分子以及使用重组双功能蛋白的相关HIV治疗和预防策略的开发的研究。 1）SCD4-17B。我们设计了这种重组双功能蛋白，并使用单个循环TZM-BL报告基因测定法表明，它具有广泛的HIV-1中和活性。 In FY2008 we greatly extended the number of isolates examined to include 12 clade B and 12 clade C isolates representing the standardized panels selected for vaccine analyses, plus 8 isolates from clades A and F. Strong potency was observed throughout, with the IC50 values generally ranging around 0.1 0.2 microgm/ml (2 4 nM) for laboratory-adapted strains and 0.5 4 microgm/ml (10 80 nM) for大多数主要分离株。尤其是戏剧性的是SCD4-17B对许多具有高度抗性（IC50高度比50微米/ml）的分离株的功效，而宽度具有良好表征的广泛中和单克隆抗体IgG B12、2G12、2G12、2F5和4E10。 SCD4-17B还显示出PBMC中传播感染的有效中和。发现不同分离株的不同SCD4-17B敏感性与CD4的功能性ENV三聚体的反应性相关，而不是与暴露的17B表位相关。这些结果支持SCD4-17B的潜在用途来防止HIV-1感染。为此，我们发现了接头组合物的修改，从而使乳酸杆菌的功能性SCD4-17B产生得多，从而支持使用这种工程的共生生物作为实时杀生型杀菌剂，以防止性传播。我们还与肯尼斯·帕尔默（Kenneth Palmer）（美国路易斯维尔）博士进行了合作，后者已经开发了重组烟草摩西病毒作为媒介，以表达烟碱植物中大量的外蛋白（例如，另一种候选杀菌剂蛋白，2周内为99％的纯度纯度为60克）。 2）3B3-PE38免疫毒素。我们正在继续开发这种双功能蛋白，以消耗HAART之后的HIV-1储层。为了扩展与IRA Pastan博士（NCI）的合作，我们检查了已知B细胞表位中有几种突变的3B3-PE38的变体，以期降低该蛋白质用于治疗应用的免疫原性。发现突变对表达HIV ENV的特异性杀死的影响取决于细胞类型。因此，在针对CHO-ENV转染细胞系进行测试时，突变没有作用，但是降低了针对HIV-1慢性感染细胞系的活性。这些结果表明，仅测试突变的子集可能适合该药物的临床使用。正在进行与Tae-Wook Chun博士进行的协作实验，以测试3B3-PE38是否可以耗尽负责在高度抑制性HAART下仍可检测到的残留病毒血症的细胞。在NIAID科学总监的支持下，我们正在与Clifford Lane博士和Richard Davey博士（NIAID）合作，以制定预先指示，目的是在受感染的Haart抑制病毒负荷的强烈抑制人群中获得3B3-PE38的FDA批准3B3-PE38。 3）我们正在继续研究HIV-1 Env Trimer的功能特征。在以前的工作的扩展中，我们观察到功能互补是在融合融合涉及的不同决定因素中含有突变的各种ENV时的功能互补。结果表明，融合不需要Env Trimer的每个成员都包含CD4或cocector的结合位点。类似，每个成员都不必包含活性融合肽，N螺旋或正常跨膜结构域。这些发现表明ENV三聚体中不同造成物之间的功能性串扰。与Susan Zolla-Pazner博士合作，我们正在研究V3环上掩盖特定表位的分子基础。我们的SCD4激活细胞融合测定法使我们能够评估仅在受体结合后才能访问的抗体的潜在中和活性。