Structure and Immunogenicity of HIV-1 gp41 Membrane Proximal Region (MPR)

HIV-1 gp41 膜近端区 (MPR) 的结构和免疫原性

• 批准号: 7592431
• 负责人: Richard Thomas Wyatt
• 金额: $ 19.12万
• 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/7592431
• 关键词: Adjuvant; Affinity; Animals; Antibodies; Antibody Formation; Antigens; Autoimmune Process; Bacteria; Binding; Biological Assay; Cardiolipins; Cellular Assay; Cloning; Collaborations; Coupled; Cysteine; DNA; Data; Databases; Epitopes; Future; HIV-1; Hepatitis B Surface Antigens; Immune response; Immunoglobulin Variable Region; Ligands; Lipids; Membrane; Molecular Conformation; Mouse Strains; Numbers; Oryctolagus cuniculus; Outcome; Particulate; Phase; Production; Proteins; Scaffolding Protein; Solid; Structural Models; Structure; Surface; Surface Antigens; System; T-Lymphocyte; Testing; Toll-like receptors; Transplantation; Universities; Vesicle; Viral; Washington; Work; Yeasts; design; gp160; immunogenic; immunogenicity; neutralizing antibody; particle; proteoliposomes; response; scaffold

Targeting the Highly Conserved gp41 MPR Neutralizing Determinant We are pursing 3 distinct means to elicit neutralizing antibodies directed against the gp41 MPR. One is the expression of MPR miniproteins captured and displayed on the surface of solid phase proteoliposomes. The second approach utilizes the highly immunogenic HepB surface antigen nanopaticles to display the MPR in selected contexts. The third approach we are pursuing is to stabilize the 2F5 epitope on heterologous protein scaffolds in collaboration with Bill Schief and David Baker at the University of Washington and Peter Kwong at the VRC. For this approach, the extended loop 2F5 epitope is precisely scaffolded onto structurally defined non-HIV proteins available in the protein data base. Once transplanted, the 2F5 epitope-scaffold will be tested for binding and crystallized (by the Kwong lab). The plan is to generate three to four scaffolds generated from the structural modeling that bind 2F5 with nanomolar affinity. Once produced, the 2F5 scaffolds will be crystallized in the free-state (without 2F5 present). Those that closely fit the 2F5 antibody-induced conformation will be tested for immunogenicity to determine their ability to generate 2F5 epitope specific responses and of course to determine if they elicit neutralizing antibodies. We will test the 2F5 protein scaffolds individually or in prime:boost combination to focus the immune response on the unique and common 2F5 epitope fold. The 2F5 epitope scaffold proteins will also be tested in prime:boost combination with the gp160 PLs or expressed in the HepB particulate context. For any of the MPR-directed approaches we will use assess if heterologous T help is required to enhance immunogenicity or if toll-like receptor (TLR) ligand adjuvants will better elicit antibody responses against the potentially self resembling determinants 13. We also plan to test a selected set of constructs in autoimmune strains of mice to determine if the cardiolipin-like antibodies commonly elicited in these animals might be a beneficial response to then drive to affinity maturation with the MPR immunogens. I. Min Tang has several projects ongoing that relate to the MPR and to improvements of the Env PLs as immunogens and prime-boost strategies with the PLs, and has been invaluable using several expression systems to generate selected cores for structure. Also she has done some cellular assays which may have some value in interpreting immune response to different immunogens. a. Immunogenicity to determine if we can elicit antibodies against the gp41 MPR employing membrane various issues are being tested; is the MPR DNA immunogenic, MPR DNA+PADRE (heterolgous T cell help since we are not sure that the short MPR sequence contains a helper epitope), MPR PLs +PADREin combination with prime-boost of EnvPL gp160. b. With Barbara Capechi, prime-boost with Barbaras bacterial expression system where large numbers of MPR repeats are presented in the outer membrane vesicles of bacteria. This is in progress in rabbits, should have some binding and neutralization data soon. c. Sequential boosting of EnvPL in order to drive what is commonly conserved between these clade isolates (CD4BS, gp41 MPR) and not the variable region-directed response. d. We have conducted studies including MPL or T helper lipopeptides into the PLs to enhance their immunogenicity. We have also changed lipids (more similar to the viral membrane composition) to enhance 2F5/4E10 binding. II. Sanjay Phogat is working with the Hepatitis B surface antigen which form 22nM particles to present the MPR for binding and immunogenicity analysis. Production of high-levels of pure particles from baclovirus expression and yeast expression. III. Javier Guenaga is examining the 2F5 epitope as defined by the Ofek-Kwong structure locked onto selected protein scaffolds (in collaboration with David Baker) to be examined by production, binding, immunogenicity and structure. Since there seems to be an indication that prime-boost, coupled with cysteine-stabilization actually works, the likelihood of significant outcome has increased. Future activities within this project include cloning, bacterial and mammalian expression, refolding, ELISAs/Biacore, neutralization assays and integrating structural information into enhanced immunogen design.

靶向高度保守的gp 41 MPR中和决定簇 我们正在寻求3种不同的方法来引发针对gp 41 MPR的中和抗体。一个是MPR微蛋白的表达，其被捕获并展示在固相脂蛋白体表面上。第二种方法利用高免疫原性HepB表面抗原纳米颗粒在选定的背景下展示MPR。我们正在研究的第三种方法是与华盛顿大学的Bill Schief和大卫贝克以及VRC的Peter Kwong合作，稳定异源蛋白支架上的2F 5表位。对于这种方法，延伸的环2F 5表位被精确地支架化到蛋白质数据库中可用的结构上定义的非HIV蛋白质上。一旦移植，2F 5表位支架将被测试结合和结晶（由Kwong实验室）。该计划是产生三到四个支架，这些支架是从以纳摩尔亲和力结合2F 5的结构建模中产生的。一旦产生，2F 5支架将在自由状态下结晶（不存在2F 5）。将测试与2F 5抗体诱导的构象紧密匹配的那些抗体的免疫原性，以确定它们产生2F 5表位特异性应答的能力，当然也确定它们是否引发中和抗体。我们将单独或以初免：加强组合测试2F 5蛋白支架，以将免疫应答集中在独特和共同的2F 5表位折叠上。2F 5表位支架蛋白也将与gp 160 PL以初免：加强组合进行测试或在HepB颗粒背景下表达。对于任何MPR导向的方法，我们将使用评估是否需要异源T辅助来增强免疫原性，或者toll样受体（TLR）配体佐剂是否会更好地引发针对潜在自相似决定簇的抗体应答13。我们还计划在自身免疫小鼠品系中测试一组选定的构建体，以确定这些动物中通常引发的心磷脂样抗体是否可能是有益的反应，然后驱动MPR免疫原的亲和力成熟。 I. Min Tang有几个正在进行的项目，涉及MPR和Env PL作为免疫原的改进以及PL的初免-加强策略，并且使用几种表达系统来产生选定的结构核心是非常宝贵的。此外，她还做了一些细胞分析，这可能对解释不同免疫原的免疫反应有一定的价值。 a.免疫原性以确定我们是否可以使用膜引发针对gp 41 MPR的抗体，正在测试各种问题; MPR DNA免疫原性、MPR DNA+PADRE（异源T细胞帮助，因为我们不确定短MPR序列包含辅助表位）、MPR PL + PADRE与EnvPL gp 160的初免-加强的组合。 B.与Barbara Capechi一起，使用Barbaras细菌表达系统进行初免-加强，其中大量MPR重复序列存在于细菌的外膜囊泡中。这是在兔子的进展，应该有一些结合和中和数据很快。 C. EnvPL的顺序加强，以驱动这些进化枝分离株之间通常保守的（CD 4 BS，gp 41 MPR）而不是可变区定向应答。 D.我们已经进行了研究，包括MPL或T辅助脂肽到PL，以提高其免疫原性。 我们还改变了脂质（更类似于病毒膜组成），以增强2F 5/4 E10结合。 二. Sanjay Phogat正在研究形成22 nM颗粒的B型肝炎表面抗原，以呈现MPR用于结合和免疫原性分析。从杆状病毒表达和酵母表达中生产高水平的纯颗粒。 三. Javier Guenaga正在研究2F 5表位，该表位由锁定在选定蛋白质支架上的Ofek-Kwong结构定义（与大卫贝克合作），以通过生产，结合，免疫原性和结构进行检查。 由于似乎有迹象表明，初免-加强，加上半胱氨酸稳定实际上工作，显着的结果的可能性增加。该项目的未来活动包括克隆、细菌和哺乳动物表达、重折叠、ELISA/Biacore、中和试验和将结构信息整合到增强的免疫原设计中。