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: 7732778
• 负责人: Richard Thomas Wyatt
• 金额: $ 46.98万
• 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/7732778
• 关键词: Adjuvant; Affinity; Animals; Antibodies; Antibody Formation; Antigens; Autoimmune Process; 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; Manuscripts; Membrane; Molecular Conformation; Mouse Strains; Outcome; Particulate; Phase; Planet Mars; Preparation; Production; Proteins; Publishing; Scaffolding Protein; Solid; Structural Models; Structure; Surface; Surface Antigens; System; T-Lymphocyte; Testing; Toll-like receptors; Transplantation; Universities; 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. 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. c. 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 was 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 were accomplished and data published. Phogat S, Svehla K, Tang M, Spadaccini A, Muller J, Mascola J, Berkower I, Wyatt R.Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles.Virology. 2008 Mar 30;373(1):72-84. Epub 2007 Dec 26 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. A manuscript is in preparation on the original scaffold design and characterization.

靶向高度保守的 gp41 MPR 中和决定簇 我们正在寻求 3 种不同的方法来引发针对 gp41 MPR 的中和抗体。一是捕获并展示在固相脂蛋白体表面的MPR小蛋白的表达。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环境中展示 MPR。我们正在寻求的第三种方法是与华盛顿大学的 Bill Schief 和 David Baker 以及 VRC 的 Peter Kwong 合作，稳定异源蛋白质支架上的 2F5 表位。对于这种方法，扩展环 2F5 表位被精确地支架到蛋白质数据库中可用的结构定义的非 HIV 蛋白质上。移植后，将测试 2F5 表位支架的结合和结晶（由 Kwong 实验室进行）。该计划是根据结构建模生成三到四个支架，以纳摩尔亲和力结合 2F5。一旦生产出来，2F5 支架将以游离状态结晶（不存在 2F5）。那些与2F5抗体诱导的构象非常吻合的细胞将进行免疫原性测试，以确定它们产生2F5表位特异性反应的能力，当然还可以确定它们是否引发中和抗体。我们将单独测试 2F5 蛋白支架或以初免：增强组合的形式测试 2F5 蛋白支架，以将免疫反应集中在独特且常见的 2F5 表位折叠上。 2F5 表位支架蛋白也将在 prime:boost 与 gp160 PL 组合中进行测试，或在 HepB 颗粒环境中表达。对于任何 MPR 导向的方法，我们将评估是否需要异源 T 帮助来增强免疫原性，或者 Toll 样受体 (TLR) 配体佐剂是否会更好地引发针对潜在的自相似决定簇的抗体反应 13。我们还计划测试在小鼠自身免疫品系中选择一组构建体，以确定这些动物中通常引发的心磷脂样抗体是否可能是一种有益的反应，然后驱动与亲和力成熟MPR 免疫原。 I. Min Tang 正在进行多个项目，这些项目涉及 MPR 和作为免疫原的 Env PL 的改进以及 PL 的初免-加强策略，并且使用多种表达系统生成选定的结构核心非常有价值。她还做了一些细胞测定，这可能对解释对不同免疫原的免疫反应有一定价值。 一个。免疫原性以确定我们是否可以使用膜引发针对 gp41 MPR 的抗体，正在测试各种问题；是 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 序列是否包含辅助表位），MPR PLs +PADRE 与 EnvPL gp160 的初免增强相结合。 b.依次增强 EnvPL，以驱动这些进化枝分离株（CD4BS、gp41 MPR）之间普遍保守的内容，而不是可变区定向反应。 c.我们进行了研究，将 MPL 或 T 辅助脂肽纳入 PL 中，以增强其免疫原性。 我们还改变了脂质（更类似于病毒膜成分）以增强 2F5/4E10 结合。 二. Sanjay Phogat 正在研究形成 22nM 颗粒的乙型肝炎表面抗原，以呈现 MPR 以进行结合和免疫原性分析。通过杆状病毒表达和酵母表达生产高水平的纯颗粒已经完成并公布了数据。 Phogat S、Svehla K、Tang M、Spadaccini A、Muller J、Mascola J、Berkower I、Wyatt R。乙型肝炎表面抗原颗粒上排列的人类免疫缺陷病毒 1 型 gp41 膜近端外部区域的分析。病毒学。 2008 年 3 月 30 日；373(1):72-84。电子版 2007 年 12 月 26 日 三． Javier Guenaga 正在检查 2F5 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 David Baker 合作），并通过生产、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与半胱氨酸稳定相结合确实有效，因此显着结果的可能性增加了。该项目未来的活动包括克隆、细菌和哺乳动物表达、重折叠、ELISA/Biacore、中和测定以及将结构信息整合到增强的免疫原设计中。关于原始支架设计和表征的手稿正在准备中。