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中和决定因素 我们正在追求3种不同的方法来引起针对GP41 MPR的中和抗体。一种是捕获并显示在固相蛋白脂质体表面上的MPR微蛋白的表达。第二种方法利用高度免疫原性的HEPB表面抗原纳米纳米剂在选定的上下文中显示MPR。我们采取的第三种方法是与华盛顿大学的Bill Schief和David Baker合作，稳定有关异源蛋白支架的2F5表位和VRC的Peter Kwong。对于这种方法，将扩展的环2F5表位精确地造成了蛋白质数据库中可用的结构定义的非HIV蛋白。一旦移植，将测试2F5表位量比结合和结晶（由Kwong Lab）进行测试。该计划是生成由与纳摩尔亲和力结合的结构建模产生的三到四个脚手架。产生后，2f5支架将在自由状态下结晶（不存在2f5）。那些非常适合2F5抗体诱导构象的人将接受免疫原性的测试，以确定它们产生2F5表位特定反应的能力，当然可以确定它们是否引起中和中和抗体。我们将单独或以Prime：增强组合进行分别测试2F5蛋白支架，以将免疫反应聚焦于独特的和常见的2F5表位折叠上。 2F5表位支架蛋白也将以Prime：Bumost与GP160 PL的组合进行测试，或在HEPB颗粒环境中表达。对于任何以MPR指导的方法，我们将使用评估是否需要异质T帮助来增强免疫原性，或者是否需要指控受体（TLR）配体佐剂（TLR）辅助剂是否会更好地引起抗体反应，以确定潜在的自我抑制剂，以确定这些动物的固定量是否能够确定这些群体的固定量。对随着MPR免疫的亲和力成熟而成为有益的反应。 I. Min Tang具有与MPR相关的几个项目，并将ENV PL的改进作为免疫原生物和PLS的原始策略，并且使用多个表达系统来生成所选核心以进行结构。她也进行了一些细胞测定，这些测定可能在解释对不同免疫原的免疫反应时具有一定价值。 一个。免疫原性，以确定我们是否可以使用膜进行各种问题来对GP41 MPR产生抗体；是MPR DNA免疫原性的MPR DNA +PADRE（异质T细胞的帮助，因为我们不确定短MPR序列包含一个辅助表位），MPR PLS +Padrein组合具有Envpl GP160的Prime-Boost。 b。 Envpl的顺序增强以驱动这些进化枝分离株（CD4B，GP41 MPR）之间通常保守的内容，而不是可变区域定向响应。 c。我们已经进行了包括MPL或T辅助脂肪肽在内的研究，以增强其免疫原性。 我们还改变了脂质（与病毒膜组成更相似）以增强2F5/4E10结合。 ii。 Sanjay Phogat正在使用丙型肝炎表面抗原，该抗原形成22nm颗粒，以呈现MPR进行结合和免疫原性分析。从巴基洛氏病毒表达和酵母表达的高水平颗粒产生并发表了数据。 Phogat S，Svehla K，Tang M，Spadaccini A，Muller J，Mascola J，Berkower I，Wyatt R.人类免疫缺陷病毒的分析1型GP41膜膜邻近外部区域，这些外部区域在肝炎表面抗原颗粒上阵列阵列。 2008年3月30日； 373（1）：72-84。 Epub 2007年12月26日 iii。哈维尔·瓜纳加（Javier Guenaga）正在研究由锁定在选定的蛋白质支架上的Ofek-kwong结构（与David Baker合作）所定义的2F5表位，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，加上半胱氨酸稳定的素升压实际上有效，因此重大预后的可能性增加了。该项目中的未来活动包括克隆，细菌和哺乳动物表达，重折叠，ELISAS/BIACORE，中和测定以及将结构信息整合到增强的免疫原设计中。手稿正在准备原始的脚手架设计和表征。