Vaccines that Replicate the Neutralization-Competent Structure of the gp41 MPER

描述（由申请人提供）：HIV gp 41的膜近端外部区域（MPER）包含参与HIV病毒融合的高度保守区域。它是抗体（Ab）介导的中和作用的重要靶点，因为它含有两种广泛（B）中和（Nt）单克隆（M）Ab（2F 5和4 E10）和两种中和大量但并非真正广泛的HIV分离株（Z13和m66.6）的MAb的表位，使得MPER成为AIDS疫苗的明显靶点。然而，所有生产靶向MPER疫苗的尝试都失败了。这些疫苗中的大多数具有三种类型：（i）合成肽;（ii）移植到蛋白质支架上的MPER序列;或（iii）移植到病毒样颗粒上展示的蛋白质上。在解释这些失败时，我们假设先前的疫苗没有忠实地模拟MPER的中和活性结构（NCS）。我们的初步工作表明，gp 41跨膜结构域（TMD）是MPER完全暴露所必需的，因为它被另一种膜蛋白的TMD取代，降低了MAb 4 E10的结合。Scott和Lu博士先前合作生产了一种DNA疫苗，该疫苗表达包含MPER和TMD的gp 41片段，以便在细胞膜的背景下呈递MPER。然而，用这种DNA疫苗重复免疫兔子引起低滴度抗体，其与MPER肽交叉反应弱，并且不中和病毒;用病毒样颗粒疫苗加强免疫没有提高抗MPER滴度，可能是因为低MPER拷贝数。我们建议设计更有效的蛋白质加强免疫原，将模仿NCS。在具体目标1中，我们计划设计脂质体和纳米颗粒免疫原，其在脂质双层中以高拷贝数呈递gp 41 MPER+TMD，以便更好地模拟MPER的NCS，而不添加可能“分散”Ab应答的蛋白质。我们将生产DNA、脂质体和纳米颗粒候选物，这些候选物已经优化了：（i）MPER、（ii）TMD和（iii）脂质环境的组成，基于bNt MAbs的相对结合亲和力、bNt MAbs的非Nt突变体的结合的不存在、它们在新型膜渗漏测定中的行为和结构稳定性。在具体目标2中，将生产优化的DNA、脂质体和纳米颗粒候选物，并测试它们引发MPR结合活性和Nt Ab的能力。然后将优化的脂质体和纳米颗粒疫苗作为蛋白质加强剂（即，在DNA引发后），目标是使MPR特异性Ab滴度和Nt效力和宽度最大化。此外，抗原性、免疫原性和结构数据将用于开发MPER NCS的分子模型，这将支持未来的疫苗设计。

DESCRIPTION (provided by applicant): The membrane proximal external region (MPER) of HIV gp41 comprises a highly conserved region involved in HIV viral fusion. It is an important target of antibody (Ab)-mediated neutralization as it contains epitopes for two broadly (b) neutralizing (Nt) monoclonal (M) Abs (2F5 and 4E10) and two MAbs that neutralize a significant, but not truly broad, range of HIV isolates (Z13 and m66.6), making the MPER an obvious target for an AIDS vaccine. However, all attempts to produce an MPER-targeting vaccine have failed. Most of these vaccines have been of three types: (i) synthetic peptides; (ii) MPER sequence grafted onto protein scaffolds; or (iii) onto proteins displayed on virus-like particles. In explaiing these failures, we hypothesize that previous vaccines have not faithfully mimicked the neutralization competent structure (NCS) of the MPER. Our preliminary work has shown that the gp41 transmembrane domain (TMD) is required for the full exposure of the MPER, since its replacement with the TMD from another membrane protein, decreases binding by MAb 4E10. Drs. Scott and Lu previously collaborated on producing a DNA vaccine that expresses a gp41 fragment comprising the MPER and TMD, so as to present the MPER in the context of the cell membrane. However, repeated immunization of rabbits with this DNA vaccine elicited low titer Abs that cross-reacted weakly with MPER peptides and did not neutralize virus; boosting immunizations with a virus-like particle vaccine did not improve anti-MPER titers, probably because of low MPER copy number. We propose to design more effective protein-boost immunogens that will mimic the NCS. In specific aim 1 we plan to design liposome and nanoparticle immunogens that present the gp41 MPER+TMD in high copy number in lipid bilayers, so as to better mimic the NCS of the MPER without added proteins that might "distract" the Ab response. We will produce DNA, liposome and nanoparticle candidates that have optimized the: (i) MPER, (ii) TMD, and (iii) composition of the lipid environment, based on relative binding affinity by bNt MAbs, the absence of binding by non-Nt mutants of the bNt MAbs, their behavior in a novel membrane leakage assay, and structural stabilization. In specific aim 2 optimized DNA, liposome and nanoparticle candidates will be produced and tested for their ability to elicit MPER-binding activities and Nt Abs. The optimized liposome and nanoparticle vaccines will then be tested as protein boosts (i.e., following DNA priming) with the goal of maximizing MPER-specific Ab titers and Nt potency and breadth. In addition, antigenicity, immunogenicity and structural data will be used to develop a molecular model of the MPER NCS, which should support future vaccine design.