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

描述（由申请人提供）：HIV gp41的膜近端外区（MPER）包括一个高度保守的参与HIV病毒融合的区域。它是抗体（Ab）介导中和的重要靶点，因为它含有两种广泛(b)中和（Nt）单克隆(M)抗体（2F5和4E10）的表位，以及两种显著但不是真正广泛的HIV分离物（Z13和m66.6）的单抗，使MPER成为艾滋病疫苗的明显靶点。然而，所有生产mper靶向疫苗的尝试都失败了。这些疫苗大多有三种类型：(i)合成多肽；（ii） MPER序列移植到蛋白支架上；或（iii）在病毒样颗粒上显示的蛋白质上。在解释这些失败时，我们假设以前的疫苗没有忠实地模仿MPER的中和能力结构（NCS）。我们的初步工作表明，gp41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，减少了MAb 4E10的结合。Drs。Scott和Lu之前合作生产了一种DNA疫苗，该疫苗表达包含MPER和TMD的gp41片段，以便在细胞膜的背景下呈现MPER。然而，用这种DNA疫苗反复免疫兔子，会产生低滴度的抗体，与MPER肽交叉反应弱，不能中和病毒；用病毒样颗粒疫苗增强免疫并没有提高抗MPER滴度，可能是因为MPER拷贝数低。我们建议设计更有效的蛋白质增强免疫原来模拟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，在脂质双层中以高拷贝数呈现gp41 MPER+TMD，以便更好地模拟MPER的NCS，而不添加可能“分散”Ab反应的蛋白质。我们将根据bNt单抗的相对结合亲和力、bNt单抗的非nt突变体不结合、它们在新型膜泄漏试验中的行为和结构稳定性，生产优化了：(i) MPER、（ii） TMD和（iii）脂质环境组成的DNA、脂质体和纳米颗粒候选物。在特定目标2中，优化的DNA、脂质体和纳米颗粒候选物将被生产并测试其引发mper结合活性和Nt抗体的能力。然后，优化的脂质体和纳米颗粒疫苗将作为蛋白质增强剂（即，在DNA引物之后）进行测试，目标是最大化mper特异性抗体滴度、Nt效价和广度。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. PUBLIC HEALTH RELEVANCE: The membrane proximal external region of the envelope protein gp41 (MPER) is a highly conserved region on the trimeric spike of infectious HIV-1, and a target of antibodies that neutralize a broad range of HIV-1 isolates. When used as a passive vaccine in monkeys, these antibodies protect against infection by HIV-like viruses; however so far there is no vaccine that can elicit such antibodies. Our goal is to produce a vaccine that will mimic the structure of the MPER on the infectious spike, and in so doing, will elicit antibodies that prevent infection by a broad spectrum of HIV-1 isolates; providing the important first steps in achieving a successful AIDS vaccine.