Structural Approaches To HIV-1 Immunogen Design For BNAb Generation

描述（由申请方提供）：HIV-1的广泛中和抗体（BNAb）主要靶向病毒gp 160包膜蛋白的近膜胞外域（MPER）。我们已经研究了MPER段在脂质环境中的核磁共振（NMR），电子顺磁共振（EPR）和表面等离子体共振（SPR）的方法相结合。结构分析揭示了倾斜的N-末端α-螺旋（aa 664-672）通过短铰链（673-674）连接到平坦的C-末端螺旋区段（675-683），其共同形成浸没在膜中的亚稳态L形结构。4 E10 BNAb浸提液在初次接触表面包埋的MPER后掩埋了W 672和F673。考虑到这种富含色氨酸的序列在HIV-1、HIV-2和SIV中的保守性，这些数据对疫苗设计具有意义，并表明BNAb如何干扰涉及MPER的色氨酸残基相关病毒融合。在这里，我们将检查其他BNAb如Z13 e1，2F 5或新产生的MPER结合抗体如何诱导W 672和F673周围或片段中其他地方的构象变化，以及抗体结合后的这种结构变化是否与病毒中和有关。此外，将比较自然HIV-1感染期间产生的抗体与疫苗接种后产生的抗体的特异性和多样性。灵敏的EPR残基深度和残基间距离测量将允许相对快速地筛选MPER构象中的可检测变化。中和效力将与MPER结构变化相关。一旦通过EPR鉴定，相互作用将随后进行详细的NMR分析。将评估病毒体的脂质成分（包括胆固醇）如何影响MPER的膜包埋结构或其在抗体结合后发生构象变化的能力。此外，将在旨在引发BNAb的鼠皮内免疫研究中测试脂质包膜纳米颗粒作为天然配置的MPER区段的载体，所述MPER区段具有生物可吸收的聚（丙交酯-共-乙交酯）（PLGA）核心，所述PLGA核心含有不变的和混杂的II类MHC分子结合表位，用于刺激CD 4 + T细胞活化。这些纳米颗粒将进一步配备免疫活化加合物，以优化特异性免疫应答的幅度。ELISA、BIAcore和抗体中和试验将评估中和反应的广度，并确定诱导能够中和进化枝B分离株的抗体所需的不同MPER序列的最佳数量。 鉴于迄今为止全球估计有6 500万人感染HIV-1，开发一种疫苗，在正常受试者中引发广泛中和抗体，将是防治艾滋病斗争中的一个巨大预防性进展。

DESCRIPTION (provided by applicant): Broadly neutralizing antibodies (BNAb) to HIV-1 primarily target the membrane proximal ectodomain region (MPER) of the viral gp160 envelope protein. We have studied the MPER segment in the lipid environment by a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and surface plasmon resonance (SPR) methodologies. Structural analyses reveal a tilted N-terminal a-helix (aa 664-672) connected via a short hinge (673-674) to a flat C-terminal helical segment (675-683) that collectively forms a metastable L-shaped structure immersed in the membrane. The 4E10 BNAb extracts buried W672 and F673 following initial encounter with the surface-embedded MPER. Given conservation of this tryptophan-rich sequence in HIV-1, HIV-2 and SIV, the data have implications for vaccine design and suggest how BNAbs perturb tryptophan residue-associated viral fusion involving the MPER. Here we shall examine how other BNAbs such as Z13e1, 2F5 or newly generated MPER binding antibodies induce conformational change around W672 and F673 or elsewhere in the segment, and whether such structural changes upon antibody binding are linked to viral neutralization. Moreover, specificity and diversity of antibodies arising during natural HIV-1 infection versus elicited upon vaccination will be compared. Sensitive EPR residue depth and inter- residue distance measurements will allow for relatively rapid screening of detectable changes in MPER conformation. Potency of neutralization will be correlated with MPER structural changes. Once identified by EPR, interaction will be followed by detailed NMR analysis. How lipid constituents of the virosome, including cholesterol, affect the membrane-embedded structure of the MPER, or its ability to undergo conformational changes upon antibody binding, will be assessed. In addition, lipid-enveloped nanoparticles as carriers of natively configured MPER segments with a bioresorbable poly (lactide-co-glycolide) (PLGA) core harboring invariant and promiscuous class II MHC molecule-binding epitopes for stimulating CD4+ T cell activation will be tested in murine intradermal immunization studies aimed at eliciting BNAbs. These nanoparticles will be further armed with immune activating adducts to optimize the magnitude of the specific immune response. ELISA, BIAcore and antibody neutralization assays will assess the breadth of neutralizing responses and determine the optimal number of distinct MPER sequences needed to induce antibodies capable of neutralizing clade B isolates. Given that globally, to date, 65 million human infections with HIV-1 have been estimated, the development of a vaccine eliciting broadly neutralizing antibodies in normal subjects will be an enormous preventive advance in the fight against AIDS.