Structural Approaches To HIV-1 Immunogen Design For BNAb Generation

描述(由申请人提供)：针对HIV-1的广谱中和抗体(BNab)主要针对病毒gp160包膜蛋白的膜近端胞外区域(MPER)。我们用核磁共振(核磁共振)、电子顺磁共振(EPR)和表面等离子体共振(SPR)相结合的方法研究了脂质环境中的MPER片段。结构分析显示，倾斜的N-末端a-螺旋(AA664-672)通过短铰链(673-674)连接到平坦的C-末端螺旋片段(675-683)，共同形成浸入膜中的亚稳态L状结构。4E10 bNab在与表面嵌入的MPER首次相遇后提取掩埋的W672和F673。考虑到HIV-1、HIV-2和SIV中这个富含色氨酸的序列的保守，这些数据对疫苗设计有影响，并表明bNAbs如何干扰涉及MPER的色氨酸残基相关病毒融合。在这里，我们将研究其他bNAb，如Z13e1，2F5或新产生的MPER结合抗体如何在W672和F673附近或片段的其他地方诱导构象变化，以及抗体结合时的结构变化是否与病毒中和有关。此外，还将比较自然感染HIV-1期间产生的抗体与接种疫苗时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构象的可检测变化。中和的效力将与MPER结构的变化相关。一旦EPR确定了相互作用，随后将进行详细的核磁共振分析。将评估病毒小体的脂质成分，包括胆固醇，如何影响MPER的膜嵌入结构，或其在抗体结合时经历构象变化的能力。此外，脂质包裹的纳米颗粒作为天然配置的MPER片段的载体，具有可生物吸收的聚(丙交酯-乙交酯)(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CD4+T细胞的激活，将在小鼠皮内免疫研究中进行测试，目的是诱导bNAbs。这些纳米粒子将进一步配备免疫激活加合物，以优化特定免疫反应的幅度。ELISA、Biaccore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分支B分离株的抗体所需的不同MPER序列的最佳数量。 鉴于到目前为止，全球估计有6500万人感染艾滋病毒-1，开发一种在正常受试者中诱发广泛中和抗体的疫苗将是防治艾滋病的巨大预防进步。

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.