Structural Approaches To HIV-1 Immunogen Design For BNAb Generation

描述（由申请人提供）：HIV-1的广泛中和抗体（BNAb）主要靶向病毒gp160包膜蛋白的膜近端外结构域（MPER）。采用核磁共振（NMR）、电子顺磁共振（EPR）和表面等离子体共振（SPR）相结合的方法对脂质环境中的MPER片段进行了研究。结构分析显示，倾斜的n端a-螺旋（aa 664-672）通过短铰链（673-674）连接到平坦的c端螺旋段（675-683），共同形成浸入膜的亚稳l形结构。4E10 BNAb提取物在与表面包埋的MPER初次接触后埋没了W672和F673。考虑到这个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启示意义，并提示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BNAbs如Z13e1, 2F5或新生成的MPER结合抗体如何诱导W672和F673周围或片段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，将比较自然感染HIV-1期间产生的抗体与接种疫苗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构象的可检测变化。中和的效力将与MPER结构变化相关。一旦通过EPR识别，相互作用将随后进行详细的核磁共振分析。病毒体的脂质成分，包括胆固醇，如何影响MPER的膜嵌入结构，或其在抗体结合时发生构象变化的能力，将被评估。此外，脂质包膜纳米颗粒作为天然配置的MPER片段的载体，具有生物可吸收的聚乳酸-羟基糖苷（PLGA）核心，具有不变和混杂的II类MHC分子结合表位，可刺激CD4+ T细胞活化，将在旨在诱导BNAbs的小鼠皮内免疫研究中进行测试。这些纳米颗粒将进一步与免疫激活加合物武装，以优化特异性免疫反应的大小。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B支分离株的抗体所需的不同MPER序列的最佳数量。

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.