Structural Approches to HIV-1 Immunogen Design for BNAb Generation

针对HIV-1的广谱中和抗体(BNab)主要针对病毒gpl60包膜蛋白保守的膜近端胞外区(MPER)。我们用核磁共振、电子顺磁共振和表面等离子体共振相结合的方法研究了脂质环境中的HxB2MPER片段。结构分析显示，一个倾斜的N-末端a-螺旋(AA664-672)通过一个短铰链(673-674)连接到一个平面末端螺旋片段(675-683)，共同形成一个浸入膜中的亚稳定的L状结构。4E10 bNab在与表面嵌入的MPER相遇后提取埋藏的W672和F673，而2F5 bNab将残基N末端向上抬到铰链区，暴露出L669和W670。BNab CDRH3与脂质的相互作用似乎对这两种bNAbs的中和活性至关重要。这些数据对疫苗设计有意义，并表明bNAbs如何干扰涉及MPER的色氨酸残留物相关的病毒融合。在这里，我们将使用几个发现的MPER片段序列来研究其他bNAb或新创建的MPER结合抗体如何在W672和F673附近或其他地方诱导构象变化。我们将确定抗体结合时的这种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过程中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构象中可检测到的变化。一旦被EPR确认，Interacfion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，或其在抗体结合时发生构象变化的能力。此外，脂质包裹的纳米颗粒作为天然配置的MPER片段的载体，包括那些具有可生物吸收的聚(丙交酯-乙交酯)(PLGA)核心的纳米颗粒，该核心含有最大限度地促进滤泡CD4T细胞帮助sfimulafion的“通用”II类MHC结合表位，将在旨在诱导bNAbs的小鼠皮内免疫研究中进行测试。在项目2中创建的这些纳米颗粒具有不同的大小和表面特征，将进一步配备靶向和免疫激活加合物，使用项目4中定义的低氧-腺苷能抑制调节剂来优化特定免疫反应的大小。ELISA、BlAcore和抗体中和试验将评估中和反应的广度。

Broadly neutralizing anfibodies (BNAb) to HIV-1 primarily target the conserved membrane proximal ectodomain region (MPER) of the viral gpl60 envelope protein. We have studied the HxB2 MPER segment in lipid environments by a combinafion of nuclear magnefic 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 fiat Cterminal helical segment (675-683), collecfively forming a metastable L-shaped structure immersed in the membrane. The 4E10 BNAb extracts buried W672 and F673 following inifial encounter with the surface embedded MPER while the 2F5 BNAb lifts up residues N-terminal to the hinge region, exposing L669 and W670. BNAb CDRH3 interactions with lipid appear critical for neutralizing activity of both BNAbs. These data have implicafions for vaccine design and suggest how BNAbs can perturb tryptophan residueassociated viral fusion involving the MPER. Here we shall examine how other BNAbs or newly created MPER-binding anfibodies induce conformafional change around W672 and F673 or elsewhere using several disfinct MPER segment sequences. We shall determine whether such structural changes upon anfibody binding are linked to viral neutralization. Moreover, specificity and diversity of antibodies arising during natural HIV-1 infecfion vs. elicited upon vaccinafion will be compared. Sensitive EPR residue depth and inter-residue distance measurements will allow for relatively rapid screening of detectable changes in MPER conformafion. Once identified by EPR, interacfion 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 conformafional changes upon anfibody binding, will be assessed. In addifion, lipid-enveloped nanoparticles as carriers of natively configured MPER segments, including those with a bioresorbable poly (lactide-co-glycolide)(PLGA) core harboring "universal" class II MHC binding epitopes for maximal sfimulafion of follicular CD4+ T cell help, will be tested in murine intradermal immunizafion studies aimed at eliciting BNAbs. These nanoparticles, created in Project 2, of differing size and surface characteristics, will be further armed with targeting and immune activating adducts to opfimize the magnitude of the specific immune response using modulators of hypoxia-adenosinergic inhibition defined in Project in Project 4. ELISA, BlAcore and antibody neutralizafion assays will assess the breadth of neutralizing responses.