Structural Approches to HIV-1 Immunogen Design for BNAb Generation

对HIV-I的广泛中和抗体（BNAb）主要靶向病毒gpl 60包膜蛋白的保守的近膜胞外域区域（MPER）。我们用核磁共振（NMR）、电子顺磁共振（EPR）和表面等离子体共振（SPR）方法研究了HxB 2 MPER片段在脂质环境中的变化。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-末端螺旋段（675-683），共同形成浸没在膜中的亚稳态L形结构。4 E10 BNAb在与表面包埋的MPER初次接触后提取包埋的W 672和F673，而2F 5 BNAb将残基N-末端提升至铰链区，暴露L 669和W 670。BNAb CDRH 3与脂质的相互作用似乎对两种BNAb的中和活性至关重要。这些数据对疫苗设计有启示，并提示BNAb如何干扰涉及MPER的色氨酸残基相关病毒融合。在这里，我们将使用几个不同的MPER片段序列来检查其他BNAb或新产生的MPER结合抗体如何诱导W 672和F673周围或其他地方的构象变化。我们将确定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引起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。一旦通过EPR鉴定，将对相互作用进行详细的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作为天然配置的MPER区段的载体的脂质包封纳米颗粒，包括具有生物可吸收的聚（丙交酯-共-乙交酯）（PLGA）核的那些，所述PLGA核含有用于滤泡CD 4 + T细胞帮助的最大刺激的“通用”II类MHC结合表位。这些纳米颗粒，在项目2中创建的，不同的大小和表面特性，将进一步配备有靶向和免疫激活加合物，以优化特定免疫应答的大小，使用项目4中定义的缺氧-腺苷能抑制调节剂。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.