Structure/function studies of immunogen recognition by anti-HIV antibody b12

有效的艾滋病疫苗可能需要诱导抗体来中和不同的HIV毒株，但基于可溶性HIV Env结构的疫苗免疫原迄今未能引发这种反应。我们的第一代尝试为bNAb 4E10设计“表位-支架”抗原，产生精致的表位特异性反应，不幸的是，这也未能有效地中和HIV。为了改进这种方法(并采用并行策略)，我们现在将重点放在广泛中和的CD4结合位点上 抗体B12。我们建议迭代地设计B12特异性的表位支架和ii)最小化的gp120结构，与成熟的B12及其生殖系前体的结合优化；后者的识别事件对于选择性激活正确的幼稚B细胞至关重要，这将导致B12等同的反应。描述BCR/抗原相互作用的生物物理参数(亲和力、动力学、热力学)已被证明对体液免疫应答过程有深远的影响。我们将测试这样的假设，即i)B12生殖系候选不会完全概括结合、结构或中和 通过描述蛋白质/蛋白质相互作用和功能的参数的某些子集来确定成熟抗体的性质；ii)为了使设计的免疫原在免疫后诱导有效的类B12反应，需要概述免疫原/B12和免疫原/生殖系相互作用。由于没有单一的B12胚系前体可以确定，我们建议研究(使用X射线结晶学和小角X射线散射(SAXS))和结合性质(使用SPR和ITC)的12个最有可能的候选集合。我们建议进行全面的生物物理研究 成熟的B12和候选的B12生殖系前体抗体的完整集合将：i)支持项目1中合理的免疫原重新设计周期；ii)详细说明B12成熟的分子机制；以及iii)为解释项目3中的体外和体内B细胞激活分析提供生物物理框架。我们的研究目标是了解生物物理对B细胞激活阈值的限制，以获得已知的最有效和最广泛的抗HLV-1中和单抗之一的表位。然后，我们将利用这些结果来指导合理设计更好的免疫原，以诱导抗艾滋病毒nab，同时做出贡献。 将基础和应用研究目标结合起来，对基础B细胞免疫学领域具有重大意义。

Effective AIDS vaccines will likely need to elicit antibodies that can neutralize diverse strains of HIV, but vaccine immunogens based on soluble HIV Env constructs have, so far, failed to elicit such responses. Our first-generation attempts to design 'epitope-scaffold' antigens for bNAb 4E10 generate exquisite epitope-specific responses that, unfortunately, also fail to potently neutralize HIV. In order to improve on this approach (and embrace parallel strategies), we will now focus on the broadly neutralizing, CD4-binding site antibody b12. We propose to iteratively engineer b12-specific i) epitope-scaffolds and ii) minimized gp120 constructs, with binding optimized to both mature b12 and its germline precursor; the latter recognition event is essential for selectively activating the correct naive B cells that will lead to b12-equivalent responses. The biophysical parameters describing BCR/antigen interactions (affinities, kinetics, thermodynamics) have been shown to have profound effects on the course of humoral immune responses. We will test the hypothesis that i) b12 germline candidates will not completely recapitulate the binding, structural or neutralization properties of the mature antibody by some subset of the parameters that describe protein/protein interactions and function and ii) that, in order for designed immunogens to induce effective b12-like responses upon immunization, both immunogen/b12 and immunogen/germline interactions will need to be recapitulated. Since no single b12 germline precursor can confidently be identified, we propose to study the structures (using x-ray crystallography and small-angle x-ray scattering (SAXS)) and binding properties (using SPR and ITC) of an ensemble of the 12 likeliest candidates. Our proposed comprehensive biophysical studies of mature b12 and the complete ensemble of candidate b12 germline precursor antibodies will: i) support the rational immunogen redesign cycle in Project 1; ii) detail the molecular mechanisms of b12 maturation; and iii) provide the biophysical framework for interpreting the in vitro and in vivo B cell activation assays in Project 3. Our research goal is to understand the biophysical constraint/s on B cell activation thresholds for an epitope of one of the most potent and broadly anti-HlV-1 neutralizing MAbs known. We will then use these results to guide efforts to rationally design better immunogens to elicit anti-HIV NAbs while contributing significantly to the field of fundamental B cell immunology, combining basic and applied research goals.