Computational design of novel antigens targeting mature and germline b12

在这里，我们将重点放在免疫原设计上，以诱导针对HIV包膜的保守cd4结合位点（cd4bs）的b12样抗体。许多不同的HIV包膜的工程变体都不能诱导出这样的抗体，但在这里，我们开发了两种以前没有测试过的新型b12抗原——将b12表位移植到非HIV蛋白支架上，以及最小化的、稳定的核心gp120变体。此外，我们采用了计算蛋白设计和酵母展示定向进化的组合，这在以前没有用于b12抗原设计。我们将检验以下假设：(i)诱导b12样抗体而不是非或窄中和的cd4bs抗体；有必要设计出结合b12而不是其他cd4bs抗体的抗原（ii），以最佳地刺激b12 B细胞并引发b12样抗体。需要抗原稳定b12表位的结构并优化抗原-b12相互作用的亲和力和动力学（iii），以刺激幼稚B细胞发育成产生成熟的广泛中和形式的b12，抗原结合两种细胞

Here we focus on immunogen design to elicit b12-like antibodies against the conserved cd4 binding site (cd4bs) of HIV Envelope. Many different engineered variants of HIV Envelope have failed to elicit such antibodies, but here we develop two novel types of b12 antigen that have not previously been tested - non-HIV protein scaffolds onto which the b12 epitope has been transplanted, and minimized, stabilized variants of core gp120. Further, we employ a combination of computational protein design and yeast display directed evolution that has not been employed for b12 antigen design previously. We will test the following hypotheses: (i) to induce b12-like antibodies rather than non- or narrowly-neutralizing antibodies against the cd4bs, it will be necessary to design antigens that bind b12 but not other cd4bs antibodies (ii) to optimally stimulate b12 B-cells and elicit b12-like antibodies will require antigens that stabilize the structure of the b12 epitope and optimize the affinity and kinetics of the antigen-b12 interaction (iii) to stimulate naive B cells to develop into those producing the mature broadly-neutralizing form of b12, antigens that bind both germline b12 and mature b12 will be required; (iv) to maximally stimulate b12 B cells by crosslinking B cell receptors, it will be necessary to multimerize b12-antigens on particles in an oriented fashion with the epitope facing outward. Our (Project 1) design efforts will be informed by structural and biophysical analysis of b12 antigens and their interactions with b12 and with mouse MAbs elicited by designed antigens (Project 2), by analysis of b12 antigen stimulation of B cells in vitro and in vivo (Project 3), and by binding specificity and neutralization analysis of rabbit sera elicited by selected b12 antigens (Core B).