Computational design of novel antigens targeting mature and germline b12

在这里，我们专注于免疫原设计，以引发针对HIV包膜的保守cd 4结合位点（cd 4 bs）的b12样抗体。HIV包膜的许多不同的工程变体都未能引发这样的抗体，但在这里，我们开发了两种以前没有测试过的新型b12抗原-非HIV蛋白支架，其中b12表位已被移植到其上，以及核心gp 120的最小化，稳定化变体。此外，我们采用了计算蛋白质设计和酵母展示定向进化的组合，这在以前还没有用于b12抗原设计。我们将检验以下假设：（i）诱导B12样抗体而不是针对CD 4 B的非中和性或窄中和性抗体，有必要设计结合B12但不结合其它CD 4 B抗体的抗原（ii）以最佳地刺激B12 B细胞并引发B12-类似的抗体将需要稳定B12表位的结构并优化抗原-B12相互作用的亲和力和动力学的抗原（iii）刺激幼稚B细胞发育成产生成熟的广泛中和形式的b12的细胞，b12是结合两种种系的抗原， 需要b12和成熟的b12;（iv）为了通过交联B细胞受体最大程度地刺激b12 B细胞，必须以表位朝外的定向方式使颗粒上的b12抗原多聚化。我们（项目1）的设计工作将通过b12抗原的结构和生物物理分析及其与b12和由设计的抗原引发的小鼠单克隆抗体的相互作用（项目2），通过体外和体内b12抗原刺激B细胞的分析（项目3），以及通过选定的b12抗原（核心B）引发的兔血清的结合特异性和中和分析来了解。

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).