Design of Immunogens to Elicit PGT122 like Antibodies

描述（由申请人提供）：近年来对HIV+个体的广泛研究揭示了许多抗体的例子，这些抗体可以中和广泛的HIV毒株，并在动物感染模型中保护免受病毒攻击。一个特别有前途的抗体家族，PGT 121 -123，已显示结合到涉及V3和V1肽与几种聚糖（可能是高甘露糖和复合物类型）的组合的表位。该项目的目标是开发模拟这些糖肽结构的免疫原，并测试它们引发具有广泛中和性、PGT 122样特异性的抗体的能力。由于PGT 122表位的聚糖和肽组分在gp 120一级序列内是不连续的，因此设计能够以其天然3D取向重建所有表位元件的单段糖肽是相当具有挑战性的。为了应对这一挑战，我们的团队开发了一种通过定向进化设计糖肽表位模拟物的方法。在目标1中，我们将采用这种方法来进化含有两种不同聚糖的糖肽，这将有助于将复杂的和高甘露糖聚糖掺入到我们的糖肽文库中。在目标2中，我们将产生约10^13个随机糖肽的文库，然后选择并扩增那些与PGT 122结合最好的糖肽。在多轮选择/扩增后，我们将获得被PGT 122非常紧密识别的糖肽。我们还将进行类似的选择以获得由种系（gl）PGT 122识别的糖肽/肽，并且将对所有有希望的构建体进行生物药理学和结构表征。在目的3中，我们将在兔中测试PGT 122表位模拟物的免疫原性。为了解决bnAb可能仅通过从正确的前体种系抗体进化而产生的可能性，我们还将使用种系靶向免疫原进行免疫原性研究。与Scripps的大卫·内马齐合作，我们将研究这些构建体激活gl-PGT 122 B细胞和刺激敲入小鼠中gl-PGT 122抗体应答的能力。

DESCRIPTION (provided by applicant): Extensive study of HIV+ individuals in recent years has brought to light many examples of antibodies which can neutralize a broad range of HIV strains and protect against viral challenge in animal models of infection. One particularly promising antibody family, PGT121-123, has been shown to bind to an epitope involving a combination of V3 and V1 peptide together with several glycans, possibly of both high mannose and complex type. The goal of this project is to develop immunogens which mimic these glycopeptide structures, and test their ability to elicit antibodies with broadly-neutralizing, PGT122-like specificity. Because the glycan and peptide components of the PGT122 epitope are discontinuous within the gp120 primary sequence, it is quite challenging to design a single stretch of glycopeptide which could reconstitute all of the epitope elements in their native 3D orientation. To address this challenge, our group has developed a way to design glycopeptide epitope mimics by directed evolution. In Aim 1, we will adapt this method to the evolution of glycopeptides containing two different glycans, which will be useful for incorporation of both complex- and high mannose glycans into our glycopeptide libraries. In Aim 2, we will generate a library of ~10^13 random glycopeptides, then select and amplify those which bind best to PGT122. After multiple rounds of selection/amplification we will obtain glycopeptides which are very tightly recognized by PGT122. We will also do an analogous selection to obtain glycopeptides/peptides which are recognized by germline (gl) PGT122, and all promising constructs will be biophysically and structurally characterized. In Aim 3, we will then test the immunogenicity of PGT122 epitope mimics in rabbits. To address the possibility that bnAbs may only arise by evolving from the correct precursor germline antibodies, we will also conduct immunogenicity studies with the germline-targeted immunogens. In collaboration with David Nemazee at Scripps, we will investigate the ability of these constructs to activate gl-PGT122 B cells and stimulate a gl-PGT122 antibody response in knock-in mice.