Design of Immunogens to Elicit PGT122 like Antibodies

描述(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预防病毒挑战。一个特别有希望的抗体家族，PGT121-123，已被证明与一个表位结合，该表位包括V3和V1多肽以及几个可能同时具有高甘露糖和复合型的糖链。该项目的目标是开发模拟这些糖肽结构的免疫原，并测试它们诱导具有广谱中和的、类似PGT122的特异性抗体的能力。由于PGT122表位的糖链和肽组分在gp120一级序列中是不连续的，因此设计一段能够以天然3D取向重组所有表位元件的糖肽是非常具有挑战性的。为了应对这一挑战，我们团队开发了一种通过定向进化设计糖肽表位模拟的方法。在目标1中，我们将使这种方法适用于含有两种不同糖链的糖肽的进化，这将有助于将复杂和高甘露糖结合到我们的糖肽库中。在目标2中，我们将建立一个包含~10^13个随机糖肽的文库，然后选择和扩增那些与PGT122结合最好的糖肽。经过多轮筛选/扩增，我们将获得PGT122紧密识别的糖肽。我们还将进行类似的选择，以获得可被生殖系(Gl)PGT122识别的糖肽/肽，所有有希望的构建物都将进行生物物理和结构表征。在目标3中，我们将测试PGT122表位模拟物在兔体内的免疫原性。为了解决只有从正确的种系前体抗体进化而来的bNAbs的可能性，我们还将进行种系靶向免疫原的免疫原性研究。与斯克里普斯的David Nemazee合作，我们将研究这些构建体激活gl-PGT122 B细胞并刺激敲入鼠的gl-PGT122抗体反应的能力。

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.