Use of Discontinuous Peptide Display for Generation of HIV Antigen Mimics

描述(由申请人提供)：HIV疫苗开发的一个主要障碍是我们无法诱导识别HIV-1包膜的广泛中和抗体(BNA)。这在一定程度上反映了HIV-1env上的关键中和表位的隔离。解决这一问题的一种方法是开发这些隔离的构象表位的抗原性模拟物，然后将其用作免疫原来诱导BNA。以前生产这种模拟表位的努力都没有成功，很大程度上是因为使用传统的噬菌体展示技术选择的线性模拟表位未能重现Env上不连续的病毒中和表位的结构。我们为这个问题提出了一种创新的解决方案，通过利用一种独特的分子支架，这种支架可以在三个不连续但相互作用的表面暴露环中多样化。我们的假设是，有必要使用不同的、不连续的分子文库来鉴定构象gp120表位的抗原模拟物，例如B12单抗识别的广谱中和表位，然后这些抗原模拟物可以用来诱导针对HIV-1的广谱中和抗体。 将进行实验，以确定在HIV-1包膜的CD4结合部位的不连续中和表位的模拟物，由单抗B12识别。为此，我们将筛选不同的不连续蛋白质文库，这些文库使用独特的展示支架构建，并呈现在噬菌体的表面。然后，我们将通过丙氨酸扫描突变和亲和选择，获得与单抗B12高亲和力结合的模拟表位。我们将进行竞争ELISA实验，以测试这些蛋白是否能与寡聚体HIV-1 Env竞争与单抗B12结合。然后将在兔子身上评估这些蛋白的免疫原性，以确定它们是否引发环境特异性抗体反应(包括病毒中和抗体)。这些实验有望为我们的假设提供一个概念验证评估。 利用一种新型的支架展示多个不连续的多肽，可以更好地保持B12表位的构象，从而诱导B12样BNA，这是一种以前从未探索过的强大的创新方法。此外，如果这项工作成功，除了B12，同样的支架还可以用于其他构象表位。因此，这项建议描绘了一种高风险但非常高回报的疫苗战略--与R21赠款机制的意图一致。 公共卫生相关性：该应用程序寻求开发一种改进的方法来生产人类免疫缺陷病毒1型(HIV-1)疫苗，采用一种创新的方法来生成识别HIV-1包膜的广谱中和抗体(BNA)。我们建议使用一种新的方法来产生HIV-1包膜上关键构象表位的新型抗原性模拟。然后，我们将使用这些抗原性模拟物来诱导能够抑制HIV-1传染性的广泛中和抗体。

DESCRIPTION (provided by applicant): A major roadblock in HIV vaccine development is our inability to elicit broadly neutralizing antibodies (BNA) that recognize the HIV-1 envelope. This reflects, in part, the sequestration of key neutralizing epitopes on HIV-1 Env. One way of solving this problem is to develop antigenic mimics of these sequestered, conformational epitopes, which can then be used as immunogens to elicit BNA. Previous efforts to produce such mimics have been unsuccessful, largely because the linear mimotopes that were selected using conventional phage display technology failed to recapitulate the structure of discontinuous, virus-neutralizing epitopes on Env. We propose an innovative solution to this problem, by exploiting a unique molecular scaffold that can be diversified in three discontinuous, but interacting, surface exposed loops. Our hypothesis is that it is necessary to use a diverse, discontinuous molecular library to identify antigenic mimics of conformational gp120 epitopes such as the broadly neutralizing epitope recognized by the b12 Mab, and that these antigenic mimics can then be used to elicit broadly neutralizing antibodies against HIV-1. Experiments will be conducted to identify mimics of a discontinuous neutralizing epitope in the CD4 binding site of the HIV-1 envelope, recognized by Mab b12. To do this, we will screen diversified discontinuous protein libraries, constructed using a unique display scaffold, and presented on the surface of bacteriophage. We will then derive mimotopes that bind with high affinity to Mab b12, by using alanine-scanning mutagenesis and affinity selection. We will perform competition ELISA experiments to test whether these proteins can compete with oligomeric HIV-1 Env for binding to Mab b12. The immunogenicity of these proteins will then be assessed in rabbits, to determine if they elicit Env-specific antibody responses (including virus-neutralizing antibodies). These experiments are expected to provide a proof-of-concept evaluation of our hypothesis. The use of a novel scaffold to display multiple discontinuous peptides that can better preserve the conformation of the b12 epitope to induce b12-like BNA is a powerful, innovative approach that has not been explored previously. Moreover, if this work is successful, the same scaffold could be used for other conformational epitopes, in addition to b12. Thus, this proposal delineates a high risk but very high reward vaccine strategy - consistent with the intent of the R21 grant mechanism. PUBLIC HEALTH RELEVANCE: This application seeks to develop an improved method to produce vaccines for human immunodeficiency virus type-1 (HIV-1), by taking an innovative approach to the generation of broadly neutralizing antibodies (BNA) that recognize the HIV-1 envelope. We propose to use a novel method to produce novel antigenic mimics of key conformational epitopes on the HIV-1 envelope. We will then use these antigenic mimics to elicit broadly neutralizing antibodies capable of inhibiting the infectivity of HIV-1.