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引发识别HIV-1包膜的广泛中和抗体（BNA）。这部分反映了HIV-1 Env上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模拟物，然后可以将其用作引发BNA的免疫原。先前产生此类模拟物的努力一直不成功，主要是因为使用常规噬菌体展示技术选择的线性模拟表位未能概括Env上不连续的病毒中和表位的结构。我们提出了一个创新的解决方案，这个问题，通过利用一个独特的分子支架，可以在三个不连续的，但相互作用，表面暴露的循环多样化。我们的假设是，有必要使用一个多样化的，不连续的分子库，以确定构象gp 120表位，如广泛的中和表位识别的b12单克隆抗体的抗原模拟物，这些抗原模拟物，然后可以用来引发广泛的中和抗体抗HIV-1。 将进行实验以鉴定HIV-1包膜的CD 4结合位点中由Mab b12识别的不连续中和表位的模拟物。为此，我们将筛选多样化的不连续蛋白质文库，使用独特的展示支架构建，并呈现在噬菌体表面。然后，我们将通过使用丙氨酸扫描诱变和亲和力选择来获得与Mab b12高亲和力结合的模拟表位。我们将进行竞争ELISA实验，以测试这些蛋白质是否可以与寡聚体HIV-1 Env竞争结合Mab b12。然后在家兔中评估这些蛋白质的免疫原性，以确定它们是否引发Env特异性抗体应答（包括病毒中和抗体）。这些实验有望为我们的假设提供概念验证评估。 使用一种新的支架来展示多个不连续的肽，这些肽可以更好地保留b12表位的构象以诱导b12样BNA，这是一种强大的创新方法，以前没有探索过。此外，如果这项工作是成功的，相同的支架可以用于其他构象表位，除了b12。因此，该提案描绘了一种高风险但回报非常高的疫苗战略-与R21赠款机制的意图一致。 公共卫生相关性：本申请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）的疫苗，通过采用创新的方法来产生识别HIV-1包膜的广泛中和抗体（BNA）。我们建议使用一种新的方法来产生新的抗原模拟物的关键构象表位的HIV-1包膜。然后，我们将使用这些抗原模拟物来引发能够抑制HIV-1感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.