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，然后将其用作引发BNA的免疫原。以前生产这种模拟物的努力都是不成功的，很大程度上是因为使用传统噬菌体展示技术选择的线性拟位未能再现Env上不连续的病毒中和表位的结构。我们提出了一种创新的解决方案，通过开发一种独特的分子支架，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，有必要使用多样化、不连续的分子文库来鉴定构象gp120表位的抗原模拟物，如b12单抗识别的广泛中和表位，然后这些抗原模拟物可以用来引发针对HIV-1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.