Yeast Genetic Approach to Enhance the Immunogenicity of HIV Envelope Glycoprotein

描述（由申请人提供）：迄今为止，研制一种有效预防全球艾滋病毒大流行的疫苗尚未成功。然而，一种有用的疫苗的最终可行性表明，在实验系统中，一些感染HIV的个体产生针对HIV病毒包膜糖蛋白（Env）的广泛中和抗体，提供对HIV感染的保护。目前的申请描述了使用随机诱变和筛选方法来更好地了解包膜糖蛋白的抗原特性并开发具有改进免疫原性的Env形式的建议。鉴于Env对种系前体对中和抗体的亲和力较差是不同形式的Env引发保护性免疫反应能力的主要限制，该项目的一个主要目标是鉴定对这些前体具有较高亲和力的Env变体形式。为了便于诱变和筛选，将在面包酵母Saccharomyces cerevisiae表面表达Env的gp140外部片段。该系统的选择是基于酵母作为表达异种蛋白的真核宿主的优势，因为酵母在疫苗生产中的有用性，以及酵母中大规模随机诱变和表面显示蛋白筛选的工具。该项目的最初目标是优化gp140表达结构，以达到在酵母细胞表面显示真实抗体和受体结合能力的有用蛋白表达水平。一旦这些参数确定，第一轮诱变和筛选将用于选定中和抗体的表位序列决定因子的详细定位，使用荧光活化细胞分选（FACS）识别对抗体亲和力降低的Env变体。此外，酵母表达的Env与种系前体结合以广泛中和抗体的能力将被评估。在这种亲和力较低的情况下（预计是大多数情况），将通过FACS筛选随机改变形式的Env文库，以确定对种系抗体具有增强亲和力的糖蛋白形式。如果有必要实现亲和力的初步改善，将使用部分成熟形式的中和抗体的单个例子或文库进行筛选，然后进行进一步的筛选，以获得对实际种系抗体具有更高亲和力的Env变体。对种系抗体具有增强亲和力的候选Env将用于兔免疫。所得到的血清将被评估其结合特异性，并在体外系统中测试其中和多种HIV毒株的能力。总的来说，该提案使用了一种新的技术和方法来增强HIV Env的免疫原性，为当前基于结构的设计提供了一种补充选择。

DESCRIPTION (provided by applicant): Development of a vaccine that is effective against the worldwide HIV pandemic has, to date, been unsuccessful. However, the ultimate feasibility of a useful vaccine is indicated by the fact that some individuals infected with HIV develop broadly neutralizing antibodies against the HIV viral envelope glycoprotein (Env) that provide protection against HIV infection in experimental systems. The current application describes a proposal to use random mutagenesis and screening approaches to provide better understanding of the antigenic properties of the envelope glycoprotein and to develop forms of Env with improved immunogenic properties. Based on the idea that poor affinity of Env for germline precursors to neutralizing antibodies is a major limitation on the ability of different forms of Env to elicit protective immune responses, a major goal of the project is the identification of variant forms of Env with the elevated affinity for binding such precursors. To facilitate mutagenesis and screening, the gp140 external segment of Env will be expressed at the surface of the baker's yeast Saccharomyces cerevisiae. This system was chosen based on the advantages of this yeast as a eukaryotic host for expression of heterologous proteins, because of the demonstrated usefulness of yeast for vaccine production, and because of the tools available for large-scale random mutagenesis and screening of surface-displayed proteins in yeast. The initial aim of the project is to optimize gp140 expression constructs to achieve useful levels of expression of protein exhibiting authentic antibody and receptor-binding capability at the yeast cell surface. Once these parameters are established, an initial round of mutagenesis and screening will be used for detailed mapping of the sequence determinants of epitopes for selected neutralizing antibodies, using Fluorescence Activated Cell Sorting (FACS) to identify Env variants with reduced affinity for the antibodies. In addition, the ability of yeast-expressed Env to bind to germline precursors to broadly neutralizing antibodies will be evaluated. In cases where such affinity is low (expected to be the majority of cases), libraries of randomly altered forms of Env will be screened by FACS to identify forms of the glycoprotein exhibiting enhanced affinity for the germline antibodies. If necessary to achieve an initial improvement in affinity, either single examples or libraries of partially matured forms of the neutralizing antibodies will e used for screening, followed by further iterations of screening to obtain Env variants exhibiting elevated affinities for actual germline antibodies. Candidate forms of Env exhibiting enhanced affinity for germline antibodies will be used to immunize rabbits. The resulting sera will be evaluated for their binding specificities and tested for the ability to neutralize diverse strains f HIV in an in vitro system. Overall, the proposal uses a new technology and approach for enhancing the immunogenicity of HIV Env that provides a complementary alternative to current structure-based design.