Yeast Genetic Approach to Enhance the Immunogenicity of HIV Envelope Glycoprotein

描述（由申请人提供）：迄今为止，有效对抗全球艾滋病毒流行的疫苗的开发尚未成功。然而，一些感染 HIV 的个体会产生针对 HIV 病毒包膜糖蛋白 (Env) 的广泛中和抗体，从而在实验系统中提供针对 HIV 感染的保护，这一事实表明了有用疫苗的最终可行性。当前的申请描述了使用随机诱变和筛选方法的建议，以更好地理解包膜糖蛋白的抗原特性，并开发具有改进的免疫原性特性的 Env 形式。基于 Env 对种系前体与中和抗体的亲和力差是不同形式的 Env 引发保护性免疫反应的能力的主要限制这一观点，该项目的主要目标是鉴定与此类前体结合的亲和力较高的 Env 变体形式。为了促进诱变和筛选，Env 的 gp140 外部片段将在面包酵母酿酒酵母的表面表达。选择该系统是基于该酵母作为表达异源蛋白质的真核宿主的优势，因为酵母已被证明可用于疫苗生产，并且因为可用于大规模随机诱变和筛选酵母中表面展示蛋白质的工具。该项目的最初目标是优化 gp140 表达构建体，以达到有效的蛋白质表达水平，从而在酵母细胞表面表现出真正的抗体和受体结合能力。一旦这些参数确定，将使用第一轮诱变和筛选来详细绘制所选中和抗体表位的序列决定簇，并使用荧光激活细胞分选 (FACS) 来识别对抗体亲和力降低的 Env 变体。此外，还将评估酵母表达的 Env 与种系前体结合以广泛中和抗体的能力。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，以鉴定对种系抗体表现出增强的亲和力的糖蛋白形式。如果有必要实现亲和力的初步提高，则将使用中和抗体的单个实例或部分成熟形式的文库进行筛选，然后进行进一步的迭代筛选以获得对实际种系抗体表现出升高的亲和力的Env变体。对种系抗体表现出增强亲和力的候选形式的 Env 将用于免疫兔子。将评估所得血清的结合特异性，并测试其在体外系统中中和多种 HIV 病毒株的能力。 总体而言，该提案使用了一种新技术和方法来增强 HIV 包膜的免疫原性，为当前基于结构的设计提供了补充替代方案。

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.