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HIV大流行的疫苗的开发尚未成功。然而，有用的疫苗的最终可行性由以下事实表明：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，其在实验系统中提供针对HIV感染的保护。本申请描述了使用随机诱变和筛选方法以提供对包膜糖蛋白的抗原性质的更好理解并开发具有改善的免疫原性性质的Env形式的建议。基于Env对生殖系前体与中和抗体的亲和力差是不同形式Env引发保护性免疫应答的能力的主要限制的想法，该项目的主要目标是鉴定对结合此类前体具有升高亲和力的Env变体形式。为了便于诱变和筛选，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的真核宿主的优点，因为酵母对疫苗生产的证明有用，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的工具。该项目的最初目的是优化gp140表达构建体，以实现在酵母细胞表面展示真实抗体和受体结合能力的蛋白质的有用表达水平。一旦建立了这些参数，将使用初始轮的诱变和筛选来详细绘制所选中和抗体的表位的序列决定簇，使用荧光激活细胞分选（FACS）来鉴定对抗体具有降低的亲和力的Env变体。此外，还将评价酵母表达的Env与广泛中和抗体的生殖系前体结合的能力。在这种亲和力低的情况下（预期是大多数情况），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亲和力的糖蛋白形式。如果需要实现亲和力的初始改善，则将中和抗体的部分成熟形式的单个实例或文库用于筛选，随后进一步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。表现出对生殖系抗体增强的亲和力的Env的候选形式将用于免疫兔。将评价所得血清的结合特异性，并检测其在体外系统中中和不同HIV毒株的能力。 总体而言，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.