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大流行的疫苗的开发尚未成功。然而，有用疫苗的最终可行性是这样一个事实，即一些感染艾滋病毒的人在实验系统中产生针对艾滋病毒病毒包膜糖蛋白(Env)的广泛中和抗体，提供对艾滋病毒感染的保护。目前的应用描述了一种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性并开发具有改进的免疫原性的Env形式的建议。基于胚系前体对中和抗体的低亲和力是不同形式的Env诱导保护性免疫应答能力的主要限制这一想法，该项目的一个主要目标是鉴定具有较高亲和力的变异型Env，以结合这些前体。为了便于诱变和筛选，env的gp140外部片段将在面包师酿酒酵母的表面表达。选择该系统是基于该酵母作为真核宿主表达异源蛋白的优势，因为酵母在疫苗生产中被证明是有用的，并且因为可用于大规模随机突变和筛选酵母中的表面展示蛋白。该项目的最初目标是优化gp140表达结构，以获得在酵母细胞表面展示真实抗体和受体结合能力的有用水平的蛋白质表达。一旦确定了这些参数，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因素图，使用荧光激活细胞分类(FACS)来识别与抗体亲和力降低的环境变异体。此外，酵母表达的Env与胚系前体结合广谱中和抗体的能力将被评估。在这种亲和力低的情况下(预计是大多数病例)，FACS将筛选随机改变形式的Env文库，以鉴定对生殖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，将使用单个实例或部分成熟形式的中和抗体文库进行筛选，随后进行进一步的筛选迭代，以获得与实际生殖系抗体亲和力提高的Env变异体。对种系抗体亲和力增强的候选形式的Env将用于免疫兔子。得到的血清将被评估其结合特异性，并在体外系统中测试中和不同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艾滋病毒环境病毒的免疫原性，这为目前基于结构的设计提供了一个补充选择。

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.