Yeast Genetic Approach to Enhance the Immunogenicity of HIV Envelope Glycoprotein

酵母遗传学方法增强 HIV 包膜糖蛋白的免疫原性

基本信息

批准号：
8500194
负责人：
MARK E. DUMONT
金额：
$ 36.31万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8500194
关键词：
Acquired Immunodeficiency Syndrome Affinity Animals Antibodies Antibody Affinity Antibody Formation Antibody Specificity Antigens B-Cell Activation Binding Cell surface Cells Complex Development Effectiveness Eligibility Determination Epitopes Exhibits Fc Receptor Fluorescence-Activated Cell Sorting Germ Lines Glycoproteins Goals HIV Immune response Immunization In Vitro Individual Libraries Maps Mutagenesis Mutate Mutation Oryctolagus cuniculus Phage Display Procedures Property Proteins Recombinants Saccharomyces cerevisiae Serum Solutions Specificity Structure Surface System Technology Testing Vaccination Vaccine Production Vaccines Variant Viral Viral Antigens Virus Virus Diseases Yeasts base combinatorial density design env Gene Products env Glycoproteins flexibility immunogenic immunogenicity improved mutant neutralizing antibody new technology novel strategies pandemic disease prevent protein expression receptor binding screening stem tool vaccine development virus envelope yeast genetics

项目摘要

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.

描述（由申请人提供）：开发针对全球艾滋病毒大流行有效的疫苗已经没有成功。然而，有用疫苗的最终可行性表明了以下事实：一些感染HIV的个体对针对HIV病毒包膜糖蛋白（ENV）产生了广泛中和抗体，这些抗体可为实验系统中的HIV感染提供保护。当前的应用描述了使用随机诱变和筛选方法的建议，以更好地了解包膜糖蛋白的抗原特性，并开发具有改善免疫原性特性的ENV形式。基于以下想法：Env对种系前体对中和抗体的亲和力是对不同形式的ENV引起保护性免疫反应的能力的主要限制，该项目的主要目标是确定ENV的变异形式，并具有升高与这种前体结合的亲和力。为了促进诱变和筛查，ENV的GP140外部将在酿酒酵母的贝克酵母糖疗法表面表达。该系统是基于该酵母作为表达异源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用于大规模随机诱变和表面播放的大规模随机诱变的工具酵母中的蛋白质。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表现出正宗抗体和受体结合能力的蛋白质表达水平。一旦建立了这些参数，使用荧光激活的细胞分选（FACS），将使用初始的诱变和筛选来详细绘制表位的序列决定因素，以识别具有抗体降低亲和力的ENV变体。另外，将评估酵母表达的ENV与种系前体结合广泛中和抗体的能力。如果这种亲和力较低（预计将是大多数情况），FACS将筛选随机变化形式的ENV形式的文库，以鉴定糖蛋白表现出对种系抗体的增强亲和力的形式。如有必要，为了实现亲和力的初始改善，无论是中和抗体的部分成熟形式的单个例子或文库都将用于筛选，然后进一步筛选以获取表现出对实际生殖线抗体的较高亲和力的ENV变体。表现出增强对种系抗体亲和力增强的Env的候选形式将用于免疫兔子。将评估所得的血清的结合特异性，并测试中和体外系统中各种菌株的能力。总体而言，该提案使用一种新技术和方法来增强HIV Env的免疫原性，该技术为当前基于结构的设计提供了互补的替代方案。