The influence of evolutionary landscapes on protective antibody development

进化景观对保护性抗体发育的影响

基本信息

批准号：
10531875
负责人：
Timothy Andrew Whitehead
金额：
$ 47.05万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-24 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10531875
关键词：
Affinity Antibodies Antibody Affinity Antibody Response Antigens Binding Biological Assay Biophysics Communicable Diseases Data Dengue Development Directed Molecular Evolution Epitopes Evolution Frustration Generations Genes Germ Lines Germ-Line Mutation Growth HIV Head Hemagglutinin Human Immunoglobulin Somatic Hypermutation In Vitro Influenza Influenza Hemagglutinin Libraries Link Maps Measures Methods Mission Modernization Motivation Mutagenesis Mutation National Institute of Allergy and Infectious Disease Pathway Analysis Patients Performance Play Probability Process Property Protein Engineering Proteins Public Health Reaction Refractory Research Research Project Grants Research Support Role Scientist Screening Result Sorting Structure Structure of germinal center of lymph node Surface Plasmon Resonance Testing Therapeutic Vaccine Design Vaccines Variant Viral Virus Virus Diseases Yeasts adaptive immunity analysis pipeline anti-influenza antibody libraries antigen binding deep sequencing design experimental study human pathogen improved in vivo influenza virus strain influenza virus vaccine innovation insight interest member mutation screening neutralizing antibody pandemic influenza pathogen prevent prophylactic response stem superinfection tool universal influenza vaccine vaccine candidate vaccine development vaccine strategy

项目摘要

One of the most pressing issues in modern vaccine development is the poor heterosubtypic neutralizing antibody responses elicited by highly diverse viruses that pose major threats to public health. Elicitation of these so-called broadly neutralizing antibodies (bnAbs) – or antibodies that bind and neutralize many different viral subtypes – are rare in human patients, which complicates vaccine development and allows for repeated annual viral infections (e.g., Influenza and Dengue) or even superinfection (HIV) in human patients. In particular, current vaccines elicit bnAbs at insufficient titers for long-lasting protection against all currently circulating and pandemic Influenza strains. Therefore, it is critical to understand why some Abs are selected over others (i.e., why they are immunodominant), an issue directly relevant to the design of effective vaccines for many recalcitrant infectious diseases. Our long-term objective is to map the development of antibody lineages in a way that enables the prediction of likely responses as a tool to manipulate the process of antibody selection. Our primary objective is to test the hypothesis that the antibody evolutionary landscape is more limited for anti-Influenza bnAbs than for subtype- specific Abs, with this difference explaining why these bnAbs are not often selected at high levels in humans. To test this hypothesis we will display Influenza-specific antibody libraries on yeast and use a transformative sorting and deep sequencing pipeline to evaluate each member variant of the library for its affinity and nonspecific binding properties. This massive functional data will then be used for a network analysis to reconstruct plausible evolutionary trajectories for each somatic bnAb and subtype-specific head Ab. The motivation for the proposed research is guided by the urgent need for develop methods to map and manipulate rules of in vivo antibody affinity maturation to develop vaccines against refractory pathogens of high interest to public health including Influenza, Dengue, and HIV. The proposed research project will be carried out by pursuing three specific aims: 1) Determine ontogeny from germ line to mature human antibodies for two heterosubtypic HA stem binders; 2) Determine ontogeny from germ line to mature human antibodies for four heterosubtypic and subtype- specific HA head binders; 3) Determine the number of evolutionary trajectories from a representative germline Ab. This approach is innovative because it combines a unique hypothesis with state of the art protein engineering tools needed to evaluate the hypothesis, and it is significant because the data generated here will illuminate why long-lasting bnAb responses to HA immunogens are so rare. The approach raised in this application may also expedite rational structure-based design of vaccines, prophylactics, and therapeutics against a range of human pathogens.

现代疫苗开发中最紧迫的问题之一是可怜的异质型中和高度潜水病毒引起的抗体反应对公共卫生构成了重大威胁。启发这些所谓的广泛中和抗体（BNAB）或结合许多不同的抗体病毒亚型 - 在人类患者中很少见，这使疫苗发育复杂化并允许重复人类患者的年病毒感染（例如流感和登革热）甚至超级感染（HIV）。特别的，当前的疫苗会引起bnabs的滴度不足，无法持续保护目前的所有疫苗循环和大流行性流感菌株。因此，重要的是要了解为什么选择一些腹肌在其他方面（即为什么它们是免疫降低的），这是与有效疫苗设计直接相关的问题对于许多顽固的传染病。我们的长期目标是以一种能够预测的方式来绘制抗体谱系的开发可能的响应是操纵抗体选择过程的工具。我们的主要目标是测试假设抗体进化景观对抗Influenza bnabs的限制更为限制特定的ABS，有这种差异，解释了为什么这些BNAB在人类中通常不经常被高水平选择。为了检验该假设，我们将在酵母上显示流感特定的抗体库并使用变革性分类和深层测序管道，以评估库的每个成员变体的亲和力和非特异性结合特性。然后，此庞大的功能数据将用于网络分析重建每个体细胞BNAB和亚型特异性头AB的合理进化轨迹。这拟议研究的动机是迫切需要开发绘制和操纵方法的迫切需要的体内抗体亲和力成熟的规则，以开发针对高疗法病原体的疫苗包括流感，登革热和艾滋病毒在内的公共卫生。拟议的研究项目将通过追求三个具体目标来实现： 1）确定从种系到成熟的人类抗体的个体发育，用于两个异源HA茎粘合剂； 2）确定从种系到成熟的人类抗体的本体发育特定的HA头粘合剂； 3）确定来自代表性种系AB的进化轨迹数量。这种方法具有创新性，因为它将独特的假设与最先进的蛋白质结合在一起评估假设所需的工程工具，这很重要，因为此处生成的数据将阐明了为什么持久的BNAB对HA免疫原的反应如此罕见。在此提出的方法应用还可以加快疫苗，预防和治疗的基于理性结构的设计针对一系列人类病原体。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis

DOI：
10.21769/bioprotoc.3697
发表时间：
2020-08-05
期刊：
BIO-PROTOCOL
影响因子：
0.8
作者：
Steiner, Paul J.;Baumer, Zachary T.;Whitehead, Timothy A.
通讯作者：
Whitehead, Timothy A.

Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis.

DOI：
10.1007/978-1-0716-2152-3_6
发表时间：
2022
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
通讯作者：

Optimization of multi-site nicking mutagenesis for generation of large, user-defined combinatorial libraries

DOI：
10.1093/protein/gzab017
发表时间：
2021-02-15
期刊：
PROTEIN ENGINEERING DESIGN & SELECTION
影响因子：
2.4
作者：
Kirby, Monica B.;Medina-Cucurella, Angelica, V;Whitehead, Timothy A.
通讯作者：
Whitehead, Timothy A.

Standardizing cassette‐based deep mutagenesis by Golden Gate assembly

通过 Golden Gate 组装标准化基于盒的深度诱变

DOI：
10.1002/bit.28564
发表时间：
2023
期刊：
Biotechnology and Bioengineering
影响因子：
3.8
作者：
Daffern, Nicolas;Francino‐Urdaniz, Irene M.;Baumer, Zachary T.;Whitehead, Timothy A.
通讯作者：
Whitehead, Timothy A.

The importance and future of biochemical engineering