HIV Envelope-based vaccines from superinfection to elicit neutralizing antibodies

描述（由申请人提供）：已经尝试了许多修改HIV-1包膜（Env）以提高免疫原性的尝试，成功率不同。已声称中和抗体（nab）的效力有所提高，但所取得的广度非常有限，要在有效的基于env的疫苗成分方面取得实质性进展，取得突破至关重要。重复感染（SI）为研究候选HIV Envs作为潜在疫苗成分提供了独特的免疫学环境。本项目的重点是确定具有精英中和广度特征的超感染个体的Env序列。来自这些个体的Env序列可能包含独特的决定因素，这些决定因素是SI期间双抗原呈递持续存在的结果。该项目的目标是进行免疫原性研究，使用精心挑选的候选疫苗组与新出现的疫苗方案相结合，这将大大改善抗体反应——在反应时间和中和的广度方面。为了实现这个项目的目标，将使用单基因组扩增技术克隆一组从血浆中分离出来的gp160 Env基因，并针对两个亚型间超感染的精英中和者（他们是一个特征明显的非洲性工作者群体的成员）产生有效中和广度相关的时间点。一组选择标准定义了SI期间的关键时间点，从这些时间点克隆Envs，预计这些Envs最有可能包含导致峰值的关键决定因素

DESCRIPTION (provided by applicant): Many attempts to modify HIV-1 Envelope (Env) for improved immunogenicity have been attempted, with varying rates of success. Some increased potency in neutralizing antibodies (NAbs) has been claimed, but the breadth achieved is very limited and a breakthrough is crucial to make substantial progress towards an effective Env-based vaccine component. Superinfection (SI) provides a unique immunological setting for studying candidate HIV Envs as potential vaccine components. This project focuses on Env sequences derived from superinfected individuals identified to have characteristics of elite neutralization breadth. The Env sequences from these individuals may contain unique determinants that have developed as the result of the persistence of dual antigen presentation during SI. The project objective is to conduct immunogenicity studies using carefully selected panels of vaccine candidates combined with newly emerging vaccine regimens that will result in substantially improved antibody responses - both in time to response and in breadth of neutralization. To achieve the goal of this project, Single Genome Amplification will be used to clone a panel of gp160 Env genes isolated from plasma and targeting time points associated with development of potent neutralization breadth from two intersubtype superinfected elite neutralizers who are members of a well-characterized African sex-worker cohort. A set of selection criteria has defined key time points during SI from which to clone Envs that are expected to have the greatest probability of containing critical determinants that led to a peak in neutralization breadth. In silico analysis will be carried out to assess the evolution of the diversity and divergence of the cloned Envs from each subject by generating a maximum-likelihood phylogenetic tree. Mutations post SI will be mapped, including recombination events between the two infecting viral strains. Potential N-linked glycosylation sites will be examined and other mutations that are known to cause resistance to several broadly neutralizing monoclonal antibodies. Effects of directional selection pressure (dN/dS) will also be evaluated. Following a full in silico analysis of all clones, vaccine candidates will be selected and made as functional pseudoviruses to test for autologous and heterologous neutralization sensitivity. Nucleotide sequences of selected vaccine candidates will be motif optimized and synthesized in preparation for full-length gp160 constructs. Gene Gun bullets will be prepared and site-directed mutagenesis will generate soluble gp140 trimeric protein for expression and purification. Comparative immunogenicity studies will be performed in rabbits using gp160 DNA combined with gp140 trimeric protein to determine which combinations of Envs derived from SI subjects are most effective in generating neutralization breadth. In the process, we expect to define novel, key epitopes that direct the development of neutralizing antibodies. This study will result in a new collection of Env-based immunogens with the potential to advance vaccine research.