Selecting HIV recombination libraries for stable Env trimer immunogens

描述（由申请人提供）：一个更好的解决方案，艾滋病毒/艾滋病全球流行病是一种有效的疫苗。HIV-1疫苗开发的一个主要优先事项是鉴定一种免疫原，该免疫原可以引发针对循环初级病毒上的包膜糖蛋白（Env）刺突的高滴度中和抗体（nAb）。Env在病毒体和感染细胞表面的功能（天然）形式是gp 120-gp 41异源三聚体。引发HIV-I nAb的关键障碍不是一般地引发抗Env Ab，而是抗Env应答不成比例地被不相关形式的Env及其亚基引发，而不是被天然Env三聚体引发。可能导致对HIV免疫原产生弱和/或分离特异性nAb应答的因素是Env中的序列变异性和异质性，以及膜表面上发现的三聚体天然形式的不稳定性和低拷贝数。 该项目的目标是鉴定、表征和开发病毒样颗粒（VP或VLP）免疫原，其展示比野生型HIV-1更稳定、更均匀的天然和更高拷贝数的Env三聚体。从使用DNA重组方法产生的不同Env文库中，Env VP将经受各种去稳定化条件，然后选择用于结合天然和受体活化构象的三聚体特异性nAb和nAb前体，同时针对非中和Ab进行反筛选。为了鉴定具有提高的产率和天然Env三聚体含量的VP，我们将再次使用nAb从不同的gp 41胞质尾区和Gag文库中工程化和选择。我们将使用具有稳定、高拷贝数Env三聚体的选定VLP免疫家兔，并在HIV-1中和试验中测定针对相关菌株和异源分离株的血清nAb滴度。我们将使用血清Ab和一组mAb在各种结合和中和测定中探测前导Env免疫原，以确定野生型Env三聚体的抗原模拟程度。稳定的Env三聚体和抗血清的详细分子表征应允许进一步优化先导免疫原和免疫方案，以引发针对不同HIV-1原代分离株的交叉反应性nAb应答。我们期望所提出的研究对基于Env三聚体的疫苗的开发做出积极贡献。

DESCRIPTION (provided by applicant): A preferable solution to the HIV/AIDS global pandemic is an effective vaccine. A major priority for HIV-1 vaccine development is to identify an immunogen that can elicit high titers of neutralizing antibody (nAb) against the envelope glycoprotein (Env) spikes on circulating primary viruses. The functional (native) form of Env on the surface of virions and infected cells is a gp120-gp41 heterotrimer. A critical barrier in eliciing HIV-1 nAb is not one of eliciting anti-Env Ab in general, but rather that anti-Env responses are disproportionately elicited to irrelevant forms of Env and its subunits, rather than to the native Env trimer. Factors that likely contribute to the elicitation of weak and/or isolate specific nAb responses to HIV immunogens are sequence variability and heterogeneity in Env, as well as lability and low copy number of the trimeric, native form that is found on membrane surfaces. The goal of this project is to identify, characterize and develop virus-like particles (VPs or VLPs immunogens that display Env trimers that are more stable, more homogeneously native and in higher copy number than that of wild-type HIV-1. From diverse Env libraries created using DNA recombination methods Env VPs will be subjected to a variety of destabilizing conditions and then selected for binding to trimer-specific nAbs and nAb precursors in native and receptor-activated conformations while counter-screening against non-neutralizing Abs. To identify VPs with improved yield and native Env trimer content, we will engineer and select from diverse gp41 cytoplasmic tail and Gag libraries, again using nAbs. We will immunize rabbits using selected VLPs with stable, high copy number Env trimers and determine serum nAb titers against related strains and heterologous isolates in HIV-1 neutralization assays. We will probe lead Env immunogens using serum Ab and a panel of mAbs in a variety of binding and neutralization assays to ascertain the extent of antigenic mimicry of wildtype Env trimers. Detailed molecular characterization of the stable Env trimers and antisera should allow further optimization of lead immunogens and immunization regimens to elicit crossreactive nAb responses against different primary isolates of HIV-1. We expect the proposed studies to positively contribute to the development of an Env trimer-based vaccine.