Engineering and sorting HIV-1 display Env libraries for vaccine design

描述（由申请人提供）：由于组成亚基gp 120和gp 41的不稳定性，引发针对HIV-1包膜糖蛋白（Env）的天然（融合）三聚体的中和Ab存在问题，这导致引发针对不相关形式Env的主要非中和抗体。因此，工程化和生产天然Env三聚体的稳定和均质制剂的方法可能有利于HIV-1疫苗设计。理性的方法已经遇到了有限的成功，部分原因是缺乏有关三聚体的结构细节的信息。我们的方法并不严格要求详细了解Env三聚体结构。在R21阶段，将采用体外诱变将序列多样性引入env内的靶向区域，并将env“文库"克隆入HIV-1表达载体中以产生”HIV-1展示Env文库“（即，HIV-1群体，其不同成员各自通过独特的env基因序列和其表面上展示的Env的同源拷贝来区分）。然后将基于对热、化学变性剂和CD 4受体不稳定性的抗性以及通过感染靶细胞拯救的存活病毒体来选择HIV-1展示Env文库。预期多轮选择可产生展示稳定性增强的天然Env三聚体的HIV-1。我们还将尝试耗尽与非中和性单克隆抗体特别反应的克隆的HIV-1 Env文库，同时富集那些免疫显性和不相关表位展示减少的克隆。体外选择的超稳定HIV-1 Env变体将用作免疫原，以确定其作为HIV-1疫苗先导的潜力。R33期免疫研究将伴随高精度血清Ab特异性图谱，以指导Env免疫原优化。最后，将使用稳定性选择的HIV-1 Env变体作为输入，对可溶性和天然折叠的gp 140进行专门定制的筛选。公共卫生相关性：我们的目标是选择HIV的突变体，其中病毒表面的关键分子比自然界中发现的更稳定。这种突变病毒的灭活形式可能引起更有效的免疫（抗体）反应，因此可能导致更好的艾滋病毒/艾滋病疫苗。

DESCRIPTION (provided by applicant): Eliciting neutralizing Abs against the native (fusogenic) trimer of the HIV-1 envelope glycoproteins (Env) is problematic due to lability in the constituent subunits, gp120 and gp41, which leads to the elicitation of mainly non-neutralizing antibodies against irrelevant forms of Env. Therefore, a means of engineering and producing a stable and homogeneous preparation of native Env trimers is likely to be beneficial for HIV-1 vaccine design. Rational approaches have been met with limited success, due in part to a lack of information about the structural details of the trimer. Our approach does not strictly require detailed knowledge of Env trimer structure. In the R21 phase, in vitro mutagenesis will be employed to introduce sequence diversity into targeted regions within env, and the env `libraries' subcloned en masse into a HIV-1 expression vector for the production of `HIV-1 display Env libraries' (i.e. HIV-1 populations whose diverse members are each distinguished by a unique env gene sequence and the cognate copies of Env it displays on its surface). The HIV-1 display Env libraries will then be selected on the basis of resistance to heat, chemical denaturants and CD4-receptor destabilization, and the surviving virions rescued by infecting target cells. Multiple rounds of selection may be expected to yield HIV-1 that display native Env trimers of enhanced stability. We will also try depleting the HIV-1 Env libraries of clones that are particularly reactive with non-neutralizing monoclonal antibodies while enriching for those clones with diminished display of immunodominant and irrelevant epitopes. The in vitro-selected, hyperstable HIV-1 Env variants will be used as immunogens to determine their potential as HIV-1 vaccine leads. R33 phase immunization studies will be accompanied by high precision serum Ab specificity mapping to guide Env immunogen optimization. Finally, a specifically tailored screen for soluble and natively folded gp140s will be performed that uses the stability-selected HIV-1 Env variants as input. PUBLIC HEALTH RELEVANCE: We aim to select mutants of HIV in which key molecules on the viral surface are more stable than that found in nature. Inactivated forms of such mutant viruses may elicit more effective immune (antibody) responses and may therefore lead to better vaccines against HIV/AIDS.