Broad Neutralization of HIV-1 from Rhinoviruses Displaying gp41 MPER Sequences

描述（由申请人提供）：面对一种危险到无法用传统的基于病原体的疫苗控制的病原体，我们开发了一种替代的、安全的活病毒疫苗系统。我们的实验室一直在制作人类鼻病毒（hrv）的组合文库，这些文库显示HIV-1免疫原通过不同长度和序列的连接物连接到它们的表面。由此产生的表现多样性增加了产生有用免疫原的机会。抗HIV-1中和抗体可以用来选择以最像HIV的方式呈现HIV表位的病毒。使用这种方法，我们已经能够产生显示HIV-1 gp41 ELDKWA表位的病毒（与广泛中和的抗HIV-1抗体2F5相对应），以引发针对不同HIV-1分离株（来自进化支A， B， D和E）的有效和交叉反应性中和反应。这一结果代表了艾滋病疫苗开发的重大进展，因为这些病毒是第一个重组基于艾滋病毒的免疫原，可引发广泛中和抗体。为了开发最有效的免疫原，我们建议：(1)从显示膜近端外区（MPER）片段的组合文库中生成和选择改进的抗hiv -1免疫原，这些片段对应于：(i) ELDKWA表位，根据从ELDKWA库中获得的有价值的免疫原设计，（ii） NWFDITNW表位（毗邻ELDKWA表位）对应于已知的最广泛中和的抗hiv -1抗体4E10，以及（iii） ELDKWA和NWFDITNW表位（在其原生的，更扩展的MPER序列中）；(2)用最广泛的免疫原性mper呈报病毒（¿peptide boost）免疫兔子和小鼠，生成并选择新的中和单克隆抗体；(3)确定最有希望的工程病毒和/或肽增强物的三维结构，游离并与中和的抗hiv -1 Fab片段络合。为此，我们将使用晶体学，低温电子显微镜和核磁共振，不知道哪些技术将最有效地表征免疫原的结构和动力学。我们从确定许多免疫原性V3环呈递病毒的结构中了解到，所显示的表位通常具有不止一种构象，这需要广泛的技术方法和思考。通过更多地了解抗原性和免疫原性的三维决定因素，我们希望能够以一种最终将与基于结构的药物设计的成功范例平行的方式接近基于结构的疫苗设计。

DESCRIPTION (provided by applicant): Faced with a pathogen too dangerous to control using traditional pathogen-based vaccines, we have developed an alternative, safe, live-virus vaccine system. Our laboratory has been producing combinatorial libraries of human rhinoviruses (HRVs) that display HIV-1 immunogens connected to their surface via linkers of varied lengths and sequences. The resulting diversity of presentations increases the chances of generating useful immunogens. Anti-HIV-1 neutralizing antibodies can be exploited to select for viruses that present their HIV epitopes in ways most like HIV. Using this approach, we have been able to generate viruses that display the HIV-1 gp41 ELDKWA epitope (that corresponds to the broadly neutralizing anti-HIV-1 antibody, 2F5) in ways that elicit potent and cross-reactive neutralizing responses against diverse isolates of HIV-1 (from clades A, B, D, and E). This result represents a significant advance toward AIDS vaccine development, as these viruses are the first recombinant HIV-based immunogens to elicit broadly neutralizing antibodies. With the goal of developing the most effective immunogens possible, we propose to: (1) generate and select for improved anti-HIV-1 immunogens from combinatorial libraries displaying membrane-proximal external region (MPER) segments corresponding to: (i) the ELDKWA epitope, designed in response to what was learned from the ELDKWA library that yielded valuable immunogens, (ii) the NWFDITNW epitope (adjacent to the ELDKWA epitope) that corresponds to the most broadly neutralizing anti-HIV-1 antibody known, 4E10, and (iii) both the ELDKWA and NWFDITNW epitopes (in their native, more extended MPER sequence); (2) generate and select for new neutralizing mAbs from rabbits and mice immunized with the most broadly immunogenic MPER-presenting viruses (¿ peptide boosts), and (3) determine the three-dimensional structures of our most promising engineered viruses and/or peptide boosts, free and complexed with neutralizing anti-HIV-1 Fab fragments. For this, we will use crystallography, cryoelectron microscopy, and NMR, not knowing in advance which techniques will be most fruitful for characterizing the structures and dynamics of the immunogens. We have learned from determining the structures of a number of immunogenic V3 loop-presenting viruses that the epitopes displayed are often in more than one conformation, demanding broad technical approaches and thinking to go with it. By learning more about the three-dimensional determinants of antigenicity and immunogenicity, we hope to be able to approach structure-based vaccine design in a way that eventually will parallel the successful paradigm of structure-based drug design.