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

描述(申请人提供)：面对一种危险到无法使用传统病原体疫苗控制的病原体，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生产人鼻病毒(HRV)的组合库，这些库显示HIV-1免疫原通过不同长度和序列的连接子连接到它们的表面。由此产生的演示内容的多样性增加了产生有用免疫原的机会。可以利用抗HIV-1中和抗体来选择以最像HIV的方式呈现其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(对应于广泛中和的抗HIV-1抗体2F5)的病毒，其方式可以激发对不同的HIV-1分离株(来自A、B、D和E分支)的有效和交叉反应的中和反应。这一结果代表着艾滋病疫苗开发的重大进展，因为这些病毒是第一种能够诱导广泛中和抗体的基于艾滋病毒的重组免疫原。为了开发最有效的免疫原，我们建议：(1)从显示膜-近端外部区域(MPER)片段的组合文库中产生和选择改进的抗HIV-1免疫原，所述MPER片段对应于：(I)ELDKWA表位，根据从ELDKWA文库中获得的有价值的免疫原而设计；(Ii)NWFDITNW表位(与ELDKWA表位相邻)，对应于已知的最广泛的中和抗HIV-1抗体4E10，以及(Iii)ELDKWA和NWFDITNW表位(在其天然的、更广泛的MPER序列中)；(2)从免疫原性最广的MPER呈递病毒免疫的兔和小鼠身上制备和筛选新的中和性单抗，以及(3)确定我们最有希望的工程病毒和/或多肽Boost的三维结构，这些病毒和/或多肽Boost是免费的，并与中和抗HIV-1 Fab片段复合。为此，我们将使用结晶学、低温电子显微镜和核磁共振，事先不知道哪些技术将在表征免疫原的结构和动力学方面最有效。通过对一些免疫原性V3环状递呈病毒结构的测定，我们了解到，所显示的表位往往具有多种构象，需要广泛的技术方法和思维来配合。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类似于基于结构的药物设计的成功范例的方式来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.