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

描述（由申请人提供）：面对一种使用传统的基于病原体的疫苗无法控制的危险病原体，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类鼻病毒（HRV）的组合文库，这些文库显示通过不同长度和序列的接头连接到其表面的HIV-1免疫原。由此产生的呈现多样性增加了产生有用免疫原的机会。抗HIV-1中和抗体可用于选择以最像HIV的方式呈现其HIV表位的病毒。使用这种方法，我们已经能够产生展示HIV-1 gp 41 ELDKWA表位（对应于广泛中和的抗HIV-1抗体2F 5）的病毒，其方式是引发针对不同HIV-1分离株（来自进化枝A、B、D和E）的有效和交叉反应性中和应答。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引发广泛中和抗体的重组艾滋病毒免疫原。为了开发可能的最有效的免疫原，我们提出：（1）从展示膜近端外部区域（MPER）区段的组合文库中产生和选择改进的抗HIV-1免疫原，所述区段对应于：（i）ELDKWA表位，其设计为响应从ELDKWA文库中获得的产生有价值的免疫原的内容，（ii）NWFDITNW表位（iii）ELDKWA和NWFDITNW表位（2）从用最广泛免疫原性的MPER呈递病毒免疫的兔和小鼠产生和选择新的中和mAb（肽加强），和（3）确定我们最有前途的工程化病毒和/或肽加强的三维结构，游离和与中和抗HIV-1 Fab片段复合。为此，我们将使用晶体学，冷冻电子显微镜和NMR，事先不知道哪些技术将是最富有成效的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，所展示的表位通常具有不止一种构象，需要广泛的技术方法和思考。通过了解更多关于抗原性和免疫原性的三维决定簇，我们希望能够以一种最终将平行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.