Flipped Germinal Centers

项目总结 开发针对流感和HIV-1的通用疫苗是一项紧迫的全球目标。一个关键的挑战是 对天然HIV-1包膜(Env)和流感血凝素(HA)的免疫应答主要由非 中和性和高度毒株特异性抗体。一些个人广泛产生的发现 中和抗体(BNAbs)激发了人们的希望，尽管不是天然的优势抗体，但具有广泛保护性的抗体 回应是可能的。抗体成熟通过体细胞超突变(SHM)和亲和力为基础 在生发中心(GC)与其他识别不同部位的抗体竞争中的选择 同样的病毒。人们普遍认为，一种初级和加强疫苗策略可以有效地诱导bNab前体和 战略制导SHM弹道可以产生bNAbs。这一过程面临的挑战是原生信封 蛋白质可能不能很好地与bNab前体抗体结合，并且在抗体中的表现可能很差 曲目。战略优质和助推战略需要生成设计的病毒包膜变体， 与bNab祖先抗体很好地结合，作为引子，然后是修饰的变异体，作为增强作用 免疫基因(S)引导bNab成熟。这种有希望的方法因所需的时间和努力而受阻。 在体外鉴定Env或HA变异体为免疫原，这传统上需要产生突变文库 静态选择、克隆、表达和验证测试。这一广泛的动手试验和错误过程 极大地阻碍了进展的步伐。这里提出了一种具有爆炸性加速能力的新技术 通过创造性地利用全自动突变和免疫原发现的速度 自然界在超进化中的创新之一所固有的选择--即GC、SHM和亲和力 成熟系统-与平行选择活动相结合的体内自动动态突变过程， 动态地往返于优秀的结合变体之间，以进一步多样化和选择。除了……之外 通过显著提高结合亲和力，可以对GC系统进行改造以产生新的识别。这个 目标是创建翻转GC系统，其中抗体基因被病毒包膜蛋白取代- 并将其用于免疫原设计。与针对病毒包膜蛋白的动态抗体进化不同 正常GC、翻转GC向用户定义的抗体(例如SELECT)动态进化病毒包膜蛋白 BNab前体和中间体)。总体假设是，在关键修改的背景下， GC/亲和成熟系统足够灵活，允许生物工程病毒包膜蛋白亲和 向用户定义的bNab前体和中间体发展。实现这一目标将有两个目标： 1)建立参数，使GCs成为非Ig蛋白进化的平台。和2)产生HIV-1 而流感包膜来自翻转的GC小鼠的变种。这项工作的完成有可能导致 科技突破具有广泛影响，因为预计它将定义参数 使GC进化的能力从免疫球蛋白扩展到基本上任何蛋白质-蛋白质相互作用。

PROJECT SUMMARY Developing universal vaccines to influenza and HIV-1 is an urgent global goal. A critical challenge is that immune responses to native HIV-1 envelope (Env) and influenza hemagglutinin (HA) are dominated by non- neutralizing and highly strain-specific antibodies. Discoveries that some individuals produce broadly neutralizing antibodies (bnAbs) invigorated hope that, while not naturally dominant, broadly protective antibody responses are possible. Antibodies mature during through somatic hypermutation (SHM) and affinity-based selection in germinal centers (GCs) in competition with other antibodies that recognize different parts of the same virus. It is widely believed that a prime and boost vaccine tactic can effectively elicit bnAb precursors and strategically guide SHM trajectory can produce bnAbs. Challenges to this process are that native envelope proteins may not bind well to the bnAb precursor antibodies and may be poorly represented in the antibody repertoire. A strategic prime and boost strategy requires generation of designer viral envelope variants that bind well to bnAb ancestor antibodies acting as a primer, followed by modified variants to function as boosting immunogen(s) to shepherd bnAb maturation. This promising approach is hindered by time and effort required to identify Env or HA variants as immunogens, which traditionally require mutation library generation, in vitro static selection, cloning, expression, and validation testing. This extensive hands-on trial and error process greatly hinders the pace of progress. Here a new technology is proposed with power to explosively accelerate the pace of immunogen discovery by creatively harnessing the full spectrum of automated mutation and selection inherent in one of nature’s innovations in hyperevolution—namely the GC SHM and affinity maturation system—an automated in vivo dynamic mutation process coupled to parallel selection activity that dynamically shuttles superior binding variants back for further diversification and selection. In addition to dramatically improving binding affinity, the GC system can be engineered to generate new recognition. The objective is to create flipped GC systems in which antibody genes are replaced with viral envelope proteins— and deploy them for immunogen design. In contrast to dynamic antibody evolution to viral envelop protein in normal GCs, flipped GCs dynamically evolve viral envelop protein toward user-defined antibodies (e.g. select bnAb precursors and intermediates). The overall hypothesis is that, in the context of key modifications, the GC/affinity maturation system is sufficiently flexible to permit bioengineered viral envelope proteins to affinity mature toward user-defined bnAb precursors and intermediates. The objective will be pursued with two aims: 1) to establish parameters to engineer GCs as a platform for non-Ig protein evolution. And 2) to generate HIV-1 and influenza envelop variants from flipped GC mice. Completion of this work has potential to result in both scientific and technological breakthroughs of broad impact because it is expected to define parameters enabling the extension of the power of GC evolution beyond Ig to essentially any protein-protein interaction.