Flipped Germinal Centers

项目摘要 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）的免疫应答由非 中和和高度菌株特异性抗体。一些个体广泛产生的发现 中和抗体（bnAbs）的研究希望，虽然不是天然显性的，但广泛的保护性抗体 答案是可能的。抗体在通过体细胞超突变（SHM）和基于亲和力的 在与其他抗体的竞争中，在生殖中心（GC）中的选择，这些抗体识别细胞的不同部分， 同样的病毒普遍认为初免和加强疫苗策略可以有效地引发bnAb前体， 策略性引导SHM轨迹可以产生bnAb。这个过程的挑战是， 蛋白质可能不能很好地与bnAb前体抗体结合， 保留曲目。策略性初免和加强策略需要产生设计的病毒包膜变体， 与作为引物的bnAb祖先抗体结合良好，随后是修饰的变体以作为加强免疫 免疫原与shepherd bnAb成熟。这一有希望的方法受到所需时间和精力的阻碍 为了在体外鉴定Env或HA变体作为免疫原，其传统上需要突变文库产生 静态选择、克隆、表达式和验证测试。这种广泛的动手试验和错误的过程 这大大阻碍了进步的步伐。这里提出了一种新技术， 通过创造性地利用全方位的自动突变， 自然界的一项超级革命性创新--即GC SHM和亲和力--中所固有的选择 成熟系统-一种与平行选择活性偶联的自动体内动态突变过程， 动态地穿梭上级结合变体以进一步多样化和选择。除了 通过显著提高结合亲和力，GC系统可以被工程化以产生新的识别。的 目的是创建翻转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。相反，动态抗体进化到病毒包膜蛋白， 正常GC、翻转GC向用户定义的抗体动态进化病毒包膜蛋白（例如选择 bnAb前体和中间体）。总的假设是，在关键修改的背景下， GC/亲和力成熟系统足够灵活，以允许生物工程化的病毒包膜蛋白与亲和素结合。 向用户定义的bnAb前体和中间体成熟。为实现这一目标，将有两个目标： 1)以建立参数来工程化GC作为非Ig蛋白进化的平台。第二，产生HIV-1 和来自翻转GC小鼠的流感病毒包膜变体。这项工作的完成有可能导致这两个 科学和技术突破的广泛影响，因为它预计将确定参数 使GC进化的力量能够超越IG而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.