Broad Spectrum Neutralizing Human Abs to SARS and Related Coronaviruses

描述（申请人提供）：人单克隆抗体（mAb）疗法在病毒爆发环境中的预防、先发制人和急性治疗方面提供了相当大的优势，但由于长期存在的障碍，例如循环病毒株的抗原变异性和中和逃逸，特别是对于RNA呼吸道病原体如冠状病毒（CoV）和流感。重要的是，近年来，制造成本（历史上认为的基于mAb的免疫疗法的限制）已显著降低。在这里，Marasco，Baric和Liddington实验室建议开发快速鉴定广谱中和人单克隆抗体（BnAbs）的平台，这些单克隆抗体可有效对抗新出现的流行病和人畜共患病池菌株，使用SARS-CoV作为模型。该模型的主要优势包括流行病和人畜共患病分离株的强大序列数据库，合成基因组学和反向遗传学以及重现严重人类终末期肺病和ARDS的年轻和老年小鼠体内发病机制模型; SARS-CoV受体ACE 2和与其Spike蛋白结合的人BnAb的原子结构;以及我们合作团队开发的完善的方法和新型试剂。我们的方法是基于我们发表的数据，即BnAb可以被设计为防止病毒中和逃逸和/或减弱病毒进化。从体外研究中获得的信息将用于平行开发新策略，以最大限度地提高疫苗对更广泛的动物菌株异质性库的效力。这些研究的影响将是高的，为其他重要的人类新兴病原体提供类似的设计平台模板。具体而言，我们将进一步建立在我们发现的有效的人抗SARS mAb 80 R，其针对刺突蛋白上的受体结合基序（RBM），以及80 R的BnAb衍生物，其随后被工程化以针对来自人类和动物（蝙蝠除外）的所有SARS CoV分离株。现在已知许多人类致病性CoV的天然储存库是蝙蝠，其中祖先CoV，包括蝙蝠SARS CoV，可以复制，重组并重新出现为新的人类病原体。SARS-CoV的蝙蝠宿主已经牢固确立，从公共卫生的角度来看，新出现和重新出现的SARS-CoV对人类健康构成了真实的威胁。我们建议同时开发针对SARS-CoV的保守S2结构域的BnAb，其可能具有更大的广度（包括针对新的SARS-like-CoV和未来可能从蝙蝠中出现的其他致病性CoV的活性）、中和效力和对中和逃逸的抗性。在4个具体目标中，我们将测试12个假设，这些假设与定义针对SARS-CoV的新nAb、其中和表位、逃逸机制和防止中和逃逸的策略有关。在这些研究期间解决的BnAbs-Spike复合物的共晶体结构将指导我们的病毒学研究和抗体工程努力。从体外中和逃逸研究中获得的信息也将用于设计原型RBM和S2定向疫苗研究，以确定我们是否可以控制引发的nAb类型、其中和宽度及其对中和逃逸的抗性，目的是引发可以阻断病毒进化的BnAb应答。

DESCRIPTION (provided by applicant): Human monoclonal antibody (mAb) therapy offers considerable advantages for prophylaxis, preemptive and acute treatment in viral outbreak settings, yet its' use as a therapeutic agent against emerging viral pathogens has been protracted because of longstanding barriers such as antigenic variability of circulating viral strains and neutralization escape, particularly for RNA respiratory pathogens such as coronaviruses (CoVs) and influenza. Importantly, cost of manufacturing, a historically perceived limitation of mAb-based immunotherapy has dramatically decreased in recent years. Here, the Marasco, Baric and Liddington labs propose to develop platforms for rapidly identifying broad-spectrum neutralizing human mAbs (BnAbs) that are effective against emerging epidemic and zoonotic pool strains, using SARS-CoV as a model. Major strengths of this model include robust sequence database of epidemic and zoonotic isolates, synthetic genomics and reverse genetics and in vivo pathogenesis models in young and aged mice that recapitulate severe human end-stage lung disease and ARDS; atomic structures of SARS-CoV receptor ACE2 and human BnAbs bound to its Spike protein; and well developed methodologies and novel reagents developed by our collaborative team. Our approach is based on our published data that BnAbs can be designed to either prevent virus neutralization escape and/or attenuate virus evolution. Information gained from in vitro studies will be used to develop, in parallel, new strategies for maximizing vaccine efficacy against the broader heterogeneous pool of animal strains. The impact of these studies will be high, providing a template for similar design platforms for other important human emerging pathogens. Specifically, we will further build on our discovery of the potent human anti-SARS mAb 80R that is directed to the receptor binding motif (RBM) on Spike protein, and BnAb derivatives of 80R that have been subsequently engineered to be active against all SARS-CoV isolates from humans and animals (except bats). It is now known that the natural reservoir of many human pathogenic CoVs is the bat, where ancestral CoVs, including the bat-SARS-CoV, can replicate, recombine and reemerge as new human pathogens. The bat reservoir of SARS-CoVs is firmly established, and from the public health standpoint, emerging and re-emerging SARS-CoVs represent a real threat to human health. We propose to simultaneously develop BnAbs against the conserved S2 domain of SARS-CoVs, which may have even greater breadth (including activity against new SARS-like-CoVs and other pathogenic CoVs that might emerge from bats in the future), potency of neutralization and resistance to neutralization escape. In 4 specific aims, we will test 12 hypotheses that relate to defining new nAbs against SARS-CoVs, their neutralizing epitopes, escape mechanisms and strategies to prevent neutralization escape. Co-crystal structures of BnAbs-Spike complexes solved during these studies will guide our virologic studies and antibody engineering efforts. The information gained from in vitro neutralization escape studies will also be used to design prototypic RBM- and S2-directed vaccine studies to determine if we can control the type of nAbs that are elicited, their breadth of neutralization and their resistance to neutralization escape, with the goal of eliciting BnAb responses that can block virus evolution.