Broad Spectrum Neutralizing Human Abs to SARS and Related Coronaviruses

描述(申请人提供)：人类单抗(MAb)疗法在病毒暴发环境中的预防、预防性和急性治疗方面具有相当大的优势，但由于长期存在的障碍，如循环病毒株的抗原性变化和中和逃逸，特别是对冠状病毒(CoV)和流感等RNA呼吸道病原体，它作为新出现的病毒病原体的治疗剂已被长期使用。重要的是，制造成本，历史上被认为是基于单抗的免疫疗法的局限性，在最近几年已经大幅下降。在这里，Marasco、Baric和Liddington实验室建议以SARS-CoV为模型，开发快速识别广谱中和人单抗(BNAbs)的平台，这些单抗对新出现的流行病和人畜共患病株有效。该模型的主要优势包括强大的流行病和人畜共患病分离株序列数据库，合成基因组学和反向遗传学，以及年轻和老年小鼠的体内发病模型(概括了严重的人类终末期肺部疾病和ARDS)；SARS-CoV受体ACE2和与其Spike蛋白结合的人bNAbs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公布的数据，即bNAbs可以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被用来同时开发新的策略，以最大限度地提高疫苗对更广泛的异质动物品系的效力。这些研究的影响将是巨大的，为其他重要的人类新出现的病原体提供了类似设计平台的模板。具体地说，我们将进一步建立在我们发现的针对Spike蛋白上的受体结合基序(RBM)的有效的人类抗SARS单抗80r和80r的bNab衍生物的基础上，这些80r的衍生物随后被设计成对来自人和动物(蝙蝠除外)的所有SARS-CoV分离株具有活性。现已知，许多人类致病冠状病毒的天然储存库是蝙蝠，包括BAT-SARS-CoV在内的祖先冠状病毒可以作为新的人类病原体复制、重组和重新出现。SARS冠状病毒的蝙蝠蓄水池已经牢固地建立起来，从公共卫生的角度来看，新出现和重新出现的SARS冠状病毒对人类健康构成了真正的威胁。我们建议同时开发针对SARS-CoV保守的S2结构域的bNAb，该结构域可能具有更大的广度(包括对新的SARS-like-CoV和未来可能从蝙蝠中出现的其他致病CoV的活性)、中和效力和抵抗中和逃逸。在4个具体目标中，我们将测试12个假设，这些假设与定义新的抗SARS-CoV的NAB、它们的中和表位、逃逸机制和防止中和逃逸的策略有关。在这些研究中解决的bNAbs-Spike复合体的共晶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被用于设计RBM和S2指导的疫苗原型研究，以确定我们是否可以控制所诱导的NAB的类型、它们的中和广度和它们对中和逃逸的抵抗力，目的是激发能够阻止病毒进化的bNab反应。 与公共卫生相关：严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV)是一种新病毒，它在2002/03年度引发了SARS的全球暴发，导致10%的死亡率和2003/04年度的聚集性病例。最近，人们发现蝙蝠携带SARS冠状病毒的祖先，以及许多(如果不是全部)人类冠状病毒呼吸道病原体。目前尚无公认的预防和治疗病原性冠状病毒感染的方法，这种感染随时可能再次出现，严重威胁人类健康。该项目将开发一种人类抗体鸡尾酒，以满足这一未得到满足的需求。

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. PUBLIC HEALTH RELEVANCE: Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a novel virus that caused the global outbreak of SARS in 2002/03 with 10% mortality and a cluster of cases in 2003/04. Recently, it was discovered that bats harbor the ancestors of SARS-CoV and many if not all human CoV respiratory pathogens. Currently, there are no approved therapies for prevention and treatment of pathogenic CoV infections, which could reemerge at anytime and become a serious threat to human health. This project will develop a human antibody cocktail for this unmet need.