The origin and future protective activity of SARS-CoV-2 RBD specific neutralizing antibodies

项目摘要 到目前为止，SARS-CoV-2已经感染了超过1.38亿人，并导致超过280万人死亡， 预计大流行将持续数月，病毒将持续数年， 新出现的关切变异（VoC）加剧了这一问题。虽然有几种疫苗正在广泛使用， 目前尚不清楚它们是否能够诱导对新出现的VoC的有效长期免疫。高效抗- 尽管有几种针对SARS-CoV-2的单克隆抗体（mAb），但SARS-CoV-2的病毒治疗方法仍然难以捉摸。 刺突（S）蛋白的受体结合结构域（RBD）已被授予轻度至中度感染的EUA， 它们对严重疾病的有效性还不明显。随着全球疫苗接种步伐的缓慢， 有限的抗病毒使用/功效，以及抗原漂移变体的出现，这种大流行的轨迹， 该病毒今后的再度出现令人极为关切。对诱导机制的基本认识 维持对SARS-CoV-2的保护性体液免疫将是缓解其的关键。病毒现在 分类成几个分支，众多的VoC出现，包括我们的工作和其他一些迹象表明， 这种抗原漂移的原因是病毒逃避免疫压力和增加传播性的结果。漂移 RBD内的病毒是最令人关注的，因为它可以增强病毒的感染性，并消除病毒的活性。 可能由既往疫苗接种或感染产生的NAb。许多报道称 患者中的反复SARS-CoV-2感染，以及完全接种疫苗的个体中的突破性感染， 强调了自然获得的SARS-CoV-2免疫力的缺陷。利用我们合理设计的 RBD/RBD-ACE 2融合蛋白变体，我们已经鉴定了表位和表型异质性， RBD特异性人B细胞，并已分离出几种有效的RBD特异性人中和单克隆抗体 （NmAbs）（IC 50 <50 ng/ml），正在进入使用吸入的1/2期临床试验 在未来几个月内交付。我们假设在RBD中，高度保守的区域（RBD-CR）， 介导广泛和有效的体液保护所需的表位被可变区（RBD-1）包围。 VR），其在结构上是动态的并且对抗原漂移高度敏感。此外，我们假设RBD-VR 通过它们的作用，减轻有效和广泛的RBD-CR特异性体液应答的发展。 免疫优势和RBD-CR的直接闭塞。这种RBD-CR/RBD-VR的进化动态可能会 调节针对未来病毒变体的体液应答的持续保护（或失败）。我们将1）定义 人RBD特异性中和抗体应答的本体和表型多样性，2）定义 ACE 2结合的维持动力学和免疫压力对限制RBD进化的影响，以及3） 确定RBD Ab对SARS-CoV-2漂移的耐受性和贡献。确定自然感染的限度， 疫苗接种诱导的RBD中和抗体驱动抗原漂移，并提供保护， SARS-CoV-2病毒将为下一代SARS-CoV-2疫苗和治疗药物的开发提供信息。

PROJECT SUMMARY SARS-CoV-2 has infected over 138 million people and resulted in over 2.8 million deaths so far, with the expectation the pandemic will continue for many more months, and the virus will persist endemically for years, exacerbated by emerging variants of concern (VoC). Although several vaccines are being used wide-spread, it is unclear if they will be able to induce effective long-term immunity against emerging VoC. Highly effective anti- viral therapeutics for SARS-CoV-2 remain elusive, although several monoclonal antibodies (mAbs) targeting the Receptor Binding Domain (RBD) of the Spike (S) protein have been granted EUA for mild to moderate infection, their effectiveness against severe disease has not yet been evident. With the slow pace of global vaccination, limited anti-viral use/efficacy, and the emergence of antigenic drift variants, the trajectory of this pandemic and future resurgences of the virus is of great concern. Fundamental understanding of the mechanisms of inducing and sustaining protective humoral immunity to SARS-CoV-2 will be critical to its mitigation. The virus is now classified into several clades, numerous VoC emerging, and indications including our work and others that some of this antigenic drift is the result of the virus escaping from immune pressure and increased transmissibility. Drift within the RBD is of the utmost concern as it can enhance the infectivity of the virus and negate the activity of NAbs that may have developed from previous vaccination or infection. Numerous reports have emerged of repeated SARS-CoV-2 infections in patients, and breakthrough infections in fully vaccinated individuals, highlighting the imperfection of naturally acquired SARS-CoV-2 immunity. Utilizing our rationally designed RBD/RBD-ACE2 fusion protein variants, we have identified epitopic and phenotypic heterogeneity amongst RBD-specific human B cells and have isolated several potent RBD-specific human neutralizing monoclonal Abs (NmAbs) (IC50<50 ng/ml) against SARS-CoV-2 which are entering into a Phase 1/2 clinical trial using inhaled delivery in the coming months. We hypothesize that within RBD, the highly conserved regions (RBD-CR), epitopes desirable for mediating broad and potent humoral protection, are surrounded by variable regions (RBD- VR) that are structurally dynamic and highly susceptible to antigenic drift. Further, we hypothesize that RBD-VR mitigate the development of potent and broad RBD-CR specific humoral responses through their immunodominance and direct occlusion of RBD-CR. This RBD-CR/RBD-VR evolutionary dynamic is likely to regulate the sustained protection (or failure) of humoral responses against future viral variants. We will 1) define the ontological and phenotypic diversity of the human RBD-specific neutralizing antibody response, 2) define the dynamics of maintenance of ACE2 binding and immunological pressure on constraining RBD evolution, and 3) determine RBD Ab tolerance for and contribution to SARS-CoV-2 drift. Defining the limits of natural infection and vaccination induced RBD neutralizing antibodies to drive antigenic drift and confer protection from divergent SARS-CoV-2 viruses will inform the development next generation SARS-CoV-2 vaccines and therapeutics.