DataSheet1_Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.PDF

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的变异株仍持续引发疾病，并削弱各类治疗手段的有效性。来自完全康复患者的趋同中和抗体（NAbs）的治疗潜力，已在多款新药的早期研发阶段得到探索。本研究通过对单中心重症监护病房（ICU）收治的新冠感染患者的外周血开展深度RNA测序（deep RNA sequencing，测序读长超1亿条），鉴定出感染初期诱导产生的中和抗体（NAbs，包括免疫球蛋白重链、免疫球蛋白λ链、免疫球蛋白κ链）；该测序手段既可作为预测疾病严重程度的诊断工具，也可用于精准定位特定的补偿性中和抗体治疗方案。研究人员在患者入住重症监护病房期间，于多个时间点前瞻性收集临床数据，并鉴定出中和抗体互补决定区3（CDR3）片段的氨基酸序列。与非存活患者相比，重症新冠患者中的存活者体内针对SARS-CoV-2的3类抗体C135的水平显著更高（分别为15059.4与1412.7，p=0.016）。本研究不仅阐明了RNA测序在揭示不同转归新冠患者独特中和抗体谱中的应用价值，还通过全原子建模（atomistic modeling）结合分子动力学模拟（molecular dynamics simulations），为研究发现提供了物理层面的机制依据。本研究明确了3类中和抗体C135与SARS-CoV-2刺突蛋白（spike protein）的相互作用机制，并提出其通过多种构象发挥抑制作用的分子基础：尽管C135并未直接阻断血管紧张素转换酶2结合基序，但可有效阻止血管紧张素转换酶2与刺突蛋白的结合。综上，本研究证实，将深度RNA测序与结构建模相结合，可为因内源性抗体产生不足而缺乏特定免疫应答的人群，识别并解析新型治疗性中和抗体提供新的可行路径。研究结果提示，当此类中和抗体的完整序列被获取后，可通过其开展针对性治疗，存在明确的临床应用窗口期。在感染早期对感染SARS-CoV-2或其变异株的患者开展快速深度RNA测序的方法，可助力基于已鉴定的特异性中和抗体开发个性化治疗方案。