Although human antibodies elicited by severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-directed antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAb) from a quick recovery coronavirus disease-19 (COVID-19) convalescent, who had dominant antibody responses to SARS-CoV-2 N protein rather than to Spike protein. The complex structure of N protein RNA binding domain with the highest binding affinity mAb nCoV396 reveals the epitopes and antigen's allosteric changes. Functionally, a virus-free complement hyper-activation analysis demonstrates that nCoV396 specifically compromises N protein-induced complement hyper-activation, a risk factor for morbidity and mortality in COVID-19, thus paving the way for functional anti-N mAbs identification.
Discussion
From a quickly recovered COVID-19 patient, we isolated 32 mAbs specifically targeting to SARS CoV-2 N protein. The binding affinity of mAbs ranged from 1 nM to 25 nM, comparable with mature spike protein-directed antibodies(20, 23-27) and the other mature antibodies identified during acute infections(30, 31). Characteristics of the isolated N-reactive mAbs are different from the isolated S-reactive mAbs in the early recovery COVID-19 patients suggested that sampling (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292318. this version posted September 11, 2020. The copyright holder for this preprint 11 time is pivotal for identifying differential immune responses to different SARS-CoV-2 viral proteins. The crystal structure of nCoV396 bound to SARS-CoV-2 N-NTD elucidates the interaction mechanism of the complex between the first reported N protein-directed human mAb and its targeted N protein. Three conservative amino acids (Q163, L167, K169) in N protein are responsible for nCoV396 recognition, which provided a clue of cross-reactivity to SARS-CoV or MERS-CoV N protein for nCoV396. Intriguingly, the nCoV396 binding of SARS-CoV-2 N-NTD undergoes several conformational changes, resulting in a change in N-NTD RNA binding pocket enlargement and partial unfolding of basic palm region. More importantly, this conformational change occurs in the C-terminal tail of the N-NTD, which may alter the positioning of individual domains in context of full-length protein and lead to a potential allosteric effect for protein functions. Complement is one of the first lines of defense in innate immunity and is essential for cellular integrity, tissue homeostasis, and modifying the adaptive immune response(32). Emerging evidence suggests that the complement system plays a vital role in a subset of COVID-19 critical patients, with features of atypical acute respiratory distress syndrome, disseminated intravascular coagulation, and multiple organs failure(9, 10, 33). A few pieces of evidence show that highly pathogenic coronavirus (i.e., SARS-CoV-2 and SARS-CoV) N protein is involved in the initiated MASP-2 dependent complement activation(15, 34). Encouragingly, COVID-19 critical patients treated with complement inhibitors, including small molecules to complement component C3 (AMY-101) and antibody targeting to complement component C5 (Eculizumab), show remarkable therapeutic outcomes(15). Currently, there are 11 clinical trials relative to targeting the complement pathway (https://clinicaltrials.gov). In order to avoid adverse effects of human (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.10.292318. this version posted September 11, 2020. The copyright holder for this preprint 12 complement component targeting therapy, a viral protein-specific approach is warranted. The antibody nCoV396 isolated from COVID-19 convalescents is an excellent potential candidate with high binding affinity to N protein and high potency to inhibit the complement hyper-activation. As revealed by atomic structural information, the binding may allosterically change the full-length N protein conformation. To determine the role of nCoV396 in the suppression of complement hyper activation, we monitor the MASP-2 protease activity based on its specific fluorescent quenched C2 substrate in serums from autoimmune disease patients. The complete complement components in sera of patients with autoimmune disorders allow us to monitor the activating effects of SARS CoV-2 N protein and its specific mAbs. Although we cannot calculate the other steady-state enzymatic reaction constants as the precisely concentration of MASP-2 in serum is unknown, we identified the Vmax of the specific C2 substrate for the enzymatic reaction. We demonstrated that SARS-CoV-2 N protein elevated the Vmax of the reaction, up to 40 folds, in serum of all 7 individuals tested, while nCoV396 effectively suppress Vmax of the reaction mixture. These results indicated that the autoimmune disease patient serum-based complement activation analysis is a virus-free and effective method for examining complement activation mediated by coronavirus protein. Although precise interaction of SARS-CoV-2 N protein with MASP-2 remains to be elucidated,our work defined the region on the SARS-CoV-2 N protein recognized by mAb nCoV396 that plays an important role on complement hyper-activation, and indicates that human mAbs from the convalescents could be a promising potential therapeutic candidate for the treatment of COVID 19
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