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    Home > Active Ingredient News > Immunology News > Nature Sub-Journal | Sun Yat-sen University Chen Shoudeng/Shan Hong/Xiao Fei/Liao Huaxin discovered the first fully human new coronavirus N protein antibody that can inhibit COVID-19 complement overactivation

    Nature Sub-Journal | Sun Yat-sen University Chen Shoudeng/Shan Hong/Xiao Fei/Liao Huaxin discovered the first fully human new coronavirus N protein antibody that can inhibit COVID-19 complement overactivation

    • Last Update: 2021-06-02
    • Source: Internet
    • Author: User
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    Editor’s note iNature is China’s largest academic official account.
    It is jointly created by the doctoral team of Tsinghua University, Harvard University, Chinese Academy of Sciences and other units.
    The iNature Talent Official Account is now launched, focusing on talent recruitment, academic progress, scientific research information, and interested parties Long press or scan the QR code below to follow us.

    The global pandemic of COVID-19 caused by the new coronavirus (SARS-CoV-2) in iNature has brought severe challenges to human health and life.

    Although the overall mortality rate of COVID-19 is about 2%, according to the epidemiological analysis of the Chinese Center for Disease Control and Prevention and Lombardy, the hardest-hit area in Italy, the mortality rate of critically ill patients is as high as 49%1,2.

    At the same time, autopsy results of severe COVID-19 patients found that a large number of complement components (C3, C4, C5, MASP-2) deposits 3, 4 can be seen in the lungs of severe patients, causing alveolar membrane damage, increased capillary permeability, and microthrombosis ; It can cause alveolar epithelial damage, decrease or disappearance of surfactants, aggravate pulmonary edema and atelectasis, and ultimately lead to acute respiratory distress syndrome (ARDS).

    Data from multiple clinical trials indicate that inhibiting the excessive activation of complement will help improve the clinical prognosis of critically ill patients5-8.

    It has been pointed out in the literature that the SARS-CoV-2 nucleocapsid (N) protein can activate the complement system through MASP-2 to aggravate lung damage in patients with COVID-199.

    Although there are many researches on the progress of the severe disease, there are few researches on the development of macromolecular-specific drugs targeting the virus N protein and the rapid analysis system for N protein-mediated abnormal activation of complement.

    On May 11, 2021, a cooperative team composed of Professor Chen Shoudeng's research group, Professor Shan Hong's research group, Professor Xiao Fei's research group, and Jinan University's Professor Liao Huaxin's research team from the Fifth Affiliated Hospital of Sun Yat-Sen University published an online publication titled "A SARS-" in Nature Communications on May 11, 2021.
    CoV-2 antibody curbs viral nucleocapsid protein-induced complement hyperactivation" research paper, which uses a fully human antibody screening method based on blood samples of clinical recoverers to cure and discharge the blood of COVID-19 convalescent patients from the Fifth Affiliated Hospital of Sun Yat-sen University Human-derived SARS-CoV-2 N protein antibody was isolated from the test, and the antigen-antibody complex crystal structure was analyzed to analyze the characteristics of the epitope and conformational changes of the antigen.

    In addition, the team independently developed an in vitro, virus-free complement over-activation detection system, and screened out fully humanized specific antibodies that can effectively inhibit the over-activation of complement caused by N protein.

    Figure 1: The first page of the research work paper.
    Researchers first screened the sera of convalescent patients with COVID-19 in different time periods (7-25 days), and found that the antibody titers against N protein were generally higher than the antibody titers against S protein, during the recovery period The titers of N protein antibodies are higher in patients in the early stage (9 days).

    In the follow-up study, the researchers selected the blood of patients with the highest antibody titers (9 days of recovery) for PBMC separation, and further separated memory B cells and plasma cells specific to N and S proteins by flow cytometry.

    The researchers identified 5 plasma cell antibodies and 3 memory B cell antibodies for S protein; 20 plasma cell antibodies and 12 memory B cell antibodies for N protein.

    From the point of view of the mutation length of the antibody and the number of mutated amino acids, the researchers found that the maturity of the N protein antibody is higher than that of the S protein [Figure 2].

    Figure 2: Antibody screening and its characteristics.
    In addition, through preliminary epitope identification (NTD, CTD, N-FL) and the determination of antigen-antibody affinity, the researchers screened out the full-length binding N protein and the most affinity to the antigen.
    Strong antibody nCoV396 (KD=1.
    02 nM) [Figure 3].

    Figure 3: The reactivity and affinity of the N protein antibody to the antigen.
    Next, the researchers used X-ray diffraction to analyze the high-resolution crystal structure of the N-NTD (49-173aa) and nCoV396Fab complex (PDB: 7CR5) .

    The results show that the antibody uses its light chain CDR1, CDR3 and heavy chain CDR1, CDR2, CDR3 to recognize the C-terminal (159-163aa) region of N-NTD through hydrophilic and hydrophobic interactions.

    The Q163, L167 and K169 sites that NTD binds to antibodies are relatively conserved in coronaviruses, suggesting that this antibody can bind to the N proteins of other highly pathogenic coronaviruses and has potential broad-spectrum application prospects.

    SPR analysis also confirmed that nCoV396Fab can also bind to SARS (KD=7.
    26nM) and MERSN protein (KD=12.
    6nM).

    By further comparing the structure with the NTD monomer (PDB: 6M3M), the researchers found that after the N protein binds to the antibody, the C-terminus of the NTD swings about 80º away from the palm area to expose the epitope, which may cause N Changes in the relative positions of protein NTD and CTD.

    At the same time, the finger area of ​​NTD moved 7.
    4 Å from the RNA binding area, resulting in the expansion of the N protein binding RNA pocket, suggesting that nCoV396 may affect the ability of the N protein to bind RNA [Figure 4].

    Figure 4: The structure of the mAb nCoV396 and SARS-CoV-2 N-NTD complex.
    In order to further verify the function of the N protein antibody nCoV39, the researchers independently developed an in vitro method to detect the excessive activation of complement.

    By using the serum of patients with autoimmune diseases and elevated C3 or C4, adding MASP-2 specific fluorescence quenching substrate (2Abz-SLGRKIQI-Lys(Dnp)-NH2), using a microplate reader to detect the initial reaction rate (V0 ), calculate the maximum reaction rate (Vmax) and Michaelis constant (Km) to reflect the complement activation status.

    When SARS-CoV-2 N-FL is added to the system, as the concentration of N-FL increases, Vmax also increases, indicating that the complement system is activated by SARS-CoV-2 N protein.

    Satisfactorily, when an excessive amount of nCoV396Fab is added to the system, the activation of complement will be inhibited, and its complement inhibition effect is positively correlated with the concentration of antibody [Figure 5].

    The results of this experiment were repeated in the blood of multiple patients with different complement activations.

    Figure 5: mAb nCoV396 inhibits overactivation of complement induced by SARS-CoV-2 N protein.
    In general, this study isolated the first fully human SARS-CoV-2 N protein antibody and confirmed that the antibody recognizes N The epitope of the protein, through the in vitro complement activation experiment, determined the role of N protein in the overactivation of complement, and identified the function of nCoV396 to inhibit the overactivation of complement induced by the N protein.

    It provides a theoretical basis for new candidate treatment options for severe COVID-19 patients with excessive complement activation.

    The postdoctoral fellow of the Fifth Affiliated Hospital of Sun Yat-sen University, Kang Si, Ph.
    D candidates Yang Mei and He Suhua, and Wang Yueming of Jinan University are the co-first authors of the article.

    Professor Chen Shoudeng, Professor Shan Hong, Professor Xiao Fei and Professor Liao Huaxin from the School of Life Science and Technology of Jinan University are the co-corresponding authors of the paper.

    This work was supported by the Special Fund for Emergency Research (Social Development) for the Prevention and Control of the Novel Coronavirus Epidemic in Zhuhai City.

    At the same time, thanks to the staff of Shanghai synchrotron radiation source BL18U/19U/17U for their help in screening and collecting X-ray diffraction data.

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