Potent mouse monoclonal antibodies that block SARS-CoV-2 infection

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies. The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a threat to global public health and economic development (1Li Q. Guan X. Wu P. Wang X. Zhou L. Tong Y. Ren R. Leung K.S.M. Lau E.H.Y. Wong J.Y. Xing X. Xiang N. Wu Y. Li C. Chen Q. et al.Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia.N. Engl. J. Med. 2020; 382: 1199-1207Crossref PubMed Scopus (10229) Google Scholar, 2Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395: 497-506Abstract Full Text Full Text PDF PubMed Scopus (31105) Google Scholar). Vaccine and therapeutic discovery efforts are paramount to restrict the spread of the virus. Passive immunization could have a major effect on controlling the virus pandemic by providing immediate protection, complementing the development of prophylactic vaccines (3Walker L.M. Burton D.R. Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray.Nat. Rev. Immunol. 2018; 18: 297-308Crossref PubMed Scopus (188) Google Scholar, 4Klasse P.J. Moore J.P. Antibodies to SARS-CoV-2 and their potential for therapeutic passive immunization.Elife. 2020; 9: 1-11Crossref Google Scholar, 5Graham B.S. Ambrosino D.M. History of passive antibody administration for prevention and treatment of infectious diseases.Curr. Opin. HIV AIDS. 2015; 10: 129-134Crossref PubMed Scopus (92) Google Scholar). With the development of humanized mouse antibodies and subsequent generation of fully human antibodies by various techniques, monoclonal antibodies have become widely used in therapy and prophylaxis for cancer, autoimmune diseases, and viral pathogens (3Walker L.M. Burton D.R. Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray.Nat. Rev. Immunol. 2018; 18: 297-308Crossref PubMed Scopus (188) Google Scholar). Indeed, a humanized mouse monoclonal antibody neutralizing respiratory syncytial virus (RSV), palivizumab, is widely used in clinical settings prophylactically to protect vulnerable infants (6Connor E.M. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in hieh-risk infants. The IMpact-RSV study group.Radiology. 1999; 210: 295-296Google Scholar). In recent years, highly specific and often broadly active neutralizing monoclonal antibodies have been developed against several viruses (3Walker L.M. Burton D.R. Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray.Nat. Rev. Immunol. 2018; 18: 297-308Crossref PubMed Scopus (188) Google Scholar, 7Caskey M. Klein F. Nussenzweig M.C. Broadly neutralizing anti-HIV-1 monoclonal antibodies in the clinic.Nat. Med. 2019; 25: 547-553Crossref PubMed Scopus (159) Google Scholar, 8Corti D. Passini N. Lanzavecchia A. Zambon M. Rapid generation of a human monoclonal antibody to combat Middle East respiratory syndrome.J. Infect. Public Health. 2016; 9: 231-235Crossref PubMed Scopus (38) Google Scholar, 9Corti D. Misasi J. Mulangu S. Stanley D.A. Kanekiyo M. Wollen S. Ploquin A. Doria-Rose N.A. Staupe R.P. Bailey M. Shi W. Choe M. Marcus H. Thompson E.A. Cagigi A. et al.Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody.Science. 2016; 351: 1339-1342Crossref PubMed Scopus (303) Google Scholar, 10Corti D. Cameroni E. Guarino B. Kallewaard N.L. Zhu Q. Lanzavecchia A. Tackling influenza with broadly neutralizing antibodies.Curr. Opin. Virol. 2017; 24: 60-69Crossref PubMed Scopus (95) Google Scholar). Passive immunization with a monoclonal antibody is currently under consideration as a treatment for COVID-19 caused by SARS-CoV-2 (4Klasse P.J. Moore J.P. Antibodies to SARS-CoV-2 and their potential for therapeutic passive immunization.Elife. 2020; 9: 1-11Crossref Google Scholar, 11Dhama K. Sharun K. Tiwari R. Dadar M. Malik Y.S. Singh K.P. Chaicumpa W. COVID-19, an emerging coronavirus infection: Advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics.Hum. Vaccin. Immunother. 2020; 16: 1232-1238Crossref PubMed Scopus (380) Google Scholar, 12Jawhara S. Could intravenous immunoglobulin collected from recovered coronavirus patients protect against COVID-19 and strengthen the immune system of new patients?.Int. J. Mol. Sci. 2020; 212272Crossref Scopus (128) Google Scholar, 13Jiang S. Hillyer C. Du L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses.Trends Immunol. 2020; 41: 355-359Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 14Ni L. Ye F. Cheng M.L. Feng Y. Deng Y.Q. Zhao H. Wei P. Ge J. Gou M. Li X. Sun L. Cao T. Wang P. Zhou C. Zhang R. et al.Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals.Immunity. 2020; 52: 971-977.e973Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Isolation of multiple human neutralizing monoclonal antibodies against SARS-CoV-2 has been reported (15Robbiani D.F. Gaebler C. Muecksch F. Lorenzi J.C.C. Wang Z. Cho A. Agudelo M. Barnes C.O. Gazumyan A. Finkin S. Hagglof T. Oliveira T.Y. Viant C. Hurley A. Hoffmann H.H. et al.Convergent antibody responses to SARS-CoV-2 in convalescent individuals.Nature. 2020; 584: 437-442Crossref PubMed Scopus (1179) Google Scholar, 16Liu L. Wang P. Nair M.S. Yu J. Rapp M. Wang Q. Luo Y. Chan J.F. Sahi V. Figueroa A. Guo X.V. Cerutti G. Bimela J. Gorman J. Zhou T. et al.Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.Nature. 2020; 584: 450-456Crossref PubMed Scopus (902) Google Scholar, 17Cao Y. Su B. Guo X. Sun W. Deng Y. Bao L. Zhu Q. Zhang X. Zheng Y. Geng C. Chai X. He R. Li X. Lv Q. Zhu H. et al.Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients' B cells.Cell. 2020; 182: 73-84.e16Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 18Chen X. Li R. Pan Z. Qian C. Yang Y. You R. Zhao J. Liu P. Gao L. Li Z. Huang Q. Xu L. Tang J. Tian Q. Yao W. et al.Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor.Cell Mol. Immunol. 2020; 17: 647-649Crossref PubMed Scopus (251) Google Scholar, 19Chi X. Yan R. Zhang J. Zhang G. Zhang Y. Hao M. Zhang Z. Fan P. Dong Y. Yang Y. Chen Z. Guo Y. Zhang J. Li Y. Song X. et al.A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2.Science. 2020; 655eabc6952Google Scholar, 20Wang C. Li W. Drabek D. Okba N.M.A. van Haperen R. Osterhaus A.D.M.E. van Kuppeveld F.J.M. Haagmans B.L. Grosveld F. Bosch B.J. A human monoclonal antibody blocking SARS-CoV-2 infection.Nat. Commun. 2020; 11: 1-6PubMed Google Scholar, 21Wu Y. Wang F. Shen C. Peng W. Li D. Zhao C. Li Z. Li S. Bi Y. Yang Y. Gong Y. Xiao H. Fan Z. Tan S. Wu G. et al.A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2.Science. 2020; 368: 1274-1278Crossref PubMed Scopus (685) Google Scholar, 22Ju B. Zhang Q. Ge J. Wang R. Sun J. Ge X. Yu J. Shan S. Zhou B. Song S. Tang X. Yu J. Lan J. Yuan J. Wang H. et al.Human neutralizing antibodies elicited by SARS-CoV-2 infection.Nature. 2020; 584: 115-119Crossref PubMed Scopus (1093) Google Scholar, 23Zeng X. Li L. Lin J. Li X. Liu B. Kong Y. Zeng S. Du J. Xiao H. Zhang T. Zhang S. Liu J. Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy.Antibody Ther. 2020; 3: 95-100Crossref PubMed Scopus (44) Google Scholar, 24Wan J. Xing S. Ding L. Wang Y. Gu C. Wu Y. Rong B. Li C. Wang S. Chen K. He C. Zhu D. Yuan S. Qiu C. Zhao C. et al.Human-IgG-neutralizing monoclonal antibodies block the SARS-CoV-2 infection.Cell Rep. 2020; 32107918Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Shi R. Shan C. Duan X. Chen Z. Liu P. Song J. Song T. Bi X. Han C. Wu L. Gao G. Hu X. Zhang Y. Tong Z. Huang W. et al.A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.Nature. 2020; 584: 120-124Crossref PubMed Scopus (855) Google Scholar, 26Pinto D. Park Y.J. Beltramello M. Walls A.C. Tortorici M.A. Bianchi S. Jaconi S. Culap K. Zatta F. De Marco A. Peter A. Guarino B. Spreafico R. Cameroni E. Case J.B. et al.Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.Nature. 2020; 583: 290-295Crossref PubMed Scopus (1096) Google Scholar, 27Rogers T.F. Zhao F. Huang D. Beutler N. Burns A. He W.-t. Limbo O. Smith C. Song G. Woehl J. Yang L. Abbott R.K. Callaghan S. Garcia E. Hurtado J. et al.Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model.Science. 2020; 7520eabc7520Google Scholar, 28Hassan A.O. Case J.B. Winkler E.S. Thackray L.B. Kafai N.M. Bailey A.L. McCune B.T. Fox J.M. Chen R.E. Alsoussi W.B. Turner J.S. Schmitz A.J. Lei T. Shrihari S. Keeler S.P. et al.A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies.Cell. 2020; 182: 744-753.e744Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 29Zost S.J. Gilchuk P. Case J.B. Binshtein E. Chen R.E. Nkolola J.P. Schafer A. Reidy J.X. Trivette A. Nargi R.S. Sutton R.E. Suryadevara N. Martinez D.R. Williamson L.E. Chen E.C. et al.Potently neutralizing and protective human antibodies against SARS-CoV-2.Nature. 2020; 584: 443-449Crossref PubMed Scopus (654) Google Scholar). These antibodies can avoid the potential risks of human–anti-mouse antibody responses and other side effects (30Hansel T.T. Kropshofer H. Singer T. Mitchell J.A. George A.J. The safety and side effects of monoclonal antibodies.Nat. Rev. Drug Discov. 2010; 9: 325-338Crossref PubMed Scopus (751) Google Scholar). However, since they are recombinant human antibodies produced in HEK293 cell lines derived from human embryonic kidney, they have a disadvantage compared with conventional hybridoma-produced antibodies in terms of their lot-to-lot quality control and manufacturing costs (31Cohen J. The race is on for antibodies that stop the new coronavirus.Science. 2020; 368: 564-565Crossref PubMed Scopus (11) Google Scholar). Instead, monoclonal antibodies produced by hybridomas are secreted into the culture supernatant, thus their production is straightforward and of low cost, and their quality is stable. In addition to the impact of monoclonal antibodies on therapy and prophylaxis, they significantly impact the characterization of SARS-CoV-2. To overcome the long-term battle with the virus, we need a detailed understanding of the replication mechanisms underlying its life cycle, including viral entry, genome replication, budding from the cellular membrane, and interaction with host immune systems. These essential pieces of information are required for drug discovery, vaccine design, and therapy development. Despite the large number of neutralizing antibodies reported to inhibit infection, there is an overwhelming lack of data on a well-characterized antibody available for basic research techniques such as western blotting (WB), immunofluorescence, and immunoprecipitation to study the viral life cycle. Here, we established six monoclonal antibodies against the spike glycoprotein of SARS-CoV-2. The trimeric spike glycoproteins of SARS-CoV-2 play a pivotal role in viral entry into human target cells the enzyme 2 as M. H. S. N. T. S. G. Wu A. M.A. C. S. SARS-CoV-2 cell entry on and and is by a 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). antibodies for in molecular pathology research. antibodies to the interaction of spike with and infection of VeroE6/TMPRSS2 cells by SARS-CoV-2. antibodies will research on SARS-CoV-2 and to new therapies and prophylaxis. The SARS-CoV-2 spike glycoprotein is a protein of and infection, the receptor-binding domain on binds to in of the spike the spike protein is into the N-terminal and by host such as and from the to the M. H. S. N. T. S. G. Wu A. M.A. C. S. SARS-CoV-2 cell entry on and and is by a 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, W. M. Wang X. Xiang Y. of the coronavirus spike glycoprotein in with its host cell receptor 2018; PubMed Scopus Google Scholar, X. Liu Y. Lei X. Li P. D. Ren L. Guo L. Guo R. Chen T. Hu J. Xiang Z. Z. Chen X. Chen J. Hu K. et of spike glycoprotein of SARS-CoV-2 on virus entry and its immune with 2020; 11: PubMed Scopus Google an by the A.C. Park Y.J. Tortorici M.A. A. D. and of the SARS-CoV-2 spike 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, W. Zhang X. He Y. S. Du L. of SARS-CoV monoclonal antibodies with neutralizing activity against 2020; PubMed Scopus Google Scholar). The to be to spike for the reported by et D. Wang N. J.A. O. B.S. J.S. of the spike in the 2020; PubMed Scopus Google in the SARS-CoV-2 spike protein to a that to protein In our recombinant spike protein and A into the of the recombinant spike to Cheng N. A.C. trimeric on 11: PubMed Scopus Google with produced using an E. system recombinant spike protein and produced using a system that protein to that virus replication and with recombinant spike to antibodies against the SARS-CoV-2 virus, by cell to a by and immunoprecipitation and six monoclonal hybridomas isolated and To antibodies in they first from the culture and in terms of and monoclonal antibodies derived from the produced by E. and and from cells and In the binding six with against spike glycoprotein and could not be from that to have binding required for antibodies to and glycoproteins the low In target are and established by E. and of and and and in In of not only spike glycoprotein but also spike glycoprotein in cells on However, could be by antibody established by the and on a for also An antibody of the of spike to research the molecular of SARS-CoV-2 infection, cell entry, play a The activity of antibodies can be with the activity of the of the target protein and neutralizing the the performance of our monoclonal antibodies. of and and for and for glycoprotein and in our antibodies the spike protein in a and the of and and are not of a for them performance in and glycoproteins in their we that and could under highly experimental we our antibodies could be used in the An antibody for also have activity in of spike is essential for the of and of from the cellular our performance in using cells spike protein with the with their performance in the A and and could spike on the side of cells with a and However, their is from that for spike are in the infection S. M. M. T. L. A. F. G. The of replication and budding of PubMed Scopus Google also coronavirus spike protein in the in a K. M.C. P.J. glycoprotein in coronavirus PubMed Scopus Google Scholar). the effect of protein on cellular of spike in and protein not to have impact on of spike in that mechanisms of viral in SARS-CoV-2 are from that of and mouse The in antibodies and spike glycoproteins in an the of interaction is by competitive binding neutralizing antibodies and spike they of binding neutralizing SARS-CoV-2 we a spike in the spike glycoprotein by in the of monoclonal antibodies and and as by the spike in To the we a by the of to blocking with monoclonal antibodies Antibodies and of and robust of binding with of and and and to inhibit binding with their performance in monoclonal antibodies derived from the produced by E. and to of spike protein In an of antibodies from cells and could not be determined The that they to that they an of the spike we our antibodies inhibit SARS-CoV-2 infection in VeroE6/TMPRSS2 is to SARS-CoV-2 infection compared with the cell by S. N. K. M. S. N. T. H. F. M. M. S. N. M. et of SARS-CoV-2 by Sci. S. A. 2020; PubMed Scopus Google Scholar). In antibodies and but not and could spike glycoprotein with the of SARS-CoV-2 infection in cells the other and to in infected cells. Spike a in the and J. Singh M. of coronavirus spike protein has 2017; PubMed Scopus Google The of spike that of the protein in cells infected with S. M. M. T. L. A. F. G. The of replication and budding of PubMed Scopus Google of SARS-CoV-2 in the a virus to and inhibit the virus to protect from SARS-CoV-2 infection, and SARS-CoV-2 infection significantly with of and of and A of and neutralizing activity that and an mouse antibodies not be for in clinical not chimeric and to their (30Hansel T.T. Kropshofer H. Singer T. Mitchell J.A. George A.J. The safety and side effects of monoclonal antibodies.Nat. Rev. Drug Discov. 2010; 9: 325-338Crossref PubMed Scopus (751) Google Scholar, J.M. L.B. M.C. antibody in the clinic.Nat. PubMed Scopus Google Scholar). In the hope of applying the antibodies of clinical the variable regions of the antibodies determined by the production of recombinant antibodies on to cell antibodies from and humanized chimeric antibodies as and by them with the of human for and of spike glycoprotein and spike glycoprotein in cells on as as and In could spike in cells and and robust of binding with of and SARS-CoV-2 is a global public health threat to is to be long-term for several C. E. M. the transmission dynamics of SARS-CoV-2 the 2020; 368: PubMed Scopus Google Scholar). there are multiple to neutralizing vaccines, and against the virus E. The race for coronavirus A 2020; PubMed Google Scholar, L. Yuan S. X. L. N. L. S. De P. Chan J.F. Cao J. et of SARS-CoV-2 2020; PubMed Scopus Google the lack of the need for a detailed and understanding of the molecular mechanisms underlying the of the virus A. biology of 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). has for developing against the virus, including vaccine design, and drug Due to the and our with antibody K. H. A. Y. M.C. H. K. by multiple including to 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, K. H. A. A. S. S. S. K. T. M.C. H. J. 2019; PubMed Scopus Google we have established and mouse monoclonal antibodies that can be used to the molecular of the virus life cycle. These antibodies as a for basic research the and of spike glycoprotein viral entry, replication, and These antibodies could to novel host with spike glycoprotein used in in with in basic research the discovery of virus and have been to the interaction of spike with and neutralize infection of cells by SARS-CoV-2. is that their neutralizing of and to be of human antibodies reported and the of experimental virus of to neutralizing of our antibodies compared with other research we used a of SARS-CoV-2 virus to are to virus infection the commonly cell is to antibody them R.S. The and future of vaccines, and therapies against emerging 2020; 11: PubMed Scopus Google Scholar). In addition to in infection, their neutralizing activity in be in animal that SARS-CoV-2 be for the of immune responses to the virus passive immunization using mouse for SARS-CoV-2 infection A.O. Case J.B. Winkler E.S. Thackray L.B. Kafai N.M. Bailey A.L. McCune B.T. Fox J.M. Chen R.E. Alsoussi W.B. Turner J.S. Schmitz A.J. Lei T. Shrihari S. Keeler S.P. et al.A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies.Cell. 2020; 182: 744-753.e744Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, L. Deng W. Huang B. Gao H. Liu J. Ren L. Wei Q. Yu P. Xu Y. F. Y. Li F. Lv Q. Wang W. J. et of SARS-CoV-2 in 2020; 583: PubMed Scopus Google Scholar, Liu Chen Y. Shan C. Zhou Shen Li Q. Zhang L. Zhu Y. Wang Q. J. Wang X. Zhang W. Li B. et of SARS-CoV-2 in mice human enzyme 2020; 182: Full Text Full Text PDF PubMed Scopus Google Scholar, Schafer A. Martinez D.R. A. B.L. Y.J. L.E. A.J. Huang E. et al.A model of SARS-CoV-2 to COVID-19 2020; PubMed Scopus Google Scholar, E.S. Bailey A.L. Kafai N.M. Nair S. McCune B.T. Yu J. Fox J.M. Chen R.E. Keeler S.P. S. A. R. et infection of human mice severe and Immunol. 2020; PubMed Scopus Google Scholar, B. Song E. T. P. A. Liu F. Wei J. Dong H. A. A. model of SARS-CoV-2 role of Med. 2020; PubMed Google Scholar). could performance to lot-to-lot and as for other antibodies and drug are as data are the information L. T. R. C. A for monoclonal antibody 2019; PubMed Scopus Google Scholar, M. J. P. of the antibody from hybridomas by the PubMed Scopus Google Scholar). The that they have of with the of A. M. and Y. for and on the production of antibodies. Y. H. and K. M. the the and the Y. A. M. and K. M. the the the data and to the of the by the COVID-19 M. H. and K. and the is to the of of study also in by to A. in of the and A. and to COVID-19 of A.

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