Citation: Qianran Wang, Lu Peng, Yanqiu Nie, Yanni Shu, Huajun Zhang, Zidan Song, Yufeng Li, Hengrui Hu, Liushuai Li, Xi Wang, Jia Liu, Jiang Li, Zhengli Shi, Fei Deng, Yu Guo, Yiwu Zhou, Bing Yan, Zhihong Hu, Manli Wang. Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo .VIROLOGICA SINICA, 2023, 38(2) : 257-267.  http://dx.doi.org/10.1016/j.virs.2022.12.007

Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo

  • Corresponding author: Manli Wang, wangml@wh.iov.cn
  • Received Date: 20 November 2022
    Accepted Date: 29 December 2022
    Available online: 31 December 2022
  • Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

  • 加载中
    1. Barnes CO, Jette CA, Abernathy ME, Dam KA, Esswein SR, Gristick HB, Malyutin AG, Sharaf NG, Huey-Tubman KE, Lee YE, Robbiani DF, Nussenzweig MC, West AP, Jr., Bjorkman PJ, 2020. SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature. 588, 682-687.

    2. Baum A, Ajithdoss D, Copin R, Zhou A, Lanza K, Negron N, Ni M, Wei Y, Mohammadi K, Musser B, Atwal GS, Oyejide A, Goez-Gazi Y, Dutton J, Clemmons E, Staples HM, Bartley C, Klaffke B, Alfson K, Gazi M, Gonzalez O, Dick E, Jr., Carrion R, Jr., Pessaint L, Porto M, Cook A, Brown R, Ali V, Greenhouse J, Taylor T, Andersen H, Lewis MG, Stahl N, Murphy AJ, Yancopoulos GD, Kyratsous CA, 2020. REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters. Science. 370, 1110-1115.

    3. Cao Y, Wang J, Jian F, Xiao T, Song W, Yisimayi A, Huang W, Li Q, Wang P, An R, Wang J, Wang Y, Niu X, Yang S, Liang H, Sun H, Li T, Yu Y, Cui Q, Liu S, Yang X, Du S, Zhang Z, Hao X, Shao F, Jin R, Wang X, Xiao J, Wang Y, Xie XS, 2022. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature. 602, 657-663.

    4. Chen P, Nirula A, Heller B, Gottlieb RL, Boscia J, Morris J, Huhn G, Cardona J, Mocherla B, Stosor V, Shawa I, Adams AC, Van Naarden J, Custer KL, Shen L, Durante M, Oakley G, Schade AE, Sabo J, Patel DR, Klekotka P, Skovronsky DM, BLAZE-1 Investigators, 2021. SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid-19. N Engl J Med. 384, 229-237.

    5. Chen RE, Winkler ES, Case JB, Aziati ID, Bricker TL, Joshi A, Darling TL, Ying B, Errico JM, Shrihari S, VanBlargan LA, Xie X, Gilchuk P, Zost SJ, Droit L, Liu Z, Stumpf S, Wang D, Handley SA, Stine WB, Jr., Shi PY, Davis-Gardner ME, Suthar MS, Knight MG, Andino R, Chiu CY, Ellebedy AH, Fremont DH, Whelan SPJ, Crowe JE, Jr., Purcell L, Corti D, Boon ACM, Diamond MS, 2021. In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains. Nature. 596, 103-108.

    6. Corti D, Purcell LA, Snell G, Veesler D, 2021. Tackling COVID-19 with neutralizing monoclonal antibodies. Cell. 184, 3086-3108.

    7. Davies NG, Abbott S, Barnard RC, Jarvis CI, Kucharski AJ, Munday JD, Pearson CAB, Russell TW, Tully DC, Washburne AD, Wenseleers T, Gimma A, Waites W, Wong KLM, van Zandvoort K, Silverman JD, Group CC-W, Consortium C-GU, Diaz-Ordaz K, Keogh R, Eggo RM, Funk S, Jit M, Atkins KE, Edmunds WJ, 2021. Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science. 372, eabg3055.

    8. Group AC-TfIwC-S, 2021. Efficacy and safety of two neutralising monoclonal antibody therapies, sotrovimab and BRII-196 plus BRII-198, for adults hospitalised with COVID-19 (TICO):a randomised controlled trial. Lancet Infect Dis. 22, 622-635.

    9. Hansen J, Baum A, Pascal KE, Russo V, Giordano S, Wloga E, Fulton BO, Yan Y, Koon K, Patel K, Chung KM, Hermann A, Ullman E, Cruz J, Rafique A, Huang T, Fairhurst J, Libertiny C, Malbec M, Lee WY, Welsh R, Farr G, Pennington S, Deshpande D, Cheng J, Watty A, Bouffard P, Babb R, Levenkova N, Chen C, Zhang B, Romero Hernandez A, Saotome K, Zhou Y, Franklin M, Sivapalasingam S, Lye DC, Weston S, Logue J, Haupt R, Frieman M, Chen G, Olson W, Murphy AJ, Stahl N, Yancopoulos GD, Kyratsous CA, 2020. Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail. Science. 369, 1010-1014.

    10. Hassan AO, Case JB, Winkler ES, Thackray LB, Kafai NM, Bailey AL, McCune BT, Fox JM, Chen RE, Alsoussi WB, Turner JS, Schmitz AJ, Lei T, Shrihari S, Keeler SP, Fremont DH, Greco S, McCray PB, Jr., Perlman S, Holtzman MJ, Ellebedy AH, Diamond MS, 2020. A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies. Cell. 182, 744-753.e4.

    11. Huang Y, Sun H, Yu H, Li S, Zheng Q, Xia N, 2020. Neutralizing antibodies against SARS-CoV-2:current understanding, challenge and perspective. Antib Ther. 3, 285-299.

    12. Ju 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, Zhao J, Zhang S, Wang Y, Shi X, Liu L, Zhao J, Wang X, Zhang Z, Zhang L, 2020. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature. 584, 115-119.

    13. Kreye J, Reincke SM, Kornau HC, Sanchez-Sendin E, Corman VM, Liu H, Yuan M, Wu NC, Zhu X, Lee CD, Trimpert J, Holtje M, Dietert K, Stoffler L, von Wardenburg N, van Hoof S, Homeyer MA, Hoffmann J, Abdelgawad A, Gruber AD, Bertzbach LD, Vladimirova D, Li LY, Barthel PC, Skriner K, Hocke AC, Hippenstiel S, Witzenrath M, Suttorp N, Kurth F, Franke C, Endres M, Schmitz D, Jeworowski LM, Richter A, Schmidt ML, Schwarz T, Muller MA, Drosten C, Wendisch D, Sander LE, Osterrieder N, Wilson IA, Pruss H, 2020. A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model. Cell. 183, 1058-1069.e19.

    14. Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, Zhang Q, Shi X, Wang Q, Zhang L, Wang X, 2020. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 581, 215-220.

    15. Ledford H, 2021. COVID antibody treatments show promise for preventing severe disease. Nature. 591, 513-514.

    16. Liu C, Ginn HM, Dejnirattisai W, Supasa P, Wang B, Tuekprakhon A, Nutalai R, Zhou D, Mentzer AJ, Zhao Y, Duyvesteyn HME, Lopez-Camacho C, Slon-Campos J, Walter TS, Skelly D, Johnson SA, Ritter TG, Mason C, Costa Clemens SA, Gomes Naveca F, Nascimento V, Nascimento F, Fernandes da Costa C, Resende PC, Pauvolid-Correa A, Siqueira MM, Dold C, Temperton N, Dong T, Pollard AJ, Knight JC, Crook D, Lambe T, Clutterbuck E, Bibi S, Flaxman A, Bittaye M, Belij-Rammerstorfer S, Gilbert SC, Malik T, Carroll MW, Klenerman P, Barnes E, Dunachie SJ, Baillie V, Serafin N, Ditse Z, Da Silva K, Paterson NG, Williams MA, Hall DR, Madhi S, Nunes MC, Goulder P, Fry EE, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR, 2021. Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum. Cell. 184, 4220-4236.e13.

    17. Liu H, Wei P, Kappler JW, Marrack P, Zhang G, 2022. SARS-CoV-2 Variants of Concern and Variants of Interest Receptor Binding Domain Mutations and Virus Infectivity. Front Immunol. 13, 825256.

    18. McCallum M, Bassi J, De Marco A, Chen A, Walls AC, Di Iulio J, Tortorici MA, Navarro MJ, Silacci-Fregni C, Saliba C, Sprouse KR, Agostini M, Pinto D, Culap K, Bianchi S, Jaconi S, Cameroni E, Bowen JE, Tilles SW, Pizzuto MS, Guastalla SB, Bona G, Pellanda AF, Garzoni C, Van Voorhis WC, Rosen LE, Snell G, Telenti A, Virgin HW, Piccoli L, Corti D, Veesler D, 2021. SARS-CoV-2 immune evasion by the B.1.427/B.1.429 variant of concern. Science. 373, 648-654.

    19. Mittal A, Khattri A, Verma V, 2022. Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants. PLoS Pathog. 18, e1010260.

    20. Pan X, Zhou P, Fan T, Wu Y, Zhang J, Shi X, Shang W, Fang L, Jiang X, Shi J, Sun Y, Zhao S, Gong R, Chen Z, Xiao G, 2020. Immunoglobulin fragment F(ab')2 against RBD potently neutralizes SARS-CoV-2 in vitro. Antiviral Res. 182, 104868.

    21. Pan Y, Du J, Liu J, Wu H, Gui F, Zhang N, Deng X, Song G, Li Y, Lu J, Wu X, Zhan S, Jing Z, Wang J, Yang Y, Liu J, Chen Y, Chen Q, Zhang H, Hu H, Duan K, Wang M, Wang Q, Yang X, 2021. Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries. Cell Discov. 7, 57.

    22. Pinto D, Park YJ, Beltramello M, Walls AC, Tortorici MA, Bianchi S, Jaconi S, Culap K, Zatta F, De Marco A, Peter A, Guarino B, Spreafico R, Cameroni E, Case JB, Chen RE, Havenar-Daughton C, Snell G, Telenti A, Virgin HW, Lanzavecchia A, Diamond MS, Fink K, Veesler D, Corti D, 2020. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature. 583, 290-295.

    23. Planas D, Veyer D, Baidaliuk A, Staropoli I, Guivel-Benhassine F, Rajah MM, Planchais C, Porrot F, Robillard N, Puech J, Prot M, Gallais F, Gantner P, Velay A, Le Guen J, Kassis-Chikhani N, Edriss D, Belec L, Seve A, Courtellemont L, Pere H, Hocqueloux L, Fafi-Kremer S, Prazuck T, Mouquet H, Bruel T, Simon-Loriere E, Rey FA, Schwartz O, 2021. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature. 596, 276-280.

    24. Shang J, Wan Y, Luo C, Ye G, Geng Q, Auerbach A, Li F, 2020. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A. 117, 11727-11734.

    25. Sun J, Zhuang Z, Zheng J, Li K, Wong RL, Liu D, Huang J, He J, Zhu A, Zhao J, Li X, Xi Y, Chen R, Alshukairi AN, Chen Z, Zhang Z, Chen C, Huang X, Li F, Lai X, Chen D, Wen L, Zhuo J, Zhang Y, Wang Y, Huang S, Dai J, Shi Y, Zheng K, Leidinger MR, Chen J, Li Y, Zhong N, Meyerholz DK, McCray PB, Jr., Perlman S, Zhao J, 2020. Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment. Cell. 182, 734-743.e5.

    26. Tada T, Dcosta BM, Samanovic MI, Herati RS, Cornelius A, Zhou H, Vaill A, Kazmierski W, Mulligan MJ, Landau NR, 2021. Convalescent-Phase Sera and Vaccine-Elicited Antibodies Largely Maintain Neutralizing Titer against Global SARS-CoV-2 Variant Spikes. mBio. 12, e0069621.

    27. Tegally H, Wilkinson E, Giovanetti M, Iranzadeh A, Fonseca V, Giandhari J, Doolabh D, Pillay S, San EJ, Msomi N, Mlisana K, von Gottberg A, Walaza S, Allam M, Ismail A, Mohale T, Glass AJ, Engelbrecht S, Van Zyl G, Preiser W, Petruccione F, Sigal A, Hardie D, Marais G, Hsiao NY, Korsman S, Davies MA, Tyers L, Mudau I, York D, Maslo C, Goedhals D, Abrahams S, Laguda-Akingba O, Alisoltani-Dehkordi A, Godzik A, Wibmer CK, Sewell BT, Lourenco J, Alcantara LCJ, Kosakovsky Pond SL, Weaver S, Martin D, Lessells RJ, Bhiman JN, Williamson C, de Oliveira T, 2021. Detection of a SARS-CoV-2 variant of concern in South Africa. Nature. 592, 438-443.

    28. Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G, 2020. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 30, 269-271.

    29. Wang P, Nair MS, Liu L, Iketani S, Luo Y, Guo Y, Wang M, Yu J, Zhang B, Kwong PD, Graham BS, Mascola JR, Chang JY, Yin MT, Sobieszczyk M, Kyratsous CA, Shapiro L, Sheng Z, Huang Y, Ho DD, 2021. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature. 593, 130-135.

    30. Weinreich DM, Sivapalasingam S, Norton T, Ali S, Gao H, Bhore R, Musser BJ, Soo Y, Rofail D, Im J, Perry C, Pan C, Hosain R, Mahmood A, Davis JD, Turner KC, Hooper AT, Hamilton JD, Baum A, Kyratsous CA, Kim Y, Cook A, Kampman W, Kohli A, Sachdeva Y, Graber X, Kowal B, DiCioccio T, Stahl N, Lipsich L, Braunstein N, Herman G, Yancopoulos GD, Trial I, 2021. REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19. N Engl J Med. 384, 238-251.

    31. Yu D, Li YF, Liang H, Wu JZ, Hu Y, Peng Y, Li TJ, Hou JF, Huang WJ, Guan LD, Han R, Xing YT, Zhang Y, Liu J, Feng L, Li CY, Liang XL, Ding YL, Zhou ZJ, Ji DM, Wang FF, Yu JH, Deng K, Xia DM, Dong DM, Hu HR, Liu YJ, Fu DX, He YL, Zhou DB, Yang HC, Jia R, Ke CW, Du T, Xie Y, Zhou R, Li CS, Wang ML, Yang XM, 2022. Potent Anti-SARS-CoV-2 Efficacy of COVID-19 Hyperimmune Globulin from Vaccine-Immunized Plasma. Adv Sci (Weinh). 9, e2104333.

    32. Yurkovetskiy L, Wang X, Pascal KE, Tomkins-Tinch C, Nyalile TP, Wang Y, Baum A, Diehl WE, Dauphin A, Carbone C, Veinotte K, Egri SB, Schaffner SF, Lemieux JE, Munro JB, Rafique A, Barve A, Sabeti PC, Kyratsous CA, Dudkina NV, Shen K, Luban J, 2020. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. Cell. 183, 739-751.e8.

    33. Zhang J, Zhang H, Sun L, 2022. Therapeutic antibodies for COVID-19:is a new age of IgM, IgA and bispecific antibodies coming? MAbs. 14, 2031483.

    34. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL, 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 579, 270-273.

  • 加载中

Article Metrics

Article views(187) PDF downloads(7) Cited by()

Related
Proportional views

    Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo

      Corresponding author: Manli Wang, wangml@wh.iov.cn
    • a. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China;
    • b. State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071, China;
    • c. Department of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430010, China;
    • d. Hubei Jiangxia Laboratory, Wuhan, 430200, China

    Abstract: Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

    Reference (34) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return