Nanobody targeting glycan cap confers broad orthoebolavirus neutralization

Xinhui Zhang; Yi Liao; Xiuying Liu; Jingya Zhou; Peixiang Gao; Xuemeng Dong; Shengnan Pan; Huarui Duan; Junyu Liu; Xiaojing Chi; Wei Yang

doi:10.1016/j.virs.2026.03.018

April 2026

. doi: 10.1016/j.virs.2026.03.018

Citation: Xinhui Zhang, Yi Liao, Xiuying Liu, Jingya Zhou, Peixiang Gao, Xuemeng Dong, Shengnan Pan, Huarui Duan, Junyu Liu, Xiaojing Chi, Wei Yang. Nanobody targeting glycan cap confers broad orthoebolavirus neutralization .VIROLOGICA SINICA, 2026, 41(2) : 413-422. http://dx.doi.org/10.1016/j.virs.2026.03.018

靶向埃博拉病毒糖帽结构的广谱中和性纳米抗体

张心慧 ^a,b ,
廖顗 ^a,b ,
刘秀英 ^a,b ,
周靖雅 ^a,b ,
高培翔 ^a,b ,
董雪蒙 ^a,b ,
潘胜男 ^a,b ,
段花蕊 ^a,b ,
刘峻雨 ^a,b ,
迟晓静 ^a,b,, ,
杨威 ^a,b,c,,

a 中国医学科学院&北京协和医学院病原生物学研究所

通讯作者： 迟晓静, chixiaojing@ipbcams.ac.cn; 杨威, wyang@ipb.pumc.edu.cn
收稿日期： 2025-09-15
录用日期： 2026-03-31

摘要

扎伊尔埃博拉病毒（EBOV）和邦迪布焦病毒（BDBV）会引发高致死率的严重出血热，凸显了开发广谱抗病毒治疗策略的迫切需求。纳米抗体因其分子量小、结构稳定及能够识别空间受限的表位等优势，成为极具潜力的抗病毒手段。本研究基于全合成噬菌体展示库，筛选获得一株高亲和力纳米抗体BDBV-Nb02，该抗体表现出优异的结合动力学特性（KD≈ 1 nM），并对BDBV及EBOV假病毒均具有显著中和活性，其半数抑制浓度（IC50）达纳摩尔水平。将该纳米抗体直接构建为二价的形式可显著增强其中和活性，其IC90较单价形式最高降低56倍。表位竞争实验与分子对接分析表明， BDBV-Nb02识别保守的EBOV GP1蛋白的糖基裂隙，其中F248和NP278/279为关键中和位点。相较而言，Fc融合形式的BDBV-Nb02纳米抗体活性反而降低，提示纳米抗体的分子尺寸与延伸的CDR3结构对于进入糖基屏蔽表位至关重要。综上，BDBV-Nb02是一种具有广谱中和潜力的埃博拉病毒候选抗体，有望成为未来抗病毒联合用药的重要组成部分。
- 纳米抗体
- , 埃博拉病毒
- , 中和抗体
- , 糖帽结构
- , 广谱性

Nanobody targeting glycan cap confers broad orthoebolavirus neutralization

Xinhui Zhang ^a,b ,
Yi Liao ^a,b ,
Xiuying Liu ^a,b ,
Jingya Zhou ^a,b ,
Peixiang Gao ^a,b ,
Xuemeng Dong ^a,b ,
Shengnan Pan ^a,b ,
Huarui Duan ^a,b ,
Junyu Liu ^a,b ,
Xiaojing Chi ^a,b,, ,
Wei Yang ^a,b,c,,

a. Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China;
b. NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China;
c. State Key Laboratory of Respiratory Health and Multimorbidity, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China

Corresponding author: Xiaojing Chi, chixiaojing@ipbcams.ac.cn Wei Yang, wyang@ipb.pumc.edu.cn
Received Date: 15 September 2025
Accepted Date: 31 March 2026

Abstract

The Zaire Ebola virus (EBOV) and Bundibugyo virus (BDBV) cause severe hemorrhagic fever with high mortality, highlighting the urgent need for broad-spectrum antiviral therapies. Neutralizing nanobodies, with their small size, structural stability, and ability to access sterically restricted epitopes, represent a promising antiviral modality. Here, we identified a high-affinity nanobody, BDBV-Nb02, from a fully synthetic phage display library targeting the glycan cap of BDBV glycoprotein (GP1). BDBV-Nb02 demonstrated strong binding kinetics (KD ≈ 1 nM) and potent neutralizing activity against both BDBV and EBOV pseudoviruses, with half-maximal inhibitory concentration (IC₅₀) values in the nanomolar range. Engineering a bivalent format significantly enhanced neutralization potency, achieving up to a 56-fold reduction in the 90% inhibitory concentration (IC₉₀) compared with the monovalent form. Epitope competition assays and molecular docking revealed that BDBV-Nb02 targets a conserved glycan cleft, with residues F248 and NP278/279 identified as critical neutralization sites. In contrast, Fc-fusion constructs impaired the nanobody's efficacy, highlighting the importance of preserving the structural features that enable access to glycan-shielded epitopes. Our findings demonstrate that BDBV-Nb02 is a promising candidate for broad-spectrum orthoebolavirus therapy and may serve as a valuable component in future antiviral cocktail formulations.
- Nanobody
- , Orthoebolavirus
- , Neutralizing antibody
- , Glycan cap
- , Broad-spectrum

References
1. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, Bodenstein SW, Evans DA, Hung CC, O'Neill M, Reiman D, Tunyasuvunakool K, Wu Z, Zemgulyte A, Arvaniti E, Beattie C, Bertolli O, Bridgland A, Cherepanov A, Congreve M, Cowen-Rivers AI, Cowie A, Figurnov M, Fuchs FB, Gladman H, Jain R, Khan YA, Low CMR, Perlin K, Potapenko A, Savy P, Singh S, Stecula A, Thillaisundaram A, Tong C, Yakneen S, Zhong ED, Zielinski M, Zidek A, Bapst V, Kohli P, Jaderberg M, Hassabis D, Jumper JM. 2024. Addendum: Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 636:E4.
2. Bornholdt ZA, Herbert AS, Mire CE, He SH, Cross RW, Wec AZ, Abelson DM, Geisbert JB, James RM, Rahim N, Zhu WJ, Borisevich V, Banadyga L, Gunn BM, Agans KN, Wirchnianski AS, Goodwin E, Tierney K, Shestowsky WS, Bohorov O, Bohorova N, Velasco J, Ailor E, Kim D, Pauly MH, Whaley KJ, Alter G, Walker LM, Chandran K, Zeitlin L, Qiu XG, Geisbert TW, Dye JM. 2019. A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates. Cell Host & Microbe, 25:49-58.e5.
3. Caballero IS, Honko AN, Gire SK, Winnicki SM, Mele M, Gerhardinger C, Lin AE, Rinn JL, Sabeti PC, Hensley LE, Connor JH. 2016. In vivo Ebola virus infection leads to a strong innate response in circulating immune cells. Bmc Genomics, 17:707.
4. Chi XJ, Liu XY, Wang CH, Zhang XH, Li X, Hou JH, Ren LL, Jin Q, Wang JW, Yang W. 2020. Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain. Nature Communications, 11:4528.
5. Chi XJ, Zhang XH, Pan SN, Yu YY, Shi YJ, Lin TL, Duan HR, Liu XY, Chen WF, Yang XH, Chen L, Dong XQ, Ren LL, Ding Q, Wang JW, Yang W. 2022. An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants. Signal Transduction and Targeted Therapy, 7:44.
6. Corti D, Misasi J, Mulangu S, Stanley DA, Kanekiyo M, Wollen S, Ploquin A, Doria-Rose NA, Staupe RP, Bailey M, Shi W, Choe M, Marcus H, Thompson EA, Cagigi A, Silacci C, Fernandez-Rodriguez B, Perez L, Sallusto F, Vanzetta F, Agatic G, Cameroni E, Kisalu N, Gordon I, Ledgerwood JE, Mascola JR, Graham BS, Muyembe-Tamfun JJ, Trefry JC, Lanzavecchia A, Sullivan NJ. 2016. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science, 351:1339-1342.
7. Divine R, Dang H, Ueda G, Fallas JA, Vulovic I, Sheffler W, Saini S, Zhao YT, Raj IX, Morawski PA, Jennewein MF, Homad LJ, Wan YH, Tooley MR, Seeger F, Etemadi A, Fahning ML, Lazarovits J, Roederer A, Walls AC, Stewart L, Mazloomi M, King NP, Campbell DJ, McGuire AT, Stamatatos L, Ruohola-Baker H, Mathieu J, Veesler D, Baker D. 2021. Designed proteins assemble antibodies into modular nanocages. Science, 372:eabd9994.
8. Escudero-Perez B, Volchkova VA, Dolnik O, Lawrence P, Volchkov VE. 2014. Shed GP of Ebola Virus Triggers Immune Activation and Increased Vascular Permeability. Plos Pathogens, 10:e1004509.
9. Fan PF, Sun BJ, Liu ZX, Fang T, Ren Y, Zhao XF, Song ZW, Yang YL, Li JM, Yu CM, Chen W. 2024. A pan-orthoebolavirus neutralizing antibody encoded by mRNA effectively prevents virus infection. Emerging Microbes & Infections, 13:2432366.
10. Feldmann H, Geisbert TW. 2011. Ebola haemorrhagic fever. Lancet, 377:849-862.
11. Gilchuk P, Murin CD, Cross RW, Ilinykh PA, Huang K, Kuzmina N, Borisevich V, Agans KN, Geisbert JB, Zost SJ, Nargi RS, Sutton RE, Suryadevara N, Bombardi RG, Carnahan RH, Bukreyev A, Geisbert TW, Ward AB, Crowe J. 2021. Pan-ebolavirus protective therapy by two multifunctional human antibodies. Cell, 184:5593-5607.e18.
12. Gilchuk P, Murin CD, Milligan JC, Cross RW, Mire CE, Ilinykh PA, Huang K, Kuzmina N, Altman PX, Hui S, Gunn BM, Bryan AL, Davidson E, Doranz BJ, Turner HL, Alkutkar T, Flinko R, Orlandi C, Carnahan R, Nargi R, Bombardi RG, Vodzak ME, Li S, Okoli A, Ibeawuchi M, Ohiaeri B, Lewis GK, Alter G, Bukreyev A, Saphire EO, Geisbert TW, Ward AB, Crowe JE. 2020. Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization. Immunity, 52:388-403.e12.
13. Han Q, Wang S, Wang Z, Zhang C, Wang X, Feng N, Wang T, Zhao Y, Chi H, Yan F, Xia X. 2023. Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models. Virol Sin, 38:787-800.
14. Howell KA, Qiu XG, Brannan JM, Bryan C, Davidson E, Holtsberg FW, Wec AZ, Shulenin S, Biggins JE, Douglas R, Enterlein SG, Turner HL, Pallesen J, Murin CD, He SH, Kroeker A, Vu H, Herbert AS, Fusco ML, Nyakatura EK, Lai JR, Keck ZY, Foung SKH, Saphire EO, Zeitlin L, Ward AB, Chandran K, Doranz BJ, Kobinger GP, Dye JM, Aman MJ. 2016. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site. Cell Reports, 15:1514-1526.
15. Hutchinson KL, Rollin PE. 2007. Cytokine and chemokine expression in humans infected with sudan Ebola virus. Journal of Infectious Diseases, 196 Suppl 2:S357-S363.
16. Lee JE, Fusco ML, Hessell AJ, Oswald WB, Burton DR, Saphire EO. 2008. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature, 454:177-182.
17. Liu GD, He SH, Chan MC, Zhang ZR, Schulz H, Cao WG, Rahim MN, Audet J, Garnett L, Wec A, Chandran K, Qiu XG, Banadyga L. 2023. A Pan-Ebolavirus Monoclonal Antibody Cocktail Provides Protection Against Ebola and Sudan Viruses. Journal of Infectious Diseases, 228(Suppl 7):S691-S700.
18. Millet, JK, Tang, T, Nathan, L, et al., 2019. Production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level 2 setting. J Vis Exp (145) Published 2019 Mar 1 doi:10.3791/59010.
19. Mulangu S, Dodd LE, Davey RT, Mbaya OT, Proschan M, Mukadi D, Manzo ML, Nzolo D, Oloma AT, Ibanda A, Ali R, Coulibaly S, Levine AC, Grais R, Diaz J, Lane HC, Muyembe-Tamfum JJ, Sivahera B, Camara M, Kojan R, Walker R, Dighero-Kemp B, Cao HY, Mukumbayi P, Mbala-Kingebeni P, Ahuka S, Albert S, Bonnett T, Crozier I, Duvenhage M, Proffitt C, Teitelbaum M, Moench T, Aboulhab J, Barrett K, Cahill K, Cone K, Eckes R, Hensley L, Herpin B, Higgs E, Ledgerwood J, Pierson J, Smolskis M, Sow Y, Tierney J, Sivapalasingam S, Holman W, Gettinger N, Vallee D, Nordwall J, Grp PW, Team PCS. 2019. A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. New England Journal of Medicine, 381:2293-2303.
20. Pallesen J, Murin CD, de Val N, Cottrell CA, Hastie KM, Turner HL, Fusco ML, Flyak AI, Zeitlin L, Crowe JE, Andersen KG, Saphire EO, Ward AB. 2016. Structures of Ebola virus GP and sGP in complex with therapeutic antibodies. Nature Microbiology, 1:16128.
21. Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng ZP. 2014. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics, 30:1771-1773.
22. Qiu XG, Wong G, Audet J, Bello A, Fernando L, Alimonti JB, Fausther-Bovendo H, Wei HY, Aviles J, Hiatt E, Johnson A, Morton J, Swope K, Bohorov O, Bohorova N, Goodman C, Kim D, Pauly MH, Velasco J, Pettitt J, Olinger GG, Whaley K, Xu BL, Strong JE, Zeitlin L, Kobinger GP. 2014. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature, 514:47-53.
23. Rijal P, Donnellan FR. 2023. A review of broadly protective monoclonal antibodies to treat Ebola virus disease. Current Opinion in Virology, 61:101339.
24. Rivera A, Messaoudi I. 2016. Molecular mechanisms of Ebola pathogenesis. Journal of Leukocyte Biology, 100:889-904.
25. Schieffelin JS, Shaffer JG, Goba A, Gbakie M, Gire SK, Colubri A, Sealfon RSG, Kanneh L, Moigboi A, Momoh M, Fullah M, Moses LM, Brown BL, Andersen KG, Winnicki S, Schaffner SF, Park DJ, Yozwiak NL, Jiang PP, Kargbo D, Jalloh S, Fonnie M, Sinnah V, French I, Kovoma A, Kamara FK, Tucker V, Konuwa E, Sellu J, Mustapha I, Foday M, Yillah M, Kanneh F, Saffa S, Massally JLB, Boisen ML, Branco LM, Vandi MA, Grant DS, Happi C, Gevao SM, Fletcher TE, Fowler RA, Bausch DG, Sabeti PC, Khan SH, Garry RF, Consortium VHF, Team WCR. 2014. Clinical Illness and Outcomes in Patients with Ebola in Sierra Leone. New England Journal of Medicine, 371:2092-2100.
26. Scully M, Cataland SR, Peyvandi F, Coppo P, Knobl P, Hovinga JAK, Metjian A, de la Rubia J, Pavenski K, Callewaert F, Biswas D, De Winter H, Zeldin RK, Investigators H. 2019. Caplacizumab Treatment for Acquired Thrombotic Thrombocytopenic Purpura. New England Journal of Medicine, 380:335-346.
27. Steeland S, Vandenbroucke RE, Libert C. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discovery Today, 21:1076-1113.
28. Thomas S, Smatti MK, Ouhtit A, Cyprian FS, Almaslamani MA, Al Thani A, Yassine HM. 2022. Antibody-Dependent Enhancement (ADE) and the role of complement system in disease pathogenesis. Molecular Immunology, 152:172-182.
29. Wauquier N, Becquart P, Padilla C, Baize S, Leroy EM. 2010. Human Fatal Zaire Ebola Virus Infection Is Associated with an Aberrant Innate Immunity and with Massive Lymphocyte Apoptosis. Plos Neglected Tropical Diseases, 4:e837.
30. Younan P, Iampietro M, Bukreyev A. 2018. Disabling of lymphocyte immune response by Ebola virus. Plos Pathogens, 14:e1006932.
31. Younan P, Iampietro M, Nishida A, Ramanathan P, Santos RI, Dutta M, Lubaki NM, Koup RA, Katze MG, Bukreyev A. 2017. Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm. Mbio, 8:e00845-17.
32. Yu T, Zheng F, He WB, Muyldermans S, Wen YR. 2024. Single domain antibody: Development and application in biotechnology and biopharma. Immunological Reviews, 328:98-112.
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10.1016j.virs.2026.03.018-ESM.docx