Robust SARS-CoV-2 infection in nasal turbinates after treatment with systemic neutralizing antibodies

•Systemic HuNAb or vaccine fails full SARS-CoV-2 infection prevention in nasal turbinate•Post-exposure HuNAb suppresses SARS-CoV-2 in lungs but poorly in nasal turbinate•Live SARS-CoV-2 persists in nasal turbinate for several days despite systemic HuNAb•Robust SARS-CoV-2 infection in nasal turbinate is a mode to evade systemic HuNAb Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a burst in the upper respiratory portal for high transmissibility. To determine human neutralizing antibodies (HuNAbs) for entry protection, we tested three potent HuNAbs (IC50 range, 0.0007–0.35 μg/mL) against live SARS-CoV-2 infection in the golden Syrian hamster model. These HuNAbs inhibit SARS-CoV-2 infection by competing with human angiotensin converting enzyme-2 for binding to the viral receptor binding domain (RBD). Prophylactic intraperitoneal or intranasal injection of individual HuNAb or DNA vaccination significantly reduces infection in the lungs but not in the nasal turbinates of hamsters intranasally challenged with SARS-CoV-2. Although postchallenge HuNAb therapy suppresses viral loads and lung damage, robust infection is observed in nasal turbinates treated within 1–3 days. Our findings demonstrate that systemic HuNAb suppresses SARS-CoV-2 replication and injury in lungs; however, robust viral infection in nasal turbinate may outcompete the antibody with significant implications to subprotection, reinfection, and vaccine. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a burst in the upper respiratory portal for high transmissibility. To determine human neutralizing antibodies (HuNAbs) for entry protection, we tested three potent HuNAbs (IC50 range, 0.0007–0.35 μg/mL) against live SARS-CoV-2 infection in the golden Syrian hamster model. These HuNAbs inhibit SARS-CoV-2 infection by competing with human angiotensin converting enzyme-2 for binding to the viral receptor binding domain (RBD). Prophylactic intraperitoneal or intranasal injection of individual HuNAb or DNA vaccination significantly reduces infection in the lungs but not in the nasal turbinates of hamsters intranasally challenged with SARS-CoV-2. Although postchallenge HuNAb therapy suppresses viral loads and lung damage, robust infection is observed in nasal turbinates treated within 1–3 days. Our findings demonstrate that systemic HuNAb suppresses SARS-CoV-2 replication and injury in lungs; however, robust viral infection in nasal turbinate may outcompete the antibody with significant implications to subprotection, reinfection, and vaccine. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in about 80 million infections globally with nearly 1.7 million deaths by the end of 2020 since the discovery of the disease outbreak in December 2019 (Chan et al., 2020dChan J.F. Yuan S. Kok K.H. To K.K. Chu H. Yang J. Xing F. Liu J. Yip C.C. Poon R.W. et al.A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster.Lancet. 2020; 395: 514-523Abstract Full Text Full Text PDF PubMed Scopus (5713) Google Scholar; Zhu et al., 2020Zhu N. Zhang D. Wang W. Li X. Yang B. Song J. Zhao X. Huang B. Shi W. Lu R. et al.China Novel Coronavirus Investigating and Research TeamA Novel Coronavirus from Patients with Pneumonia in China, 2019.N. Engl. J. Med. 2020; 382: 727-733Crossref PubMed Scopus (16620) Google Scholar). The growing coronavirus disease 2019 (COVID-19) pandemic urgently requires the development of effective prophylaxis and treatment. While triple combination therapy (interferon-β1b, lopinavir/ritonavir, and ribavirin), remdesivir, and dexamethasone have all shown some clinical benefits in select patient groups (Boulware et al., 2020Boulware D.R. Pullen M.F. Bangdiwala A.S. Pastick K.A. Lofgren S.M. Okafor E.C. Skipper C.P. Nascene A.A. Nicol M.R. Abassi M. et al.A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19.N. Engl. J. Med. 2020; 383: 517-525Crossref PubMed Scopus (897) Google Scholar; Goldman et al., 2020Goldman J.D. Lye D.C.B. Hui D.S. Marks K.M. Bruno R. Montejano R. Spinner C.D. Galli M. Ahn M.Y. Nahass R.G. et al.GS-US-540-5773 InvestigatorsRemdesivir for 5 or 10 Days in Patients with Severe Covid-19.N. Engl. J. Med. 2020; 383: 1827-1837Crossref PubMed Scopus (897) Google Scholar; Hung et al., 2020bHung I.F. Lung K.C. Tso E.Y. Liu R. Chung T.W. Chu M.Y. Ng Y.Y. Lo J. Chan J. Tam A.R. et al.Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial.Lancet. 2020; 395: 1695-1704Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar), the discovery of specific anti-SARS-CoV-2 agents with higher efficacy, better safety profiles, and bioavailability remain essential for improving the clinical outcome of COVID-19 patients. In addition to some drugs identified in large-scale drug repurposing programs (Riva et al., 2020Riva L. Yuan S. Yin X. Martin-Sancho L. Matsunaga N. Pache L. Burgstaller-Muehlbacher S. De Jesus P.D. Teriete P. Hull M.V. et al.Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.Nature. 2020; 586: 113-119Crossref PubMed Scopus (477) Google Scholar), direct cloning of human neutralizing antibodies (HuNAbs) against SARS-CoV-2 has also been reported recently (Cao et al., 2020Cao Y. Su B. Guo X. Sun W. Deng Y. Bao L. Zhu Q. Zhang X. Zheng Y. Geng C. 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 (750) Google Scholar; Liu et al., 2020bLiu L. Wang P. Nair M.S. Yu J. Rapp M. Wang Q. Luo Y. Chan J.F. Sahi V. Figueroa A. et al.Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.Nature. 2020; 584: 450-456Crossref PubMed Scopus (851) Google Scholar; Robbiani et al., 2020Robbiani D.F. Gaebler C. Muecksch F. Lorenzi J.C.C. Wang Z. Cho A. Agudelo M. Barnes C.O. Gazumyan A. Finkin S. et al.Convergent antibody responses to SARS-CoV-2 in convalescent individuals.Nature. 2020; 584: 437-442Crossref PubMed Scopus (1122) Google Scholar; Shi et al., 2020Shi R. Shan C. Duan X. Chen Z. Liu P. Song J. Song T. Bi X. Han C. Wu L. et al.A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.Nature. 2020; 584: 120-124Crossref PubMed Scopus (808) Google Scholar; Sun et al., 2020Sun Z. Chen C. Li W. Martinez D.R. Drelich A. Baek D.S. Liu X. Mellors J.W. Tseng C.T. Baric R.S. Dimitrov D.S. Potent neutralization of SARS-CoV-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold.MAbs. 2020; 12: 1778435Crossref PubMed Scopus (39) Google Scholar; Wu et al., 2020aWu Y. Li C. Xia S. Tian X. Kong Y. Wang Z. Gu C. Zhang R. Tu C. Xie Y. et al.Identification of Human Single-Domain Antibodies against SARS-CoV-2.Cell Host Microbe. 2020; 27: 891-898.e5Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, Wu et al., 2020bWu Y. Wang F. Shen C. Peng W. Li D. Zhao C. Li Z. Li S. Bi Y. Yang Y. 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 (659) Google Scholar; Zost et al., 2020Zost S.J. Gilchuk P. Case J.B. Binshtein E. Chen R.E. Nkolola J.P. Schäfer A. Reidy J.X. Trivette A. Nargi R.S. et al.Potently neutralizing and protective human antibodies against SARS-CoV-2.Nature. 2020; 584: 443-449Crossref PubMed Scopus (611) Google Scholar). Furthermore, with the recent clinical trials of HuNAbs and urgent approval of COVID-19 vaccines for human use, it is necessary to determine whether or not HuNAbs may warrant sterile protection against live SARS-CoV-2 infection. Unlike SARS patients who had peak upper respiratory tract (URT) viral loads at day 10 after symptom onset (Peiris et al., 2003Peiris J.S. Chu C.M. Cheng V.C. Chan K.S. Hung I.F. Poon L.L. Law K.I. Tang B.S. Hon T.Y. Chan C.S. et al.HKU/UCH SARS Study GroupClinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study.Lancet. 2003; 361: 1767-1772Abstract Full Text Full Text PDF PubMed Scopus (1901) Google Scholar), COVID-19 patients exhibited peak salivary or URT viral loads during the first week after symptom onset that declined over time; this phenomenon could account for the fast-spreading nature of the pandemic (Hung et al., 2020aHung I.F. Cheng V.C. Li X. Tam A.R. Hung D.L. Chiu K.H. Yip C.C. Cai J.P. Ho D.T. Wong S.C. et al.SARS-CoV-2 shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series.Lancet Infect. Dis. 2020; 20: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar; To et al., 2020bTo K.K. Tsang O.T. Leung W.S. Tam A.R. Wu T.C. Lung D.C. Yip C.C. Cai J.P. Chan J.M. Chik T.S. et al.Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study.Lancet Infect. Dis. 2020; 20: 565-574Abstract Full Text Full Text PDF PubMed Scopus (2150) Google Scholar). Regarding the humoral response, SARS-CoV-2-specific IgG and neutralizing antibody responses were quickly detectable in adult and children patients only 6 days after symptom onset (Liu et al., 2020cLiu P. Cai J. Jia R. Xia S. Wang X. Cao L. Zeng M. Xu J. Dynamic surveillance of SARS-CoV-2 shedding and neutralizing antibody in children with COVID-19.Emerg. Microbes Infect. 2020; 9: 1254-1258Crossref PubMed Scopus (50) Google Scholar; Suthar et al., 2020Suthar M.S. Zimmerman M.G. Kauffman R.C. Mantus G. Linderman S.L. Hudson W.H. Vanderheiden A. Nyhoff L. Davis C.W. Adekunle O. et al.Rapid Generation of Neutralizing Antibody Responses in COVID-19 Patients.Cell Rep. Med. 2020; 1: 100040Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar; Zhou et al., 2020bZhou R. To K.K. Wong Y.C. Liu L. Zhou B. Li X. Huang H. Mo Y. Luk T.Y. Lau T.T. et al.Acute SARS-CoV-2 infection impairs dendritic cell and T cell responses.Immunity. 2020; 53: 864-877.e5Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). However, COVID-19 patients with higher amounts of anti-spike (S) and anti-nucleocapsid (NP) IgM and IgG tend to have poorer disease outcomes (Jiang et al., 2020Jiang H.W. Li Y. Zhang H.N. Wang W. Yang X. Qi H. Li H. Men D. Zhou J. Tao S.C. SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses.Nat. Commun. 2020; 11: 3581Crossref PubMed Scopus (163) Google Scholar; Tan et al., 2020Tan W. Lu Y. Zhang J. Wang J. Dan Y. Tan Z. He X. Qian C. Sun Q. Hu Q. et al.Viral Kinetics and Antibody Responses in Patients with COVID-19.medRxiv. 2020; https://doi.org/10.1101/2020.03.24.20042382Crossref Scopus (0) Google Scholar). We and others also reported that COVID-19 patients with severe disease developed significantly more robust SARS-CoV-2-specific NAb responses (Liu et al., 2020aLiu L. To K.K.-W. Chan K.-H. Wong Y.-C. Zhou R. Kwan K.-Y. Fong C.H.-Y. Chen L.-L. Choi C.Y.-K. Lu L. et al.High neutralizing antibody titer in intensive care unit patients with COVID-19.Emerg. Microbes Infect. 2020; 9: 1664-1670Crossref PubMed Scopus (107) Google Scholar; Wang et al., 2020aWang P. Liu L. Nair M.S. Yin M.T. Luo Y. Wang Q. Yuan T. Mori K. Solis A.G. Yamashita M. et al.SARS-CoV-2 neutralizing antibody responses are more robust in patients with severe disease.Emerg. Microbes Infect. 2020; 9: 2091-2093Crossref PubMed Scopus (79) Google Scholar, Wang et al., 2020bWang Y. Zhang L. Sang L. Ye F. Ruan S. Zhong B. Song T. Alshukairi A.N. Chen R. Zhang Z. et al.Kinetics of viral load and antibody response in relation to COVID-19 severity.J. Clin. Invest. 2020; 130: 5235-5244Crossref PubMed Scopus (364) Google Scholar). Nevertheless, convalescent plasma with high NAb titers from recovered patients has been reported to be beneficial in the treatment of severe COVID-19 in small case cohorts (Duan et al., 2020Duan K. Liu B. Li C. Zhang H. Yu T. Qu J. Zhou M. Chen L. Meng S. Hu Y. et al.Effectiveness of convalescent plasma therapy in severe COVID-19 patients.Proc. Natl. Acad. Sci. USA. 2020; 117: 9490-9496Crossref PubMed Scopus (1315) Google Scholar). To replace convalescent plasma, which is not readily available in most countries, HuNAbs have been recently identified and showed promising results in preclinical studies (Cao et al., 2020Cao Y. Su B. Guo X. Sun W. Deng Y. Bao L. Zhu Q. Zhang X. Zheng Y. Geng C. 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 (750) Google Scholar; Liu et al., 2020bLiu L. Wang P. Nair M.S. Yu J. Rapp M. Wang Q. Luo Y. Chan J.F. Sahi V. Figueroa A. et al.Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.Nature. 2020; 584: 450-456Crossref PubMed Scopus (851) Google Scholar; Robbiani et al., 2020Robbiani D.F. Gaebler C. Muecksch F. Lorenzi J.C.C. Wang Z. Cho A. Agudelo M. Barnes C.O. Gazumyan A. Finkin S. et al.Convergent antibody responses to SARS-CoV-2 in convalescent individuals.Nature. 2020; 584: 437-442Crossref PubMed Scopus (1122) Google Scholar; Shi et al., 2020Shi R. Shan C. Duan X. Chen Z. Liu P. Song J. Song T. Bi X. Han C. Wu L. et al.A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.Nature. 2020; 584: 120-124Crossref PubMed Scopus (808) Google Scholar; Sun et al., 2020Sun Z. Chen C. Li W. Martinez D.R. Drelich A. Baek D.S. Liu X. Mellors J.W. Tseng C.T. Baric R.S. Dimitrov D.S. Potent neutralization of SARS-CoV-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold.MAbs. 2020; 12: 1778435Crossref PubMed Scopus (39) Google Scholar; Wu et al., 2020aWu Y. Li C. Xia S. Tian X. Kong Y. Wang Z. Gu C. Zhang R. Tu C. Xie Y. et al.Identification of Human Single-Domain Antibodies against SARS-CoV-2.Cell Host Microbe. 2020; 27: 891-898.e5Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, Wu et al., 2020bWu Y. Wang F. Shen C. Peng W. Li D. Zhao C. Li Z. Li S. Bi Y. Yang Y. 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 (659) Google Scholar; Zost et al., 2020Zost S.J. Gilchuk P. Case J.B. Binshtein E. Chen R.E. Nkolola J.P. Schäfer A. Reidy J.X. Trivette A. Nargi R.S. et al.Potently neutralizing and protective human antibodies against SARS-CoV-2.Nature. 2020; 584: 443-449Crossref PubMed Scopus (611) Google Scholar). However, the in vivo efficacy of anti-SARS-CoV-2 HuNAbs in protecting against URT infection in a physiologically relevant animal model has not been thoroughly investigated. In this study, we identified a panel of candidate HuNAbs and conducted a thorough investigation of the lead candidate HuNAb ZDY20 at a dose of 10 mg/kg, which is much higher than its IC50 and IC90 values of 0.35 and 1 μg/mL, respectively, against live SARS-CoV-2 infection in both prophylactic and therapeutic settings in our established golden Syrian hamster model for COVID-19 (Chan et al., 2020aChan J.F.-W. Zhang A.J. Yuan S. Poon V.K.-M. Chan C.C.-S. Lee A.C.-Y. Chan W.-M. Fan Z. Tsoi H.-W. Wen L. et al.Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility.Clinical Infectious Diseases. 2020; 71: 2428-2446Crossref PubMed Scopus (198) Google Scholar). We also tested the prophylactic efficacy of two much stronger RBD-specific HuNAbs 2–15 (IC50 and IC90 values of 0.0007 and 0.04 μg/mL) and ZB8 (IC50 and IC90 values of 0.013 and 0.031 μg/mL) and an S-based DNA vaccine to address the possible sterile protection in the same animal model. To clone SARS-CoV-2-specific HuNAbs, we obtained peripheral blood mononuclear cells (PBMCs) from 12 convalescent COVID-19 patients in Hong Kong at a mean duration of 19 (±10.4) days after symptom onset (Table S1). These patients included seven females and five males with a mean age of 59 years (range, 21–75). Two patients had severe disease, seven had mild disease, and three were asymptomatic. The PBMCs of these 12 patients were pooled for the generation of a Fab phage library, as only small amounts of PBMCs were obtained from each patient. ELISA and pseudovirus neutralization assays were performed to measure the antibody titers in each patient’s serum prior to pooling, which confirmed that each study subject had SARS-CoV-2 RBD-specific binding (Figure 1A) and neutralizing antibody activities (Figure 1B). Consistent with the observations that we and others reported previously, the two severe patients had higher NAb titers than the mild and asymptomatic patients (Liu et al., 2020aLiu L. To K.K.-W. Chan K.-H. Wong Y.-C. Zhou R. Kwan K.-Y. Fong C.H.-Y. Chen L.-L. Choi C.Y.-K. Lu L. et al.High neutralizing antibody titer in intensive care unit patients with COVID-19.Emerg. Microbes Infect. 2020; 9: 1664-1670Crossref PubMed Scopus (107) Google Scholar; Riva et al., 2020Riva L. Yuan S. Yin X. Martin-Sancho L. Matsunaga N. Pache L. Burgstaller-Muehlbacher S. De Jesus P.D. Teriete P. Hull M.V. et al.Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.Nature. 2020; 586: 113-119Crossref PubMed Scopus (477) Google Scholar; Wang et al., 2020aWang P. Liu L. Nair M.S. Yin M.T. Luo Y. Wang Q. Yuan T. Mori K. Solis A.G. Yamashita M. et al.SARS-CoV-2 neutralizing antibody responses are more robust in patients with severe disease.Emerg. Microbes Infect. 2020; 9: 2091-2093Crossref PubMed Scopus (79) Google Scholar). The mean NAb IC50 titer was 1:1753 with a range of 1:638–1:5701 (Figure 1B). Using the pooled PBMCs, we first set up a Fab phage library consisting of 3×106 clones (Figure S1). We subsequently developed an in-solution selection method for two rounds of panning (Figure 1C). We were able to pick up 384 single reactive colonies (Figure 1D). Next, we tested the binding ability of phage-displayed Fab to recombinant SARS-CoV-2 RBD by a monoclonal phage-based ELISA followed by sequencing 18 single-phage colonies that displayed strong RBD-binding ability. Finally, we obtained four pairs of variable heavy (VH) chain/variable light (VL) chain from the top four clones (Figure 1D, color-coded), which resulted in four human monoclonal antibodies in the native form of IgG1, named ZDY20, ZDY28, ZDY49, and ZDY95. Sequence analysis revealed that ZDY28, ZDY49, and ZDY95 share high similarity with the same immunoglobulin heavy chain variable region gene 3-30 (IGHV3-30) and immunoglobulin kappa chain variable region gene 1-39 (IGKV1-39) (Table S2). They share identical complementarity-determining region 3 (CDR3) heavy (H) and CDR3 light (L) chains but with a few amino acid differences in framework region 1 (FR1) (Figure S2). Since repeated sequencing gave identical results and the IGHV/IGKV fragments were cloned from the phage library derived from 12 patients, it is possible that the small amino acid differences between ZDY49 and ZDY95 might be derived from different donors. In contrast, ZDY20 has IGHV3-53 and IGKV1-33, which were recently shown to be associated with higher antiviral activity (Dalamaga et al., 2020Dalamaga M. Karampela I. Mantzoros C.S. Commentary: Could iron chelators prove to be useful as an adjunct to COVID-19 Treatment Regimens?.Metabolism. 2020; 108: 154260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar; Yuan et al., 2020Yuan M. Liu H. Wu N.C. Lee C.-C.D. Zhu X. Zhao F. Huang D. Yu W. Hua Y. Tien H. et al.Structural basis of a shared antibody response to SARS-CoV-2.Science. 2020; 369: 1119-1123Crossref PubMed Scopus (320) Google Scholar). ZDY20 has a longer CDR3H with three additional amino acids compared with the other three antibodies (Figure S2). These four antibodies all had low rates of somatic hypermutation (SHM) in both IGHV (0.35% or 0.69%) and IGKV (0.36% or 1.08%), except that the SHM rate in IGKV of ZDY20 reached 7.89% (Table S2). We next analyzed the binding activity of these four antibodies to recombinant SARS-CoV-2 RBD and S proteins by ELISA. In these solid phase ELISAs, ZDY28, ZDY49, and ZDY95 showed similar binding abilities to RBD (EC50 0.012 μg/mL) (Figure 2A) and to S (EC50 0.1 μg/mL) (Figure 2B), which were lower than those of ZDY20 (EC50 0.135 and 0.52 μg/mL, respectively) (Table S3). However, the anti-SARS-CoV-2 neutralization activity of ZDY20 (IC90 1.24 μg/mL) was better than those of ZDY28 (IC90 7.47 μg/mL), ZDY49 (IC90 17.08 μg/mL), and ZDY95 (IC90 13.35 μg/mL) in the pseudovirus assay (Figure 2C; Table S3). Moreover, ZDY20 exhibited, relatively, a better IC90 (1 μg/mL) than those of ZDY28 (1.28 μg/mL), ZDY49 (1.24 μg/mL), and ZDY95 (1.5 μg/mL) against live SARS-CoV-2 in Vero-E6 cells (Figure 2D; Table S3). Notably, none of these HuNAbs showed cross-neutralization against the SARS-CoV pseudovirus (data not shown). Using a random yeast surface display of SARS-CoV-2 S fragments, we observed that all four HuNAbs recognized conformational determinants instead of a small linear epitope because their binding domain on viral RBD required a minimum of 179 amino acid residues (337–524 for ZDY20, 336–523 for ZDY28, and 336–515 for ZDY49 and ZDY95) (Figure S3). Next, we measured the binding affinity of each antibody to the SARS-CoV-2 RBD or S trimer using surface plasmon resonance (SPR). Similar to the weaker binding activity observed by ELISA, ZDY20 Fab showed weaker affinity binding to SARS-CoV-2 RBD with a KD value of 74.9 nM, which was larger than those of the other three antibodies (ZDY28 19.2 nM, ZDY49 42.3 nM, and ZDY95 26.2 nM) (Table S4). The complete ZDY20 antibody, however, displayed the highest avidity binding to SARS-CoV-2 S trimer with a dissociation constant (KD) value of 8.8 nM, which was lower than those of the other three antibodies (ZDY28 10.2 nM, ZDY49 24.4 nM, and ZDY95 15 nM) (Figure 2E; Table S4). Furthermore, in the human cellular receptor angiotensin converting enzyme-2 (ACE2) competition assay by SPR, ZDY20 presented the most potent activity to block S binding to ACE2 followed by ZDY95, ZDY28, and ZDY49 (Figure 2F), although ZDY20 was not the strongest competitor to block soluble RBD binding to ACE2 (Figure 2G). These results suggested that the relative higher anti-SARS-CoV-2 neutralizing potency of ZDY20 is likely due to its stronger competitive blockade of S trimer binding to ACE2, which is probably associated with avidity interaction. Based on its relatively better activities of avidity binding KD to S, blocking S binding to ACE2, and neutralization, we chose ZDY20 for subsequent in vivo experiments. To determine the potential role of HuNAb as prophylaxis for SARS-CoV-2 infection, we first administered ZDY20 intraperitoneally to golden Syrian hamsters before virus challenge in our biosafety level-3 (BSL-3) animal laboratory. Syrian hamsters typically recover from SARS-CoV-2 infection with resolution of clinical signs and clearance of virus shedding within 1 week after infection, as we previously described (Chan et al., 2020aChan J.F.-W. Zhang A.J. Yuan S. Poon V.K.-M. Chan C.C.-S. Lee A.C.-Y. Chan W.-M. Fan Z. Tsoi H.-W. Wen L. et al.Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility.Clinical Infectious Diseases. 2020; 71: 2428-2446Crossref PubMed Scopus (198) Google Scholar). Accordingly, the hamsters were sacrificed for analysis at 4 days post-infection (dpi) when high viral loads and acute lung injury were consistently observed. In this prophylaxis study, each hamster received a single intraperitoneal injection of either 10 mg/kg (n = 4) high dose or 5 mg/kg (n = 3) low dose of ZDY20 (Figure 3A) that were much higher than its IC90 value of 1 μg/mL tested by live SARS-CoV-2 in Vero-E6 cells, respectively. Another group of hamsters (n = 4) was injected with a control HIV-1-specific HuNAb VRC01 at a high dose of 10 mg/kg (Liu et al., 2020bLiu L. Wang P. Nair M.S. Yu J. Rapp M. Wang Q. Luo Y. Chan J.F. Sahi V. Figueroa A. et al.Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.Nature. 2020; 584: 450-456Crossref PubMed Scopus (851) Google Scholar). Twenty-four hours after the ZDY20 injection, each animal was challenged intranasally with 105 plaque-forming units (PFUs) of live SARS-CoV-2 (HKU-001a strain) (Chan et al., 2020aChan J.F.-W. Zhang A.J. Yuan S. Poon V.K.-M. Chan C.C.-S. Lee A.C.-Y. Chan W.-M. Fan Z. Tsoi H.-W. Wen L. et al.Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility.Clinical Infectious Diseases. 2020; 71: 2428-2446Crossref PubMed Scopus (198) Google Scholar; Liu et al., 2020bLiu L. Wang P. Nair M.S. Yu J. Rapp M. Wang Q. Luo Y. Chan J.F. Sahi V. Figueroa A. et al.Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.Nature. 2020; 584: 450-456Crossref PubMed Scopus (851) Google Scholar). At 4 dpi, nasal turbinate, trachea, and lung tissues were harvested to quantify infectious viruses by plaque assay, viral loads by real-time PCR and infected cells by immunofluorescence (IF) staining. We found that infectious PFUs were readily detected in all tissue compartments of VRC01-controlled hamsters tested but not in 75% and 0% nasal turbinates, 75% and 67% trachea, and 50% and 33% lungs of hamsters that were given high and low doses of ZDY20, respectively (Figure 3B). The more sensitive RT-PCR further demonstrated that viral RNA copy numbers were reduced in the lungs by an average of 3 logs (range, 0.7–4.5) (Figure 3C). In contrast, there was no significant viral load reduction in nasal turbinates and trachea in both dose groups, suggesting less preventive efficacy by ZDY20 in the URT of viral entry. To determine the role of ZDY20 in protection, we measured the serum antibody concentration and neutralization titer at 0 and 4 dpi. Over 40 and 9 μg/mL ZDY20 were found in 50% high-dose and 67% low-dose animals at 0 dpi (Figure 3D, solid symbol; Table S5), together with mean IC50 values of approximately 1:236 and 1:99, respectively (Figure 3E, solid symbol). At 4 dpi, over 26 and 5 μg/mL ZDY20 were found in 50% high-dose and 67% low-dose animals (Figure 3D, open symbol; Table S5), together with mean IC50 values of approximately 1:55 and 1:45, respectively (Figure 3E, open symbol). These results demonstrated that most animals maintained peripheral amounts of ZDY20 much higher than its IC90 values during the entire course of the experiment. Next, to understand whether or not ZDY20 protects against infection-induced lung injury in hamsters, we performed pathological analysis on lung specimens. Compared with uninfected healthy animals (Figure S4), hamsters treated with the high-dose ZDY20 showed mild interstitial alveoli inflammation with minor septal infiltration and congestion (Figure 3F, top left, arrows). There was no apparent peribronchiolar infiltration, and the bronchiolar epithelia appeared normal (open arrows). Hamsters treated with the low dose showed limited areas of bronchiolar epithelial cell swelling and detachment (Figure 3F, middle left, open arrows) together with mild peribronchiolar infiltration and mild alveolar septal infiltration. In contrast, control hamsters showed large patchy areas of alveolar wall and alveolar space involvement by inflammatory infiltrates and exudation (Figure 3F, bottom left, arrows). The two blood vessels (BVs) in the lung section showed vasculitis and endotheliitis. Furthermore, we performed IF staining of viral NP antigen in both lung and nasal turbinate tissues compared with uninfected healthy animals (Figure S4). In the high-dose group, little viral NP expression was observed in lung sections (Figure 3F, top right). In the low-dose group, a small amount of viral NP was observed in localized areas of alveoli (Figure 3F, middle right, arrows) and in the epithelia of a few bronchiolar sections (thin arrows). In contrast, control hamsters showed diffuse NP expression in extensive areas of alveoli (Figure 3F, bottom right, thick arrows) and in the bronchiolar epithelia (thin arrows). The mean number of NP+ cells per 50×field in the lungs was significantly lower in the high-dose group than that in the control animals (Figure 3G). Remarkably, nasal turbinate tissues showed damage to the respiratory and olfactory epithelium with extensive submucosal immune cell infiltration, together with robust and diffuse viral NP expression in extensive areas lining the stratified squamous epithelia in both the high-dose and low-dose groups of ZDY20-treated animals, similar to untreated control hamsters (Figures 4A–4C). Moreover, no significant difference in NP+ cells per 50×field was found between treated and control animals when nasal turbinate tissues were analyzed (Figure 4D). Collectively, these results demonstrated that prior administration of 10 mg/kg ZDY20 did not prevent robust SARS-CoV-2 infection in nasal turbinates but suppressed productive infection in lungs in the Syrian hamster model. Considering that HuNAbs with greater inhibitory potency may be more effective in preventing viral replication in the nasal turbinates, we tested two such RBD-specific HuNAbs, namely ZB8 and 2-15. ZB8 is a newly cloned HuNAb with IC50 and IC90 values of 0.013 and 0.031 μg/mL, whereas