Two linear epitopes on the sars-cov-2 spike protein that elicit neutralising antibodies in covid-19 patients

ABSTRACT Given the ongoing SARS-CoV-2 pandemic, identification of immunogenic targets against the coronavirus spike glycoprotein will provide crucial advances towards the development of

sensitive diagnostic tools and potential vaccine candidate targets. In this study, using pools of overlapping linear B-cell peptides, we report two IgG immunodominant regions on SARS-CoV-2

spike glycoprotein that are recognised by sera from COVID-19 convalescent patients. Notably, one is specific to SARS-CoV-2, which is located in close proximity to the receptor binding

domain. The other region, which is localised at the fusion peptide, could potentially function as a pan-SARS target. Functionally, antibody depletion assays demonstrate that antibodies

targeting these immunodominant regions significantly alter virus neutralisation capacities. Taken together, identification and validation of these neutralising B-cell epitopes will provide

insights towards the design of diagnostics and vaccine candidates against this high priority coronavirus. SIMILAR CONTENT BEING VIEWED BY OTHERS THE SARS-COV-2 SPIKE RESIDUES 616/644 AND

1138/1169 DELINEATE TWO ANTIBODY EPITOPES IN COVID-19 MRNA COMIRNATY VACCINE (PFIZER/BIONTECH) Article Open access 09 April 2022 EPITOPE MAPPING OF SARS-COV-2 SPIKE PROTEIN USING

NATURALLY-ACQUIRED IMMUNE RESPONSES TO DEVELOP MONOCLONAL ANTIBODIES Article Open access 09 May 2025 A SARS-COV-2 NEUTRALIZING ANTIBODY WITH EXTENSIVE SPIKE BINDING COVERAGE AND MODIFIED FOR

OPTIMAL THERAPEUTIC OUTCOMES Article Open access 11 May 2021 INTRODUCTION In December 2019, a cluster of pneumonia cases of unknown aetiology was reported in the city of Wuhan in the

province of Hubei. The previously unidentified pathogen, which induces symptoms resembling an infection by the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), was later identified

as a novel coronavirus, SARS-CoV-21. To date, there are more than four million laboratory-confirmed cases of human Coronavirus Disease 2019 (COVID-19), with over 280,000 deaths across 212

countries and territories (For up to date information consult https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). After being declared a pandemic by World

Health Organization (WHO) on 11th March 2020, there is a compelling need to understand and develop effective therapeutic interventions against SARS-CoV-2. SARS-CoV-2 uses the spike (S)

glycoprotein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor with a better affinity than SARS-CoV S glycoprotein for entry2. Thus, blocking the binding to ACE2, or blocking

host protease cleavage of S glycoprotein to release the fusion peptide is an efficient strategy to prevent coronavirus entry3,4,5. Several studies have assessed the immunogenicity of

structural domains of recombinant SARS-CoV-2 S glycoprotein6,7. At the time of writing, findings on SARS-CoV-2 linear epitopes remain mostly limited to bioinformatics prediction of human B-

and T-cell epitopes using SARS-CoV as a model8,9,10, and one recent pre-print described the use of a microarray of overlapping peptides to assess linear epitopes in 10 COVID-19 patients11.

Five regions on the S glycoprotein of SARS-CoV (residues 274–306, 510–586, 587–628, 784–803, and 870-893) were predicted to be associated with a robust immune response8, whereas other

studies reported candidate epitopes9,10 that require validation with patient samples. In this study, we report the antibody profiles of COVID-19 patients and the identification of two

immunodominant linear B-cell epitopes on the S glycoprotein of SARS-CoV-2. Interestingly, using S glycoprotein pseudotyped lentiviruses, we demonstrate that antibodies recognising these two

linear epitopes account for a high proportion of the anti-spike response. These epitopes can potentially be used in the design of more sensitive serological assays for epidemiological or

vaccine efficiency assessments. RESULTS A SPIKE PSEUDOTYPED LENTIVIRUS ASSAY FOR VIRUS NEUTRALISATION To investigate whether a biosafety level (BSL) 2 approved pseudotyped lentivirus

expressing SARS-CoV-2 S glycoprotein tagged with a luciferase reporter could detect neutralising antibodies, we performed an initial screen at 1:1000 dilution using sera from 25 convalescent

COVID-19 patients and from 13 SARS patients recalled in January–February 2020 as controls (Fig. 1a). Majority of the COVID-19 patients’ sera were able to neutralise >50% of SARS-CoV-2

pseudovirus entry, whereas recalled SARS patients did not show neutralisation. To validate the absence of neutralisation from the 13 recalled SARS patients, we assessed their neutralisation

capacity at a lower dilution of 1:100 against the pseudotyped lentivirus expressing SARS-CoV S or SARS-CoV-2 S glycoproteins (Fig. 1b). The results indicate that these recalled SARS patients

still possess antibodies specific to SARS-CoV albeit at low levels, making them an appropriate control group for subsequent linear B-cell epitope mapping. Next, this assay was used to

determine the IC50 values of anti-SARS-CoV-2 S-neutralising antibodies from the sera of 41 convalescent COVID-19 patients (Fig. 1c). To further validate that this safer pseudotyped

lentivirus assay is representative of live SARS-CoV-2 virus neutralisation, we performed antibody neutralisation titrations for eight patients under BSL3 conditions. IC50 values obtained

were comparable, validating the lentivirus assay (Fig. 1d). Six patients (2, 5, 6, 7, 8, 9) with sufficient amount of sera and good neutralising capacity were then selected for further

characterisation. Notably, sera from these patients showed similar IC50 values ranging from 694 to 836, except for patient 9, who showed the strongest neutralising activity with an IC50

value of 1603 (Fig. 1c, Supplementary Table 4). TWO SPECIFIC LINEAR EPITOPES ON THE SARS-COV-2 S PROTEIN We next assessed the linear antigenic targets from sera of the six selected COVID-19

patients and five recalled SARS patients using a linear B-cell peptide library spanning the entire S glycoprotein of either SARS-CoV-2 or SARS-CoV, in pools of five overlapping peptides

(Fig. 2a, Supplementary Fig. 1). Interestingly, two distinct peptide pools from SARS-CoV-2 S library, pools S14 and S21, were strongly detected by sera from COVID-19 patients (Fig. 2a) and

not by recalled SARS patients or healthy control serum (Supplementary Fig. 1a). Two COVID-19 patients could detect SARS-CoV S library pool S51, which partially overlaps with SARS-CoV-2 pool

S21 (Fig. 2a, Supplementary Fig. 1b). This region encompasses the fusion peptide, which is highly conserved among coronaviruses12,13, suggesting a potential pan-SARS epitope at this

location. Further assessment of individual peptides within pools S14 and S21 narrowed down the specific region of interest to peptides S14P5 and S21P2, respectively (Fig. 2b). Recognition of

S14P5 and S21P2 was stronger for the peptides of SARS-CoV-2 than on the corresponding SARS-CoV peptides (Fig. 2c). The use of these peptides as potential detection epitopes for serology

assessment was further validated with 41 COVID-19 patients and 28 healthy donors (collected before the pandemic). Detection for both S14P5 and S21P2 was consistently and significantly higher

in COVID-19 patients (Fig. 2d). More importantly, the level of antibodies targeting these two specific peptides determined by enzyme-linked immunosorbent assay (ELISA) significantly

correlated with sera neutralising IC50 values (Fig. 2e), suggesting that antibodies directed at these epitopes could neutralise SARS-CoV-2. ANTIBODIES AGAINST S14P5 AND S21P2 CAN NEUTRALISE

SARS-COV-2 Using a recently published structure of SARS-CoV-2 S glycoprotein in the prefusion conformation, S14P5 was shown to localise in close proximity to the receptor binding domain

(RBD) (Fig. 3a), whereas S21P2 covers part of the fusion peptide (Fig. 3b). To assess the importance of these regions in controlling SARS-CoV-2 infection, antibody depletion assays were

performed against S14P5 and S21P2 (Fig. 3c). Depletion efficiency and specificity were validated by ELISA, and results showed that only antibodies against the respective peptides were

depleted (Fig. 3d). Interestingly, sera depleted for antibodies targeting either peptides S14P5, S21P2, or S14P5 + S21P2 significantly reduced the ability to neutralise SARS-CoV-2

pseudovirus infection, as compared with the non-depleted sera controls (Fig. 3e). Taken together, these results demonstrated that antibodies targeting these two linear epitopes account for a

significant fraction of the anti-S-neutralising response. DISCUSSION In this study, we identified two immunodominant linear B-cell epitopes, S14P5 and S21P2, on the SARS-CoV-2 S

glycoprotein, and further assessed the functional capacity of COVID-19 patient sera antibodies against these regions using a pseudotyped lentivirus assay. This assay uses safer

third-generation lentiviruses, which will greatly benefit the scientific community in allowing rapid and safer assessments and characterisation of neutralising antibody titres in patient

blood, and potential monoclonal antibodies (mAbs). Depletion assays functionally validated the positive correlation between antibody levels against these epitopes and neutralisation titres

against SARS-CoV-2 pseudotyped lentiviruses. Future studies will be needed to fully understand the role and neutralisation capacity of antibodies targeting these regions. Peptide S14P5 is

localised in close proximity to the RBD. As such, it is plausible that antibodies binding to this region may sterically hinder binding to the ACE2 receptor, thereby abolishing virus

infection14. Another possibility could be an allosteric effect on ACE2 binding. Supporting our results, the partial sequence of peptide S14P5 was computationally predicted to be

immunogenic8,10. Peptide S21P2 partially overlaps with an epitope identified in a recent pre-print11 and contains a part of the fusion peptide sequence (Fig. 3b). As such, alterations to

this region may potentially affect virus fusion. Indeed, targeting the SARS-CoV and MERS-CoV fusion peptide region was demonstrated to neutralise coronavirus infection with a pan-coronavirus

fusion inhibitor peptide15. Although our findings showed a robust IgG response against the two identified linear epitopes, it is plausible that they represent a small proportion of the

total anti-S antibody response6,7. Nevertheless, antibody depletion assays against S14P5 or S21P2 led to >20% reduction in pseudotyped lentivirus neutralisation, validating that

antibodies targeting these linear S regions are important for neutralising SARS-CoV-2 infection. Surprisingly, depletion of antibodies directed against both S14P5 and S21P2 did not

significantly decrease the neutralisation as compared with single depletions, suggesting that neutralisation at these regions is not synergistic. Future studies involve the isolation of mAbs

targeting these linear epitopes to allow proper quantification and comparison of peptide-specific IgG titres with antibodies directed against the RBD domain, or to other conformational

epitopes. It would be interesting to also assess the level of persistence of these antibodies against linear and other conformational epitopes. Interestingly, IgG levels against each peptide

correlated positively with the patient neutralisation IC50 values, suggesting that quantitative serological assays against these peptides could be used as a proxy for virus exposure status

as well as protection levels. However, this will require validation with other patient cohorts. Notably, the two identified epitopes present a low-to-moderate rate to impact mutations, which

would minimise the possibility of false negatives in serological assays (Supplementary Table 5)16. Together, these results will be essential to guide the design and evaluation of efficient

and specific serological assays against linear epitopes, as well as help prioritise vaccine target designs during this unprecedented crisis. METHODS ETHICS STATEMENT Written informed consent

was obtained from participants in accordance with the tenets of the Declaration of Helsinki. For COVID-19 serum/plasma collection “A Multi-centred Prospective Study to Detect Novel

Pathogens and Characterize Emerging Infections (The PROTECT study group)”, a domain specific review board (DSRB) evaluated the study design and protocol, which was approved under study

number 2012/00917. Serum/plasma collection of recalled SARS patients “Comparison of host immune responses to coronavirus infections” was approved by DSRB under study number 2020/00091. Sera

from healthy volunteers “Study of blood cell subsets and their products in models of infection, inflammation and immune regulation” was approved under study number 2017/2806. PATIENT SERUM

AND PLASMA FRACTIONS Serum was collected in BD Vacutainer SST II Advance tubes (Fisher Scientific, #12927696). After clotting, serum was separated using centrifugation for 10 minutes at 1000

rcf, and aliquoted before storing at −80 °C. Patient serum was heat-inactivated for 30 minutes at 56 °C before usage for this study. Plasma fraction was harvested after 20 minutes

centrifugation at 1700 rcf of blood collected in BD Vacutainer CPT tubes (BD, #362753). Plasma samples were treated by solvent/detergent treatment with a final concentration of 1% Triton

X-100 (Thermo Fisher Scientific, #28314) for virus inactivation at RT for 2 hours17. Information on selected patients is provided in Supplementary Table 1. Patient demographics and clinical

characteristics are described in Supplementary Table 2. LINEAR PEPTIDE LIBRARY The sequences used for the design of biotinylated linear peptides of the S glycoprotein of SARS-CoV and

SARS-CoV-2 are under GenBank accession numbers NC_004718.3 and MN908947.3. Preliminary epitope screening was used with a library of peptides (Mimotopes, Mulgrave, VIC, Australia) consisting

of 18‐mer overlapping sequences. Peptides were used individually or as pooled sets. Five to eight peptides were combined to form one pooled peptide set. Lyophilised individual peptides were

dissolved in 200 μL of DMSO (Sigma‐Aldrich, #D8418-100ML) to obtain a stock solution. PEPTIDE-BASED ELISA B-cell linear library ELISA was performed in a similar manner to a previously

established peptide-based screen18. In brief, streptavidin‐coated plates (Thermo Fisher Scientific, #15125) were blocked with 0.1% PBST (0.1% v/v Tween‐20, Sigma-Aldrich, #P1379-500ML, in

PBS, Gibco, #20012-043) containing 1% w/v sodium caseinate (Sigma‐Aldrich, #C8654-500G, lot BCBP6469) and 1% w/v bovine serum albumin (BSA; Sigma‐Aldrich, #A7030-500G, lot SLBW5033)

overnight at 4 °C, before addition of pooled or single biotinylated peptides at 1:1000 dilution in 0.1% PBST. Heat‐inactivated patient serum samples were added at 1:1000 dilution in 0.1%

PBST. Horseradish peroxidase-conjugated goat anti-human IgG (H + L) antibody (Jackson ImmunoResearch, #109-035-088, lot 139159) prepared in 10% blocking buffer was used for detection of

peptide‐bound antibodies. In total, 100 μL of TMB substrate (Sigma‐Aldrich, #T8665, lot SLCB5343) was used for a 5 minute development and was stopped by addition of 100 μL of 0.16 M sulfuric

acid prepared from 95% to 97% Sulfuric Acid stock solution (Merck, #1.00731.1000), prior to absorbance measurements. Absorbance was measured with the following parameters: 450 nm minus 690 

nm (bandwidth of 9 nm) in five flashes after a 10 second shaking at 1 mm amplitude on an Infinite M200 plate reader (Tecan, firmware V_2.02_11/06). PEPTIDES S14P5 AND S21P2 ELISA FOR 41

COVID-19 PATIENTS Owing to the limitation of available serum samples, ELISA was performed with 1% Triton X-100 (Thermo Fisher Scientific, #28314) treated plasma fractions. ELISA was

performed in similar conditions as described above with the following modifications. Nunc Maxisorp flat-bottom 96-well plates (Thermo Fisher Scientific, #442404) were coated overnight with

50 μL per well of 0.5 μg/mL of NeutrAvidin protein (Thermo Fisher Scientific, #31050). Blocking was performed for 1 hour with 0.01% polyvinyl alcohol (PVA; Sigma-Aldrich, #341584) in 0.1%

PBST (blocking buffer) prepared from stock of 0.5% PVA w/v in distilled H2O. Peptide coating was performed at 1:2000 dilution for 1 hour. Secondary antibody was incubated for 1 hour in

blocking buffer at 1:1000 dilution. Development was performed with 50 μL of TMB and stopped with 50 μL of 0.16 M sulfuric acid. PEPTIDE AFFINITY DEPLETION OF POOLED SERA Using principles

similar to previous work19,20, we performed affinity depletion as follows. Selected synthetic biotinylated peptides were added at 1:1000 dilution in 0.1% PBST to pre-blocked

streptavidin-coated plates and incubated at room temperature for 1 hour. Plates were washed three times with 0.1% PBST followed by PBS wash to remove traces of Tween-20. Pooled patient sera

were prepared at a dilution of 1:100 in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone, #SH30243.01, lot AE29431634), and 50 μL was added to each well and incubated for 20 minutes at room

temperature for adsorption. The unbound fraction was collected after 24 rounds of adsorption. ELISA analysis was performed as described above but at 1:2000 dilution to assess the levels of

peptide-specific antibodies before and after affinity depletion. Adsorbed samples were then mixed with lentiviruses pseudotyped with SARS-CoV-2 S protein as described below. Selected peptide

sequences are given in Supplementary Table 3. CELL LINES AND CELL CULTURE The human embryonic kidney epithelial cell 293T (ATCC, CRL-3216) and VERO E6 C1008 (ATCC CRL-1586 were cultured in

DMEM (Hyclone, #SH30022.01) supplemented with 10% heat-inactivated foetal bovine serum (FBS; Gibco, #10270-106). A stable cell line expressing human ACE2, CHO-ACE2 (a kind gift from

Professor Yee-Joo Tan, Department of Microbiology, NUS & IMCB, A*STAR, Singapore)21 was maintained in DMEM supplemented with 10% heat-inactivated FBS, 1% MEM non-essential amino acids

solution (Gibco, #11140-050) and 0.5 mg/mL of Geneticin Selective Antibiotic (Gibco, #10131-027). Every 2–3 days, cells were passaged by dissociating the cells with StemPro Accutase Cell

Dissociation Reagent (Gibco, #A1110501). ACE2 surface expression on CHO-ACE2 cells was verified using anti-human ACE2 AF647 (Santa Cruz Biotech, #sc-390851, lot B0320). Cells were routinely

tested for mycoplasma contamination. SARS-COV-2 AND SARS-COV PSEUDOTYPED LENTIVIRUS PRODUCTION Based on the third-generation lentivirus system, pseudotyped viral particles expressing

SARS-CoV or SARS-CoV-2 S proteins were produced by reverse transfection of 30 × 106 of 293 T cells with 12 µg pMDLg/pRRE (Addgene, #12251), 6 µg pRSV-Rev (Addgene, #12253), 12 µg

pTT5LnX-coV-SP (SARS-CoV-2 spike) or pXJ3’-S (SARS-CoV spike, a kind gift from Professor Yee-Joo Tan, Department of Microbiology, NUS & IMCB, A*STAR, Singapore)22 and 24 µg

pHIV-Luc-ZsGreen (Addgene, #39196) using Lipofectamine 2000 transfection reagent (Invitrogen, #11668-019) and cultured in a 37 °C incubator for 3 days. Viral supernatant was harvested, spun

down by centrifugation to remove cell debris and filtered through a 0.45 µm filter unit (Sartorius, #16555). Lenti-X p24 rapid titre kit (Takara Bio, #632200) was used to quantify the viral

titres following the manufacturer’s instructions. PSEUDOTYPED LENTIVIRUS NEUTRALISATION ASSAY CHO-ACE2 cells were seeded at a density of 2.5 × 104 cells in 100 µL of complete medium without

Geneticin in 96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates (Corning, #3904). After heat-inactivation at 56 °C for 30 minutes, serially diluted patient sera were

incubated in a 96-well flat-bottom cell culture plate (Costar, #3596) with an equal volume of pseudotyped virus (12 ng of p24) at the final volume of 50 μL at 37 °C for 1 h, and the mixture

was added to the monolayer of pre-seeded CHO-ACE2 cells. After 1 hour of pseudotyped viral infection at 37 °C, 150 µL of culture medium was added to each well and the cells were further

incubated for another 48 h. Upon removal of culture medium, cells were washed twice with sterile PBS, and then lysed in 20 µL of 1 × Passive lysis buffer (Promega, #E1941) with gentle

shaking at 400 rpm at 37 °C for 30 minutes. Luciferase activity was then assessed using Luciferase Assay System (Promega, #E1510) on a Promega GloMax Luminometer. LIVE SARS-COV-2

NEUTRALISATION ASSAY IN BSL3 Using a 96-well opaque (white) plate, 25 µL of 100 TCID50 of SARS-CoV-2 isolated from a naso-pharengeal swab of a patient in Singapore23, was incubated with 25 

µL of the indicated sera dilution for 1 hour at 37 °C with 5% CO2. After incubation, 50 µL of 4 × 105 cells/ml (VERO E6 C1008) was added into each well. The plate was subsequently incubated

for 4 days at 37 °C with 5% CO2. Cell viability was then determined using Viral ToxGlo Assay (Promega, #G8941). In brief, 100 µL of the reagent was added into each well and incubated for 10 

minutes at room temperature prior to measurement of luminescence readout using microplate reader (Tecan). DATA VISUALISATION AND STATISTICAL ANALYSIS Structural data of SARS-CoV-2 S protein

was retrieved from Protein Databank (PDB ID: 6VSB) in homotrimeric prefusion conformation and visualised using PyMOL (Schrodinger, version 2.2.0). Data were analysed using Excel for Mac

16.16.8 and GraphPad Prism for macOS version 8.4.1. Statistical tests are indicated in the figure legends. IC50 values of individual patients were calculated using the [Inhibitor] vs

response variable slope four parameter of GraphPad Prism, with negative values forced to zero. Correlation between pseudovirus IC50 and OD values were analysed using the non-parametric

Spearman correlation and straight line non-linear regression robust fit functions of Prism with data from Supplementary Table 4. For the neutralisation assay with non-depleted and depleted

pooled serum, one-sample _t_ test for each experiment was perform to assess if the values were significantly different from 100. REPORTING SUMMARY Further information on research design is

available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The source data underlying Figs. 1–3 and Supplementary Fig. 1 are provided as a Source Data file.

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SARS-CoV-2 in Singapore. _JAMA_. https://doi.org/10.1001/jama.2020.3204 (2020). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Professor Yee-Joo

Tan (Department of Microbiology, NUS; Institute of Molecular and Cell Biology—IMCB, A*STAR) who kindly provided CHO-ACE2 cells and pXJ3’-S plasmid. We acknowledge the excellent technical

work in the BSL3 facility of Wong Pui San (Wong P.S.) and Chye De Ho (Chye D.H.). We also like to thank the study participants who donated their blood samples to this project, and the

healthcare workers caring for COVID-19 patients. This work was supported by core research grants provided to Singapore Immunology Network by the Biomedical Research Council (BMRC), and by

the A*ccelerate GAP-funded project (ACCL/19-GAP064-R20H-H) from the Agency of Science, Technology and Research (A*STAR). Subject recruitment and sample collection were funded by the National

Medical Research Council (NMRC) COVID-19 Research fund (COVID19RF-001). AUTHOR INFORMATION Author notes * These authors contributed equally: Chek Meng Poh, Guillaume Carissimo, Bei Wang.

AUTHORS AND AFFILIATIONS * Singapore Immunology Network, Agency of Science, Technology and Research, Immunos, Biopolis, Singapore, 138648, Singapore Chek Meng Poh, Guillaume Carissimo, Bei

Wang, Siti Naqiah Amrun, Cheryl Yi-Pin Lee, Rhonda Sin-Ling Chee, Siew-Wai Fong, Nicholas Kim-Wah Yeo, Wen-Hsin Lee, Anthony Torres-Ruesta, Cheng-I Wang, Laurent Renia & Lisa F. P. Ng *

Department of Biological Science, National University of Singapore, Singapore, Singapore Siew-Wai Fong * Department of Biochemistry, Yong Loo Lin School of Medicine, National University of

Singapore, 8 Medical Drive, Singapore, 117596, Singapore Anthony Torres-Ruesta & Lisa F. P. Ng * National Centre for Infectious Diseases, 16 Jalan Tan Tock Seng, Singapore, 308442,

Singapore Yee-Sin Leo, Mark I-Cheng Chen, Barnaby Edward Young & David C. Lye * Department of Infectious Diseases, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore, 308433,

Singapore Yee-Sin Leo, Louis Yi Ann Chai, Barnaby Edward Young & David C. Lye * Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore, 308232,

Singapore Yee-Sin Leo, Barnaby Edward Young & David C. Lye * Yong Loo Lin School of Medicine, National University of Singapore and National University Health System, 10 Medical Drive,

Singapore, 117597, Singapore Yee-Sin Leo & David C. Lye * Saw Swee Hock School of Public Health, National University of Singapore and National University Health System, 12 Science Drive

2, #10-01, Singapore, 117549, Singapore Mark I-Cheng Chen * Department of Infectious Diseases, Changi General Hospital, 2 Simei Street 3, Singapore, 529889, Singapore Seow-Yen Tan *

Department of Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore, 119074, Singapore Louis Yi Ann Chai * Department of Infectious Diseases, Singapore General Hospital,

31 Third Hospital Ave, #03-03 Bowyer Block C, Singapore, 168753, Singapore Shirin Kalimuddin & Siew-Yee Thien * Emerging Infectious Disease Program, Duke-NUS Medical School, 8 College

Road, Singapore, 169857, Singapore Shirin Kalimuddin * Biological Defence Program, DSO National Laboratories, 27 Medical Drive, Singapore, 117510, Singapore Shirley Seah Gek Kheng & 

Brendon John Hanson * Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, 8 West Derby Street, Liverpool, L7 3EA, United Kingdom Lisa F. P. Ng

Authors * Chek Meng Poh View author publications You can also search for this author inPubMed Google Scholar * Guillaume Carissimo View author publications You can also search for this

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also search for this author inPubMed Google Scholar * Lisa F. P. Ng View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS C.M.P., G.C., S.N.A.,

C.Y.P.L. conceptualised, designed, acquired, analysed, interpreted the data and wrote the manuscript. B.W. acquired, analysed, interpreted the data, and wrote the manuscript. R.S.L.C.,

S.W.F., N.K.W.Y., S.W.H.L., S.S.G.K., and A.T.R. acquired and analysed the data. Y.S.L., M.I.C.C., S.Y. Thien, L.Y.A.C., S.K., S.Y. Tan, B.E.Y., and D.C.L. designed and supervised sample

collection. B.J.H., C.I.W., L.R., L.F.P.N. conceptualised, designed, analysed, and wrote the manuscript. All authors revised and approved the final version of the manuscript. CORRESPONDING

AUTHOR Correspondence to Lisa F. P. Ng. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature

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_et al._ Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients. _Nat Commun_ 11, 2806 (2020).

https://doi.org/10.1038/s41467-020-16638-2 Download citation * Received: 04 May 2020 * Accepted: 17 May 2020 * Published: 01 June 2020 * DOI: https://doi.org/10.1038/s41467-020-16638-2 SHARE

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