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Id Subject Object Predicate Lexical cue
T1 0-109 Sentence denotes Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2
T2 111-119 Sentence denotes Abstract
T3 120-130 Sentence denotes Background
T4 131-261 Sentence denotes A novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections.
T5 262-512 Sentence denotes While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002–2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2).
T6 513-627 Sentence denotes Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.
T7 629-632 Sentence denotes Aim
T8 633-772 Sentence denotes The cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.
T9 774-781 Sentence denotes Methods
T10 782-922 Sentence denotes The SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses.
T11 923-1150 Sentence denotes Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit.
T12 1151-1241 Sentence denotes Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.
T13 1243-1250 Sentence denotes Results
T14 1251-1401 Sentence denotes An immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses.
T15 1402-1531 Sentence denotes Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines.
T16 1532-1672 Sentence denotes In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.
T17 1674-1684 Sentence denotes Conclusion
T18 1685-1817 Sentence denotes The cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.
T19 1819-1831 Sentence denotes Introduction
T20 1832-2041 Sentence denotes The severe acute respiratory syndrome coronavirus (SARS-CoV-1), a virus considered to have a zoonotic origin, is the aetiological agent for the infectious disease, SARS, which first emerged in 2002–2003 [1,2].
T21 2042-2307 Sentence denotes In December of 2019, another novel coronavirus (SARS-CoV-2), which causes coronavirus disease (COVID-19), appeared to have crossed species barriers to infect humans and was effectively transmitted from person to person, leading to an outbreak in Wuhan, China [3-5].
T22 2308-2437 Sentence denotes This virus subsequently spread worldwide, leading the World Health Organization (WHO) to declare a pandemic on 11 March 2020 [6].
T23 2438-2614 Sentence denotes To date, SARS-CoV-2 continues to pose a high global health and economy burden, and as at 3 May 2020, COVID-19 had affected 215 countries with over 3.35 million confirmed cases.
T24 2615-2738 Sentence denotes To tackle the problems caused by SARS-CoV-2, improving its detection and knowledge of its infection mechanism is important.
T25 2739-2881 Sentence denotes In this respect, the viral surface spike glycoprotein (S protein) has been demonstrated to play key role in host cell selectivity and binding.
T26 2882-3138 Sentence denotes The S protein is functionally divided into two subunits, with the S1 subunit containing the receptor binding domain (RBD), which allows attachment to host cells, and the S2 subunit mediating fusion between viral and host membranes (reviewed by Li, F.) [7].
T27 3139-3309 Sentence denotes Phylogenetic analysis revealed that like SARS-CoV-1 and bat-derived SARS-like coronaviruses (SL-CoVs), SARS-CoV-2 belongs to lineage B of the betacoronavirus genus [8,9].
T28 3310-3530 Sentence denotes A study of 56 complete and partial SARS-CoV-2 genomes isolated from COVID-19 patients showed very high sequence conservation of more than 99%, indicating a recent introduction of the virus into the human population [10].
T29 3531-3666 Sentence denotes Although the animal source of SARS-CoV-2 is not clear, SARS-CoV-1 is believed to have originated from SL-CoVs residing in bats [11-14].
T30 3667-3818 Sentence denotes For the majority of SL-CoVs, the S1 subunit has low sequence identity to that of SARS-CoV-1, which suggests species-dependent receptor binding [14,15].
T31 3819-3995 Sentence denotes On the other hand, the high amino acid sequence identity of more than 90% in the S2 subunit suggests that the fusion mechanism during virus infection is well-conserved [14,15].
T32 3996-4200 Sentence denotes While SARS-CoV-2 shares higher whole-genome sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21 (88–89%) than with SARS-CoV-1 (79–82%), the RBD of SARS-CoV-2 is more similar to SARS-CoV-1 RBD [8,9].
T33 4201-4391 Sentence denotes In line with this, several research groups have demonstrated that SARS-CoV-2 utilises the same host receptor, angiotensin-converting enzyme 2 (ACE2), as SARS-CoV-1 for viral entry [3,16-18].
T34 4392-4508 Sentence denotes Due to its role in virus entry, the S protein has been the target for the generation of monoclonal antibodies (mAb).
T35 4509-4608 Sentence denotes In our previous work, we used five different fragments of SARS-CoV-1 S protein to immunise rabbits.
T36 4609-4762 Sentence denotes A fragment corresponding to residues 1029 to 1192 in the S2 subunit of SARS-CoV-1 was found to stimulate neutralising antibodies against SARS-CoV-1 [19].
T37 4763-4919 Sentence denotes This fragment was subsequently used to generate a panel of murine mAbs with their respective binding domains characterised and described in Lip et al. [20].
T38 4920-5211 Sentence denotes One of them, mAb 1A9, which binds to the S protein through a recently identified epitope within the S2 subunit at amino acids 1111–1130, has the ability to bind and cross-neutralise pseudotyped viruses expressing the S protein of human SARS-CoV-1, civet SARS-CoV and bat SL-CoV strains [21].
T39 5212-5460 Sentence denotes In this study, we aim to verify if the sequence of the immunogen used to generate mAb 1A9, as well as three other mAbs, is conserved in different coronaviruses and if these mAbs bind to the S protein of SARS-CoV-2 expressed in mammalian cell lines.
T40 5461-5686 Sentence denotes Importantly, mAb 1A9 is investigated for its ability to detect the S protein in SARS-CoV-2 infected cells and purified S protein in a sandwich ELISA format when paired with another mAb binding to the S1 subunit of SARS-CoV-2.
T41 5688-5695 Sentence denotes Methods
T42 5697-5702 Sentence denotes Cells
T43 5703-6086 Sentence denotes Vero E6 and COS-7 cells were purchased from the American Type Culture Collection (Manassas, VA, United States) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham MA, United States) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, United States), 100 units/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific).
T44 6087-6304 Sentence denotes 293FT cells were purchased from Invitrogen (Carlsbad, CA, United States) and grown in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin and 500 µg/mL geneticin (Thermo Fisher Scientific).
T45 6305-6348 Sentence denotes Cells were maintained at 37 °C with 5% CO2.
T46 6350-6389 Sentence denotes Purification of monoclonal antibody 1A9
T47 6390-6446 Sentence denotes The hybridoma for mAb 1A9 was previously generated [20].
T48 6447-6609 Sentence denotes All mAbs were purified from cell culture supernatants using HiTrap protein G HP affinity columns (GE Healthcare, Chicago, IL, United States) and stored at −80 °C.
T49 6610-6728 Sentence denotes The purity of the mAb was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis.
T50 6729-6856 Sentence denotes The concentration of the purified mAb was determined using the Coomassie Plus protein assay reagent (Thermo Fisher Scientific).
T51 6858-6917 Sentence denotes Plasmids for expression of full spike protein and fragments
T52 6918-7098 Sentence denotes SARS-CoV-2 S-protein-expressing plasmids were codon-optimised and generated by gene synthesis (Bio Basic Asia Pacific, Singapore) according to GenBank accession number: QHD43416.1.
T53 7099-7282 Sentence denotes One plasmid is for expressing untagged full-length S protein while the other is for expressing a Myc-tagged S-protein fragment consisting of residues 1048–1206 (SARS-CoV-2 numbering).
T54 7283-7471 Sentence denotes The pXJ40-Myc expression vector was used as an empty vector control and pXJ40-Myc-HBcAg plasmid expressing Myc-tagged hepatitis B virus core antigen (HBcAg) was used as a negative control.
T55 7473-7521 Sentence denotes Transient transfection and western blot analysis
T56 7522-7717 Sentence denotes 293FT cells were seeded onto 6-cm dishes 24 hours before transient transfection using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s protocol.
T57 7718-7858 Sentence denotes At 24 hours post-transfection, cells were harvested, spun down by centrifugation and washed with cold phosphate buffered saline (PBS) twice.
T58 7859-7937 Sentence denotes Cells were then resuspended in 2× Laemmli sample buffer, boiled and sonicated.
T59 7938-8140 Sentence denotes Clarified supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13,000 rpm at 4 °C to remove the cell debris and further analysed by western blot (WB) analysis.
T60 8141-8334 Sentence denotes Equal amounts of total cell lysates were loaded per lane and resolved using electrophoresis on SDS-PAGE gels and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA, United States).
T61 8335-8522 Sentence denotes The membrane was blocked in 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 hour at room temperature (RT) and incubated with primary antibodies at 4 °C overnight.
T62 8523-8691 Sentence denotes After the membrane was washed with TBST, it was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific) at RT for 1 hour.
T63 8692-8881 Sentence denotes The membrane was then washed with TBST again and bound antibodies visualised with enhanced chemiluminescence substrate (Thermo Fisher Scientific) using ChemiDoc MP Imaging System (Bio-Rad).
T64 8883-8937 Sentence denotes Transient transfection and immunofluorescence analysis
T65 8938-9222 Sentence denotes For immunofluorescence (IF) analysis, COS-7 cells on glass coverslips were transfected as above and fixed at 24 hours post-transfection in 4% paraformaldehyde for 10 min at RT followed by permeabilisation with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States) for 5 min.
T66 9223-9298 Sentence denotes Fixed cells were then blocked with PBS containing 10% FBS for 30 min at RT.
T67 9299-9493 Sentence denotes Cells were immunolabelled for 1 hour at RT with the indicated murine mAb and 45 min with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, United States).
T68 9494-9693 Sentence denotes Immunolabelled coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and mounted using ProLong Gold Antifade Mountant (Molecular Probes, Eugene, OR, United States).
T69 9694-9872 Sentence denotes Images were acquired with Olympus CKX53 microscope using Olympus (Tokyo, Japan) LCAch N 20×/0.40 iPC objective lens and Olympus DP27 colour camera with Olympus cellSens software.
T70 9873-9946 Sentence denotes Each channel was collected separately, with images at 1024 × 1024 pixels.
T71 9948-9953 Sentence denotes ELISA
T72 9954-10064 Sentence denotes Whole ectodomain of SARS-CoV-2 S protein with His Tag (Sino Biological Inc., Beijing, China; catalogue number:
T73 10065-10287 Sentence denotes 40589-B08V1) was diluted with coating buffer (0.1 M NaHCO3, 34 mM Na2CO3) and a total of 20 ng of protein was loaded into individual wells of a 96 well plate (Nunc, Roskilde, Denmark) and allowed to coat overnight at 4 °C.
T74 10288-10522 Sentence denotes Plates were then washed four times with 0.05% Tween 20 in PBS (PBST) and blocked with 5% bovine serum albumin (BSA)/PBST for 30 min before murine antibodies serially diluted with blocking buffer were added to desired wells for 1 hour.
T75 10523-10739 Sentence denotes Plate were washed four times with PBST before incubation for 1 hour with HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) secondary antibodies diluted in blocking buffer, and washed four times with PBST.
T76 10740-10973 Sentence denotes Visualisation of bound secondary antibodies was done by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) for 5 min in the absence of light and the reaction was stopped with 2 M sulphuric acid.
T77 10974-11188 Sentence denotes Optical density at 450 nm (OD450nm) was determined by a Tecan (Männedorf, Switzerland) Infinite M1000 reader and normalised OD450nm was obtained by subtracting background absorbances determined in BSA coated wells.
T78 11190-11230 Sentence denotes Production of monoclonal antibody CR3022
T79 11231-11315 Sentence denotes The human mAb CR3022 was expressed in a similar manner as previously described [22].
T80 11316-11637 Sentence denotes The variable heavy (VH; GenBank accession number: DQ168569) and variable light (VL; GenBank accession number: DQ168570) genes of CR3022 were generated by gene synthesis (Bio Basic Asia Pacific) and cloned into pFUSEss-CHIg-hIgG1 and pFUSE2ss-CLIg-hK cloning vectors (InvivoGen, San Diego, CA, United States) respectively.
T81 11638-11824 Sentence denotes Transfection of suspension FreeStyle 293 cells (Thermo Fisher Scientific) and purification of antibodies by fast protein liquid chromatography is as described in our previous study [23].
T82 11826-11840 Sentence denotes Sandwich ELISA
T83 11841-12023 Sentence denotes Mab 1A9 was diluted with coating buffer (0.1 M NaHCO3, 34 mM Na2CO3) and 0.1 µg of antibody was coated onto individual wells of a Maxisorp flat-bottom plate (Nunc) overnight at 4 °C.
T84 12024-12130 Sentence denotes The plate was washed three times with PBST before blocking was done using 5% BSA/PBST at 37 °C for 60 min.
T85 12131-12228 Sentence denotes Dilutions of His-tagged full length SARS-CoV-2 S protein (Sino Biological Inc., catalogue number:
T86 12229-12305 Sentence denotes 40589-B08V1) and His-tagged H7N7-HA (Sino Biological Inc., catalogue number:
T87 12306-12415 Sentence denotes 11082-V08B) were added to desired wells and incubated at 37 °C for 90 min followed by three washes with PBST.
T88 12416-12645 Sentence denotes 100 µL of CR3022 antibody was added at a concentration of 1 µg/mL and incubated at 37 °C for 60 min followed by three PBST washes before HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific) was added for 60 min at 37 °C.
T89 12646-12771 Sentence denotes Finally, after three PBST washes, TMB (Sigma-Aldrich) was added for 5 min and the reaction was stopped by 2 M sulphuric acid.
T90 12772-12832 Sentence denotes The OD450nm was determined by a Tecan Infinite M1000 reader.
T91 12833-13021 Sentence denotes Statistical analyses were performed using an unpaired, one-tailed Student’s t-test with Welch’s correction for unequal variances. p values < 0.05 were considered statistically significant.
T92 13023-13061 Sentence denotes Virus infection and immunofluorescence
T93 13062-13180 Sentence denotes All works with live virus were performed in the biosafety level (BSL)3 facility at the Public Health Agency of Sweden.
T94 13181-13382 Sentence denotes Vero-E6 cells were infected with SARS-CoV-2 (SARS-CoV-2-Iso/01/human/2020/SWE; GenBank accession number: MT093571) at a multiplicity of infection (MOI) of one in DMEM 2% FBS (Thermo Fisher Scientific).
T95 13383-13501 Sentence denotes At 24 hour post-infection, cells were fixed with chilled methanol/acetone and the cells were kept at −20 °C overnight.
T96 13502-13628 Sentence denotes Cells were then stained using mAb 1A9 at 5 µg/mL at 37 °C for 30 min in IF buffer (BSA 0.2%, Triton ×100 0.1% in PBS, pH 7.4).
T97 13629-13842 Sentence denotes The cells were washed three times with PBS and incubated, subsequently with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) in IF buffer containing DAPI for an additional 30 min.
T98 13843-13953 Sentence denotes Cells were washed three times with PBS before visualisation and image acquisition with fluorescent microscopy.
T99 13955-13978 Sentence denotes Bioinformatics analysis
T100 13979-14228 Sentence denotes S protein reference sequences for SARS-CoV-1, SARS-CoV-2, batRaTG13, Middle East respiratory syndrome (MERS) and human common-cold coronaviruses 229E, NL63, OC43 and HKU1 were downloaded from the National Center for Biotechnology Information (NCBI).
T101 14229-14510 Sentence denotes A multiple sequence alignment was created with multiple alignment using fast Fourier transform (MAFFT) using the slow but accurate L-INS-I parameter settings [24] and the alignment curated, cut to the target region 1029–1192 (SARS-CoV-1 numbering) and visualised with Jalview [25].
T102 14511-14838 Sentence denotes We used Molecular Evolutionary Genetics Analysis (MEGA) X [26] to calculate the number of amino-acid differences for all sequence pairs in the alignment of the mAb target region and the full S protein normalised by the length of the aligned sequence of the respective reference protein to obtain per cent amino acid identities.
T103 14839-15034 Sentence denotes To determine SARS-CoV-2 sequence diversity in the S protein within the current pandemic, 230 human and environmental viral sequences were downloaded from GISAID’s EpiCoV database on 1 March 2020.
T104 15035-15175 Sentence denotes We gratefully acknowledge the authors, originating and submitting laboratories of the sequences on which this part of the research is based.
T105 15176-15222 Sentence denotes The list is detailed in Supplementary Table 1.
T106 15223-15334 Sentence denotes The nt sequences were searched with basic local alignment search tool (BLAST)X against the reference S protein.
T107 15335-15488 Sentence denotes 174 hits covered the full length of the S protein and amino-acid mutations were counted and tabulated using a custom Perl script (Supplementary Table 2).
T108 15490-15507 Sentence denotes Ethical statement
T109 15508-15557 Sentence denotes Ethical approval was not required for this study.
T110 15559-15566 Sentence denotes Results
T111 15568-15692 Sentence denotes An immunogenic domain in the S2 subunit of SARS-CoV-1 is highly conserved in SARS-CoV-2 but not in MERS and common cold HCoV
T112 15693-15911 Sentence denotes Sequence alignment of the S2 fragment corresponding to residues 1029 to 1192 shows that this fragment, which encompasses the heptad repeat (HR)2 but not HR1, is highly conserved in SARS-CoV-1 and SARS-CoV-2 (Figure 1).
T113 15912-16388 Sentence denotes When compared with additional reference sequences from bat RaTG13 (closest bat precursor), MERS and human common cold coronaviruses 229E, NL63, OC43 and HKU1 (Figure 1), it becomes apparent that the amino-acid identity between SARS-CoV-2 and SARS-CoV-1 is much higher in this region (93%, Table) than over the full protein length (78%, Table) and the similarity drops sharply (< 40% in this region) when considering MERS and the other coronaviruses infecting humans regularly.
T114 16389-16521 Sentence denotes Figure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses
T115 16522-16527 Sentence denotes MERS:
T116 16528-16659 Sentence denotes Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.
T117 16660-16832 Sentence denotes The name of the viruses, for which sequences are being compared figure on the left side of the alignment, together with the respective sequences’ GenBank accession numbers.
T118 16833-17077 Sentence denotes Colour schemes represent the following categories of amino acids: blue – hydrophobic, cyan – aromatic, green – polar, magenta – negative charge, orange – glycines, pink – cysteines, red – positive charge, yellow – prolines, white – unconserved.
T119 17078-17290 Sentence denotes Table Pairwise amino-acid identity across relevant coronaviruses in the sequence fragment of the spike glycoprotein S2 subunit recognised by monoclonal antibody 1A9 or the sequence of the full spike glycoprotein
T120 17291-17340 Sentence denotes Query/reference Pairwise amino-acid identity (%)
T121 17341-17404 Sentence denotes SARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63
T122 17405-17432 Sentence denotes Fragment region of spike S2
T123 17433-17479 Sentence denotes SARS-Co-V2 100.00 SB SB SB SB SB SB SB
T124 17480-17528 Sentence denotes BatRaTG13 99.40 100.00 SB SB SB SB SB SB
T125 17529-17575 Sentence denotes SARS 93.10 92.50 100.00 SB SB SB SB SB
T126 17576-17625 Sentence denotes MERS 39.00 39.00 39.00 100.00 SB SB SB SB
T127 17626-17678 Sentence denotes OC43 39.00 39.00 38.40 51.20 100.00 SB SB SB
T128 17679-17734 Sentence denotes HKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB
T129 17735-17793 Sentence denotes 229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB
T130 17794-17855 Sentence denotes NL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00
T131 17856-17874 Sentence denotes Full spike protein
T132 17875-17921 Sentence denotes SARS-CoV-2 100.00 SB SB SB SB SB SB SB
T133 17922-17970 Sentence denotes BatRaTG13 97.70 100.00 SB SB SB SB SB SB
T134 17971-18023 Sentence denotes SARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB
T135 18024-18073 Sentence denotes MERS 35.40 35.40 35.20 100.00 SB SB SB SB
T136 18074-18126 Sentence denotes OC43 37.30 37.10 36.90 39.50 100.00 SB SB SB
T137 18127-18182 Sentence denotes HKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB
T138 18183-18241 Sentence denotes 229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB
T139 18242-18303 Sentence denotes NL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00
T140 18304-18309 Sentence denotes MERS:
T141 18310-18460 Sentence denotes Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below.
T142 18461-18574 Sentence denotes High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.
T143 18575-18854 Sentence denotes The sequence identity is not affected by the order in which paired sequences are compared so only one-way comparisons are shown to avoid redundancies; the abbreviation ‘SB’ is used when the pairwise amino-acid identity in question is already shown in a further cell of the table.
T144 18855-18989 Sentence denotes We also studied the sequence diversity across 174 SARS-CoV-2 S proteins derived from nt sequences shared via the GISAID platform [27].
T145 18990-19156 Sentence denotes Only four amino-acid mutations were found within the putative antibody-binding region compared with 30 mutations over the full length protein (Supplementary Table 2).
T146 19157-19300 Sentence denotes Two of these four amino-acid mutations are from a sequence flagged in GISAID’s EpiCoV database as lower quality due to many undetermined bases.
T147 19302-19389 Sentence denotes Four murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2
T148 19390-19582 Sentence denotes Four mAbs with distinct binding profiles to SARS-CoV-1, as previously mapped by internal deletion mutagenesis study, were selected for testing to determine if they cross-react with SARS-CoV-2.
T149 19583-19789 Sentence denotes A fragment containing residues 1048 to 1206 of SARS-CoV-2 S protein was expressed in 293FT cells via transient transfection and WB analysis was performed using the four mAbs, namely 2B2, 1A9, 4B12 and 1G10.
T150 19790-19931 Sentence denotes As shown in Figure 2A, all four mAbs detected this fragment of SARS-CoV-2, which is consistent with the sequence alignment shown in Figure 1.
T151 19932-20018 Sentence denotes Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead.
T152 20019-20165 Sentence denotes IF analysis performed on transiently transfected COS-7 cells showed binding of the four mAbs to this S protein fragment of SARS-CoV-2 (Figure 2B).
T153 20166-20321 Sentence denotes These interactions are also specific for the SARS-COV-2 S protein (1048–1206) fragment as all four mAbs did not show binding to the negative control HBcAg.
T154 20322-20540 Sentence denotes Figure 2 Monoclonal antibodies expected to target a SARS-CoV-2 S protein S2 fragment, (A) hybridise to the peptide fragment in western blot and (B) recognise cells expressing the peptide as shown by immunofluorescence
T155 20541-20601 Sentence denotes aa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI:
T156 20602-20785 Sentence denotes 4′,6-diamidino-2-phenylindole; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; S protein: spike protein; vec: pXJ40-Myc expression vector used as an empty vector control.
T157 20786-20788 Sentence denotes A.
T158 20789-20900 Sentence denotes Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10).
T159 20901-21175 Sentence denotes Empty vector-transfected 293FT cell products are on the lanes labelled as ‘Vec’, while products of 293FT cells expressing Myc-tagged SARS-CoV-2 S protein fragment (aa 1048–1206; SARS-CoV-2 numbering) or Myc-tagged HBcAg are on the lanes respectively labelled ‘S’ or ‘HBcAg’.
T160 21176-21269 Sentence denotes Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies.
T161 21270-21425 Sentence denotes A ladder indicative of the molecular weights in kD of the proteins relative to their vertical position on the blots, is indicated on the left of the panel.
T162 21426-21428 Sentence denotes B.
T163 21429-21584 Sentence denotes Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10).
T164 21585-21868 Sentence denotes Immunofluorescence assay results of COS7 cells expressing Myc-tagged HBcAg (top photos) or a Myc-tagged fragment (aa1048–1206) of SARS-CoV-2 S protein (bottom photos) using the indicated primary antibodies, followed by Alexa Fluor 488-conjugated secondary antibody (green) are shown.
T165 21869-21918 Sentence denotes Cell nuclei were counterstained with DAPI (blue).
T166 21919-21937 Sentence denotes Scale bar = 50 µm.
T167 21939-22020 Sentence denotes Four murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2
T168 22021-22170 Sentence denotes Next, the full-length S protein of SARS-CoV-2 was overexpressed in 293FT and COS-7 cells and detected with each of the mAbs using WB and IF analyses.
T169 22171-22268 Sentence denotes As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).
T170 22269-22497 Sentence denotes Figure 3 Antibodies expected to target SARS-CoV-2 S protein, (A) hybridise to the denatured protein in western blot, (B) bind to the protein in ELISA and (C) recognise cells expressing the protein as shown by immunofluorescence
T171 22498-22503 Sentence denotes DAPI:
T172 22504-22734 Sentence denotes 4′,6-diamidino-2-phenylindole; mAb: monoclonal antibody: OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; S protein: spike protein; vec: pXJ40-Myc expression vector used as an empty vector control.
T173 22735-22737 Sentence denotes A.
T174 22738-22844 Sentence denotes Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10).
T175 22845-23041 Sentence denotes Empty vector-transfected 293FT cell products are on the lanes labelled as ‘Vec’, while products of 293FT cells expressing full-length SARS-CoV-2 S protein are on the lanes labelled ‘SARS-CoV-2 S’.
T176 23042-23135 Sentence denotes Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies.
T177 23136-23291 Sentence denotes A ladder indicative of the molecular weights in kD of the proteins relative to their vertical position on the blots, is indicated on the left of the panel.
T178 23292-23294 Sentence denotes B.
T179 23295-23414 Sentence denotes The abilities of 2B2, 1A9, 4B12 and 1G10 monoclonal antibodies to bind to SARS-CoV-2 S protein was determined by ELISA.
T180 23415-23533 Sentence denotes Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated.
T181 23534-23715 Sentence denotes A representative plot from three independent experiment is show for each antibody and error bars correspond to standard deviations of each mAb experiment carried out in triplicates.
T182 23716-23718 Sentence denotes C.
T183 23719-23874 Sentence denotes Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10).
T184 23875-24040 Sentence denotes Immunofluorescence analysis was performed on empty vector-transfected COS-7 cells (top photos) and cells expressing full-length SARS-CoV-2 S protein (bottom photos).
T185 24041-24150 Sentence denotes The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green).
T186 24151-24200 Sentence denotes Cell nuclei were counterstained with DAPI (blue).
T187 24201-24219 Sentence denotes Scale bar = 50 µm.
T188 24220-24388 Sentence denotes The binding of these mAbs to recombinant purified S protein was also determined using indirect ELISA where different concentrations of antibodies were used for binding.
T189 24389-24492 Sentence denotes Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B).
T190 24493-24598 Sentence denotes Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C).
T191 24599-24732 Sentence denotes Collectively our data demonstrates the ability of all four mAbs to bind full-length S protein in both its native and denatured forms.
T192 24734-24855 Sentence denotes Utility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells
T193 24856-24973 Sentence denotes Based on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs.
T194 24974-25126 Sentence denotes Hence, a sandwich ELISA was performed to determine if it can be paired with the human mAb CR3022 which is known to bind to the S1 subunit of SARS-CoV-2.
T195 25127-25292 Sentence denotes As shown in Figure 4A, recombinant S protein was detected at 15.6 ng/mL and above when 1A9 was used as a capture antibody and CR3022 was used as a detector antibody.
T196 25293-25436 Sentence denotes Since the S protein was His-tagged, a His-tagged haemagglutinin (HA) protein of influenza A virus was used to check for specificity of binding.
T197 25437-25599 Sentence denotes The absorbance readings in the presence of S protein were significantly higher than that in the presence of HA for protein concentrations of 15.6 ng/mL and above.
T198 25600-25740 Sentence denotes Figure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells
T199 25741-25926 Sentence denotes HA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.
T200 25927-25929 Sentence denotes A.
T201 25930-26073 Sentence denotes Sandwich ELISA assay to determine mAb 1A9 ability to pair with the human mAb CR3022 for the detection of a His-tagged SARS-CoV-2 spike protein.
T202 26074-26147 Sentence denotes 1A9 and CR3022 were used as capture and detector antibodies respectively.
T203 26148-26229 Sentence denotes His-tagged HA protein of influenza A (H7N7) virus was used as a negative control.
T204 26230-26297 Sentence denotes Averaged readings across three replicate experiments are presented.
T205 26298-26378 Sentence denotes Error bars represent standard deviations across the three replicate experiments.
T206 26379-26522 Sentence denotes Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p < 0.05.
T207 26523-26525 Sentence denotes B.
T208 26526-26624 Sentence denotes Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1).
T209 26625-26764 Sentence denotes At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green).
T210 26765-26840 Sentence denotes Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue).
T211 26841-26899 Sentence denotes Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells.
T212 26900-27108 Sentence denotes As shown in Figure 4B, mAb 1A9 stained a considerable number of SARS-CoV-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of S protein during infection.
T213 27110-27120 Sentence denotes Discussion
T214 27121-27237 Sentence denotes Numerous mAbs against the S protein of SARS-CoV-1 have been generated for research and diagnostic assay development.
T215 27238-27375 Sentence denotes Some of these may be able to cross-react with the S protein of SARS-CoV-2 and serve as tools to aid research on this newly emerged virus.
T216 27376-27541 Sentence denotes In this current study, an immunogenic domain in the S2 subunit of SARS-CoV-1 was found to be highly conserved in multiple strains of SARS-CoV-2 (Figure 1 and Table).
T217 27542-27720 Sentence denotes Consistently, WB and IF analyses showed that four different mAbs generated using this SARS-CoV-1 domain were cross-reactive against the S protein of SARS-CoV-2 (Figures 2 and 3).
T218 27721-27846 Sentence denotes Recent cross-reactivity studies have evaluated SARS-CoV-1 neutralising antibodies that bind to the RBD-containing S1 subunit.
T219 27847-28019 Sentence denotes Although both SARS-CoV-1 and SARS-CoV-2 use ACE2 as a receptor for viral entry [3,16], several SARS-CoV-1 RBD-directed mAbs did not cross-react with SARS-CoV-2 RBD [28,29].
T220 28020-28223 Sentence denotes Interestingly, CR3022, which was isolated from a SARS convalescent patient [22], showed cross-reactivity to SARS-CoV-2 RBD and recognises an epitope that does not overlap with the ACE2 binding site [28].
T221 28224-28356 Sentence denotes Among the four mAbs tested in this study, indirect ELISA showed that 1A9 binds strongest to the S protein of SARS-CoV-2 (Figure 3B).
T222 28357-28524 Sentence denotes To determine if 1A9 is useful for detection of S protein in a sandwich ELISA, it was paired with CR3022 since 1A9 binds to S2 subunit while CR3022 binds to S1 subunit.
T223 28525-28630 Sentence denotes As would be expected, these two antibodies can be paired to detect S protein from 15.6 ng/mL (Figure 4A).
T224 28631-28841 Sentence denotes In addition, mAb 1A9 stained a considerable number of SARS-CoV-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of S protein during infection (Figure 4B).
T225 28842-28983 Sentence denotes Thus, mAbs 1A9 will be useful for studying the kinetics of SARS-CoV-2 replication in vitro and development of diagnostic assays for COVID-19.
T226 28984-29131 Sentence denotes It is noteworthy that cytotoxic T-lymphocyte (CTL) epitopes also reside at residues 884–891 and 1116–1123 within the S2 subunit of SARS-CoV-1 [30].
T227 29132-29223 Sentence denotes Interestingly, the latter CTL epitope overlaps with the epitope recognised by mAb 1A9 [21].
T228 29224-29374 Sentence denotes Hence, the S2 subunit may serve as an important antigen for inducing both humoral as well as cell-mediated immunity against SARS-CoV-1 and SARS-CoV-2.
T229 29375-29594 Sentence denotes To our knowledge, this is the first study showing that mAbs targeting the S2 domain of SARS-CoV-1 can cross-react with SARS-CoV-2 and this observation is consistent with the high sequence conservation in the S2 subunit.
T230 29595-29801 Sentence denotes The ability of these antibodies, particularly 1A9, to detect SARS-CoV-2 S protein in indirect and sandwich ELISAs demonstrate their utility for detection of SARS-CoV-2 infections in a public health setting.
T231 29802-30022 Sentence denotes Whether or not the current sensitivity of these antibodies are sufficient for robust detection of SARS-CoV-2 infections in a clinical setting and how they compare to existing PCR-based detection remains to be determined.
T232 30023-30170 Sentence denotes Successful development of these antibodies into a point of care diagnostic kit will provide a complementary approach to existing detection methods.
T233 30171-30306 Sentence denotes Besides the mAbs characterised here, several other mAbs have been reported to bind to epitopes in the S2 subunit of SARS-CoV-1 [31-33].
T234 30307-30398 Sentence denotes Thus, it will be important to determine if these mAbs can also cross-react with SARS-CoV-2.
T235 30400-30416 Sentence denotes Acknowledgements
T236 30417-30569 Sentence denotes The work performed in NUS/NUHS was supported by NUHS Research Office under Project Number NUHSRO/2020/033/RO5+5/CORONAVIRUS/LOA (WBS R-571-000-071-733).
T237 30570-30712 Sentence denotes The work performed in IMCB and BII was also supported by A*STAR through intramural funding and an A*CRUSE gap funding (ACCL/19-GAP064-R20H-F).
T238 30714-30887 Sentence denotes Conflict of interest: WJH and YJT declare that they are involved in the licensing of mAbs 1A9, 1G10, 4B12 and 2B2 to commercial companies as research or diagnostic reagents.
T239 30888-30954 Sentence denotes The other authors have declared that no competing interests exist.
T240 30955-30978 Sentence denotes Authors’ contributions:
T241 30979-31084 Sentence denotes Zheng Z – Designed experiments, performed experiments, analysed and organised data, wrote the manuscript.
T242 31085-31194 Sentence denotes Monteil, VM - Designed experiments, performed experiments, analysed and organised data, wrote the manuscript.
T243 31195-31308 Sentence denotes Maurer-Stroh, S - Designed experiments, performed experiments, analysed and organised data, wrote the manuscript.
T244 31309-31362 Sentence denotes Yew CW - Designed experiments, performed experiments.
T245 31363-31417 Sentence denotes Leong C - Designed experiments, performed experiments.
T246 31418-31530 Sentence denotes Mohd-Ismail NK - Designed experiments, performed experiments, analysed and organised data, wrote the manuscript.
T247 31531-31607 Sentence denotes Cheyyatraivendran Arularasu S - Designed experiments, performed experiments.
T248 31608-31647 Sentence denotes Chow VTK – Analysed and organised data.
T249 31648-31686 Sentence denotes Lin RTP – Analysed and organised data.
T250 31687-31794 Sentence denotes Mirazimi A– Designed experiments, performed experiments, analysed and organised data, wrote the manuscript.
T251 31795-31832 Sentence denotes Hong WJ- Analysed and organised data.
T252 31833-31936 Sentence denotes Tan YJ- Designed experiments, performed experiments, analysed and organised data, wrote the manuscript.
T253 31938-31956 Sentence denotes Supplementary Data
T254 31957-31978 Sentence denotes Supplementary Table 1
T255 31979-32015 Sentence denotes Click here for additional data file.
T256 32017-32035 Sentence denotes Supplementary Data
T257 32036-32057 Sentence denotes Supplementary Table 2
T258 32058-32094 Sentence denotes Click here for additional data file.