LitCovid-sentences  

PMC:7376845 / 1819-30398 JSONTXT 13 Projects

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Id Subject Object Predicate Lexical cue
T19 0-12 Sentence denotes Introduction
T20 13-222 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 223-488 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 489-618 Sentence denotes This virus subsequently spread worldwide, leading the World Health Organization (WHO) to declare a pandemic on 11 March 2020 [6].
T23 619-795 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 796-919 Sentence denotes To tackle the problems caused by SARS-CoV-2, improving its detection and knowledge of its infection mechanism is important.
T25 920-1062 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 1063-1319 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 1320-1490 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 1491-1711 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 1712-1847 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 1848-1999 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 2000-2176 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 2177-2381 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 2382-2572 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 2573-2689 Sentence denotes Due to its role in virus entry, the S protein has been the target for the generation of monoclonal antibodies (mAb).
T35 2690-2789 Sentence denotes In our previous work, we used five different fragments of SARS-CoV-1 S protein to immunise rabbits.
T36 2790-2943 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 2944-3100 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 3101-3392 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 3393-3641 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 3642-3867 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 3869-3876 Sentence denotes Methods
T42 3878-3883 Sentence denotes Cells
T43 3884-4267 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 4268-4485 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 4486-4529 Sentence denotes Cells were maintained at 37 °C with 5% CO2.
T46 4531-4570 Sentence denotes Purification of monoclonal antibody 1A9
T47 4571-4627 Sentence denotes The hybridoma for mAb 1A9 was previously generated [20].
T48 4628-4790 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 4791-4909 Sentence denotes The purity of the mAb was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis.
T50 4910-5037 Sentence denotes The concentration of the purified mAb was determined using the Coomassie Plus protein assay reagent (Thermo Fisher Scientific).
T51 5039-5098 Sentence denotes Plasmids for expression of full spike protein and fragments
T52 5099-5279 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 5280-5463 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 5464-5652 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 5654-5702 Sentence denotes Transient transfection and western blot analysis
T56 5703-5898 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 5899-6039 Sentence denotes At 24 hours post-transfection, cells were harvested, spun down by centrifugation and washed with cold phosphate buffered saline (PBS) twice.
T58 6040-6118 Sentence denotes Cells were then resuspended in 2× Laemmli sample buffer, boiled and sonicated.
T59 6119-6321 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 6322-6515 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 6516-6703 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 6704-6872 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 6873-7062 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 7064-7118 Sentence denotes Transient transfection and immunofluorescence analysis
T65 7119-7403 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 7404-7479 Sentence denotes Fixed cells were then blocked with PBS containing 10% FBS for 30 min at RT.
T67 7480-7674 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 7675-7874 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 7875-8053 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 8054-8127 Sentence denotes Each channel was collected separately, with images at 1024 × 1024 pixels.
T71 8129-8134 Sentence denotes ELISA
T72 8135-8245 Sentence denotes Whole ectodomain of SARS-CoV-2 S protein with His Tag (Sino Biological Inc., Beijing, China; catalogue number:
T73 8246-8468 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 8469-8703 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 8704-8920 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 8921-9154 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 9155-9369 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 9371-9411 Sentence denotes Production of monoclonal antibody CR3022
T79 9412-9496 Sentence denotes The human mAb CR3022 was expressed in a similar manner as previously described [22].
T80 9497-9818 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 9819-10005 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 10007-10021 Sentence denotes Sandwich ELISA
T83 10022-10204 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 10205-10311 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 10312-10409 Sentence denotes Dilutions of His-tagged full length SARS-CoV-2 S protein (Sino Biological Inc., catalogue number:
T86 10410-10486 Sentence denotes 40589-B08V1) and His-tagged H7N7-HA (Sino Biological Inc., catalogue number:
T87 10487-10596 Sentence denotes 11082-V08B) were added to desired wells and incubated at 37 °C for 90 min followed by three washes with PBST.
T88 10597-10826 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 10827-10952 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 10953-11013 Sentence denotes The OD450nm was determined by a Tecan Infinite M1000 reader.
T91 11014-11202 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 11204-11242 Sentence denotes Virus infection and immunofluorescence
T93 11243-11361 Sentence denotes All works with live virus were performed in the biosafety level (BSL)3 facility at the Public Health Agency of Sweden.
T94 11362-11563 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 11564-11682 Sentence denotes At 24 hour post-infection, cells were fixed with chilled methanol/acetone and the cells were kept at −20 °C overnight.
T96 11683-11809 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 11810-12023 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 12024-12134 Sentence denotes Cells were washed three times with PBS before visualisation and image acquisition with fluorescent microscopy.
T99 12136-12159 Sentence denotes Bioinformatics analysis
T100 12160-12409 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 12410-12691 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 12692-13019 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 13020-13215 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 13216-13356 Sentence denotes We gratefully acknowledge the authors, originating and submitting laboratories of the sequences on which this part of the research is based.
T105 13357-13403 Sentence denotes The list is detailed in Supplementary Table 1.
T106 13404-13515 Sentence denotes The nt sequences were searched with basic local alignment search tool (BLAST)X against the reference S protein.
T107 13516-13669 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 13671-13688 Sentence denotes Ethical statement
T109 13689-13738 Sentence denotes Ethical approval was not required for this study.
T110 13740-13747 Sentence denotes Results
T111 13749-13873 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 13874-14092 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 14093-14569 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 14570-14702 Sentence denotes Figure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses
T115 14703-14708 Sentence denotes MERS:
T116 14709-14840 Sentence denotes Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.
T117 14841-15013 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 15014-15258 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 15259-15471 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 15472-15521 Sentence denotes Query/reference Pairwise amino-acid identity (%)
T121 15522-15585 Sentence denotes SARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63
T122 15586-15613 Sentence denotes Fragment region of spike S2
T123 15614-15660 Sentence denotes SARS-Co-V2 100.00 SB SB SB SB SB SB SB
T124 15661-15709 Sentence denotes BatRaTG13 99.40 100.00 SB SB SB SB SB SB
T125 15710-15756 Sentence denotes SARS 93.10 92.50 100.00 SB SB SB SB SB
T126 15757-15806 Sentence denotes MERS 39.00 39.00 39.00 100.00 SB SB SB SB
T127 15807-15859 Sentence denotes OC43 39.00 39.00 38.40 51.20 100.00 SB SB SB
T128 15860-15915 Sentence denotes HKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB
T129 15916-15974 Sentence denotes 229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB
T130 15975-16036 Sentence denotes NL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00
T131 16037-16055 Sentence denotes Full spike protein
T132 16056-16102 Sentence denotes SARS-CoV-2 100.00 SB SB SB SB SB SB SB
T133 16103-16151 Sentence denotes BatRaTG13 97.70 100.00 SB SB SB SB SB SB
T134 16152-16204 Sentence denotes SARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB
T135 16205-16254 Sentence denotes MERS 35.40 35.40 35.20 100.00 SB SB SB SB
T136 16255-16307 Sentence denotes OC43 37.30 37.10 36.90 39.50 100.00 SB SB SB
T137 16308-16363 Sentence denotes HKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB
T138 16364-16422 Sentence denotes 229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB
T139 16423-16484 Sentence denotes NL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00
T140 16485-16490 Sentence denotes MERS:
T141 16491-16641 Sentence denotes Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below.
T142 16642-16755 Sentence denotes High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.
T143 16756-17035 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 17036-17170 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 17171-17337 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 17338-17481 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 17483-17570 Sentence denotes Four murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2
T148 17571-17763 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 17764-17970 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 17971-18112 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 18113-18199 Sentence denotes Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead.
T152 18200-18346 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 18347-18502 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 18503-18721 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 18722-18782 Sentence denotes aa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI:
T156 18783-18966 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 18967-18969 Sentence denotes A.
T158 18970-19081 Sentence denotes Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10).
T159 19082-19356 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 19357-19450 Sentence denotes Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies.
T161 19451-19606 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 19607-19609 Sentence denotes B.
T163 19610-19765 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 19766-20049 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 20050-20099 Sentence denotes Cell nuclei were counterstained with DAPI (blue).
T166 20100-20118 Sentence denotes Scale bar = 50 µm.
T167 20120-20201 Sentence denotes Four murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2
T168 20202-20351 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 20352-20449 Sentence denotes As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).
T170 20450-20678 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 20679-20684 Sentence denotes DAPI:
T172 20685-20915 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 20916-20918 Sentence denotes A.
T174 20919-21025 Sentence denotes Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10).
T175 21026-21222 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 21223-21316 Sentence denotes Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies.
T177 21317-21472 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 21473-21475 Sentence denotes B.
T179 21476-21595 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 21596-21714 Sentence denotes Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated.
T181 21715-21896 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 21897-21899 Sentence denotes C.
T183 21900-22055 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 22056-22221 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 22222-22331 Sentence denotes The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green).
T186 22332-22381 Sentence denotes Cell nuclei were counterstained with DAPI (blue).
T187 22382-22400 Sentence denotes Scale bar = 50 µm.
T188 22401-22569 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 22570-22673 Sentence denotes Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B).
T190 22674-22779 Sentence denotes Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C).
T191 22780-22913 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 22915-23036 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 23037-23154 Sentence denotes Based on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs.
T194 23155-23307 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 23308-23473 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 23474-23617 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 23618-23780 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 23781-23921 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 23922-24107 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 24108-24110 Sentence denotes A.
T201 24111-24254 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 24255-24328 Sentence denotes 1A9 and CR3022 were used as capture and detector antibodies respectively.
T203 24329-24410 Sentence denotes His-tagged HA protein of influenza A (H7N7) virus was used as a negative control.
T204 24411-24478 Sentence denotes Averaged readings across three replicate experiments are presented.
T205 24479-24559 Sentence denotes Error bars represent standard deviations across the three replicate experiments.
T206 24560-24703 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 24704-24706 Sentence denotes B.
T208 24707-24805 Sentence denotes Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1).
T209 24806-24945 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 24946-25021 Sentence denotes Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue).
T211 25022-25080 Sentence denotes Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells.
T212 25081-25289 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 25291-25301 Sentence denotes Discussion
T214 25302-25418 Sentence denotes Numerous mAbs against the S protein of SARS-CoV-1 have been generated for research and diagnostic assay development.
T215 25419-25556 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 25557-25722 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 25723-25901 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 25902-26027 Sentence denotes Recent cross-reactivity studies have evaluated SARS-CoV-1 neutralising antibodies that bind to the RBD-containing S1 subunit.
T219 26028-26200 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 26201-26404 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 26405-26537 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 26538-26705 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 26706-26811 Sentence denotes As would be expected, these two antibodies can be paired to detect S protein from 15.6 ng/mL (Figure 4A).
T224 26812-27022 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 27023-27164 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 27165-27312 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 27313-27404 Sentence denotes Interestingly, the latter CTL epitope overlaps with the epitope recognised by mAb 1A9 [21].
T228 27405-27555 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 27556-27775 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 27776-27982 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 27983-28203 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 28204-28351 Sentence denotes Successful development of these antibodies into a point of care diagnostic kit will provide a complementary approach to existing detection methods.
T233 28352-28487 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 28488-28579 Sentence denotes Thus, it will be important to determine if these mAbs can also cross-react with SARS-CoV-2.