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LitCovid-PD-FMA-UBERON

LitCovid-PD-UBERON

{"project":"LitCovid-PD-UBERON","denotations":[{"id":"T5","span":{"begin":6360,"end":6365},"obj":"Body_part"},{"id":"T6","span":{"begin":8642,"end":8647},"obj":"Body_part"}],"attributes":[{"id":"A5","pred":"uberon_id","subj":"T5","obj":"http://purl.obolibrary.org/obo/UBERON_0002542"},{"id":"A6","pred":"uberon_id","subj":"T6","obj":"http://purl.obolibrary.org/obo/UBERON_0002542"}],"text":"Results\n\nAn 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\nSequence 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). 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 (\u003c 40% in this region) when considering MERS and the other coronaviruses infecting humans regularly.\nFigure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses\nMERS: Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.\nThe 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.\nColour 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.\nTable 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\nQuery/reference Pairwise amino-acid identity (%)\nSARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63\nFragment region of spike S2\nSARS-Co-V2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 99.40 100.00 SB SB SB SB SB SB\nSARS 93.10 92.50 100.00 SB SB SB SB SB\nMERS 39.00 39.00 39.00 100.00 SB SB SB SB\nOC43 39.00 39.00 38.40 51.20 100.00 SB SB SB\nHKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB\n229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB\nNL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00\nFull spike protein\nSARS-CoV-2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 97.70 100.00 SB SB SB SB SB SB\nSARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB\nMERS 35.40 35.40 35.20 100.00 SB SB SB SB\nOC43 37.30 37.10 36.90 39.50 100.00 SB SB SB\nHKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB\n229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB\nNL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00\nMERS: Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below. High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.\nThe 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. We also studied the sequence diversity across 174 SARS-CoV-2 S proteins derived from nt sequences shared via the GISAID platform [27]. 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). 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.\n\nFour murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2\nFour 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. 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. 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. Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead. 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). 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.\nFigure 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\naa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\n\nFour murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2\nNext, 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. As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).\nFigure 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\nDAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. The abilities of 2B2, 1A9, 4B12 and 1G10 monoclonal antibodies to bind to SARS-CoV-2 S protein was determined by ELISA. Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated. 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.\nC. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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). The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. 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. Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B). Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C). Collectively our data demonstrates the ability of all four mAbs to bind full-length S protein in both its native and denatured forms.\n\nUtility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells\nBased on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs. 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. 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. 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. 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.\nFigure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells\nHA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.\nA. 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. 1A9 and CR3022 were used as capture and detector antibodies respectively. His-tagged HA protein of influenza A (H7N7) virus was used as a negative control. Averaged readings across three replicate experiments are presented. Error bars represent standard deviations across the three replicate experiments. Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p \u003c 0.05.\nB. Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1). At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells. 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."}

LitCovid-PubTator

LitCovid-PD-MONDO

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-PD-GlycoEpitope

{"project":"LitCovid-PD-GlycoEpitope","denotations":[{"id":"T1","span":{"begin":4225,"end":4229},"obj":"GlycoEpitope"},{"id":"T2","span":{"begin":5335,"end":5339},"obj":"GlycoEpitope"},{"id":"T3","span":{"begin":6019,"end":6023},"obj":"GlycoEpitope"},{"id":"T4","span":{"begin":7279,"end":7283},"obj":"GlycoEpitope"},{"id":"T5","span":{"begin":7772,"end":7776},"obj":"GlycoEpitope"},{"id":"T6","span":{"begin":8309,"end":8313},"obj":"GlycoEpitope"}],"attributes":[{"id":"A1","pred":"glyco_epitope_db_id","subj":"T1","obj":"http://www.glycoepitope.jp/epitopes/AN0438"},{"id":"A2","pred":"glyco_epitope_db_id","subj":"T2","obj":"http://www.glycoepitope.jp/epitopes/AN0438"},{"id":"A3","pred":"glyco_epitope_db_id","subj":"T3","obj":"http://www.glycoepitope.jp/epitopes/AN0438"},{"id":"A4","pred":"glyco_epitope_db_id","subj":"T4","obj":"http://www.glycoepitope.jp/epitopes/AN0438"},{"id":"A5","pred":"glyco_epitope_db_id","subj":"T5","obj":"http://www.glycoepitope.jp/epitopes/AN0438"},{"id":"A6","pred":"glyco_epitope_db_id","subj":"T6","obj":"http://www.glycoepitope.jp/epitopes/AN0438"}],"text":"Results\n\nAn 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\nSequence 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). 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 (\u003c 40% in this region) when considering MERS and the other coronaviruses infecting humans regularly.\nFigure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses\nMERS: Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.\nThe 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.\nColour 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.\nTable 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\nQuery/reference Pairwise amino-acid identity (%)\nSARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63\nFragment region of spike S2\nSARS-Co-V2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 99.40 100.00 SB SB SB SB SB SB\nSARS 93.10 92.50 100.00 SB SB SB SB SB\nMERS 39.00 39.00 39.00 100.00 SB SB SB SB\nOC43 39.00 39.00 38.40 51.20 100.00 SB SB SB\nHKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB\n229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB\nNL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00\nFull spike protein\nSARS-CoV-2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 97.70 100.00 SB SB SB SB SB SB\nSARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB\nMERS 35.40 35.40 35.20 100.00 SB SB SB SB\nOC43 37.30 37.10 36.90 39.50 100.00 SB SB SB\nHKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB\n229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB\nNL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00\nMERS: Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below. High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.\nThe 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. We also studied the sequence diversity across 174 SARS-CoV-2 S proteins derived from nt sequences shared via the GISAID platform [27]. 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). 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.\n\nFour murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2\nFour 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. 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. 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. Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead. 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). 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.\nFigure 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\naa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\n\nFour murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2\nNext, 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. As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).\nFigure 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\nDAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. The abilities of 2B2, 1A9, 4B12 and 1G10 monoclonal antibodies to bind to SARS-CoV-2 S protein was determined by ELISA. Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated. 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.\nC. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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). The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. 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. Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B). Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C). Collectively our data demonstrates the ability of all four mAbs to bind full-length S protein in both its native and denatured forms.\n\nUtility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells\nBased on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs. 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. 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. 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. 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.\nFigure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells\nHA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.\nA. 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. 1A9 and CR3022 were used as capture and detector antibodies respectively. His-tagged HA protein of influenza A (H7N7) virus was used as a negative control. Averaged readings across three replicate experiments are presented. Error bars represent standard deviations across the three replicate experiments. Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p \u003c 0.05.\nB. Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1). At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells. 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."}

LitCovid-sentences

LitCovid-PD-HP

{"project":"LitCovid-PD-HP","denotations":[{"id":"T2","span":{"begin":5005,"end":5014},"obj":"Phenotype"}],"attributes":[{"id":"A2","pred":"hp_id","subj":"T2","obj":"http://purl.obolibrary.org/obo/HP_0012115"}],"text":"Results\n\nAn 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\nSequence 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). 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 (\u003c 40% in this region) when considering MERS and the other coronaviruses infecting humans regularly.\nFigure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses\nMERS: Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.\nThe 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.\nColour 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.\nTable 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\nQuery/reference Pairwise amino-acid identity (%)\nSARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63\nFragment region of spike S2\nSARS-Co-V2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 99.40 100.00 SB SB SB SB SB SB\nSARS 93.10 92.50 100.00 SB SB SB SB SB\nMERS 39.00 39.00 39.00 100.00 SB SB SB SB\nOC43 39.00 39.00 38.40 51.20 100.00 SB SB SB\nHKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB\n229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB\nNL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00\nFull spike protein\nSARS-CoV-2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 97.70 100.00 SB SB SB SB SB SB\nSARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB\nMERS 35.40 35.40 35.20 100.00 SB SB SB SB\nOC43 37.30 37.10 36.90 39.50 100.00 SB SB SB\nHKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB\n229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB\nNL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00\nMERS: Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below. High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.\nThe 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. We also studied the sequence diversity across 174 SARS-CoV-2 S proteins derived from nt sequences shared via the GISAID platform [27]. 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). 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.\n\nFour murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2\nFour 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. 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. 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. Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead. 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). 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.\nFigure 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\naa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\n\nFour murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2\nNext, 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. As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).\nFigure 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\nDAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. The abilities of 2B2, 1A9, 4B12 and 1G10 monoclonal antibodies to bind to SARS-CoV-2 S protein was determined by ELISA. Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated. 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.\nC. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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). The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. 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. Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B). Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C). Collectively our data demonstrates the ability of all four mAbs to bind full-length S protein in both its native and denatured forms.\n\nUtility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells\nBased on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs. 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. 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. 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. 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.\nFigure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells\nHA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.\nA. 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. 1A9 and CR3022 were used as capture and detector antibodies respectively. His-tagged HA protein of influenza A (H7N7) virus was used as a negative control. Averaged readings across three replicate experiments are presented. Error bars represent standard deviations across the three replicate experiments. Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p \u003c 0.05.\nB. Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1). At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells. 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."}

2_test

{"project":"2_test","denotations":[{"id":"32700671-31565258-29327482","span":{"begin":3426,"end":3428},"obj":"31565258"}],"text":"Results\n\nAn 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\nSequence 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). 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 (\u003c 40% in this region) when considering MERS and the other coronaviruses infecting humans regularly.\nFigure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses\nMERS: Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.\nThe 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.\nColour 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.\nTable 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\nQuery/reference Pairwise amino-acid identity (%)\nSARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63\nFragment region of spike S2\nSARS-Co-V2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 99.40 100.00 SB SB SB SB SB SB\nSARS 93.10 92.50 100.00 SB SB SB SB SB\nMERS 39.00 39.00 39.00 100.00 SB SB SB SB\nOC43 39.00 39.00 38.40 51.20 100.00 SB SB SB\nHKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB\n229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB\nNL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00\nFull spike protein\nSARS-CoV-2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 97.70 100.00 SB SB SB SB SB SB\nSARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB\nMERS 35.40 35.40 35.20 100.00 SB SB SB SB\nOC43 37.30 37.10 36.90 39.50 100.00 SB SB SB\nHKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB\n229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB\nNL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00\nMERS: Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below. High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.\nThe 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. We also studied the sequence diversity across 174 SARS-CoV-2 S proteins derived from nt sequences shared via the GISAID platform [27]. 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). 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.\n\nFour murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2\nFour 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. 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. 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. Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead. 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). 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.\nFigure 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\naa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\n\nFour murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2\nNext, 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. As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).\nFigure 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\nDAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. The abilities of 2B2, 1A9, 4B12 and 1G10 monoclonal antibodies to bind to SARS-CoV-2 S protein was determined by ELISA. Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated. 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.\nC. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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). The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. 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. Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B). Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C). Collectively our data demonstrates the ability of all four mAbs to bind full-length S protein in both its native and denatured forms.\n\nUtility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells\nBased on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs. 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. 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. 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. 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.\nFigure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells\nHA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.\nA. 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. 1A9 and CR3022 were used as capture and detector antibodies respectively. His-tagged HA protein of influenza A (H7N7) virus was used as a negative control. Averaged readings across three replicate experiments are presented. Error bars represent standard deviations across the three replicate experiments. Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p \u003c 0.05.\nB. Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1). At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells. 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."}

MyTest

{"project":"MyTest","denotations":[{"id":"32700671-31565258-29327482","span":{"begin":3426,"end":3428},"obj":"31565258"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Results\n\nAn 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\nSequence 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). 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 (\u003c 40% in this region) when considering MERS and the other coronaviruses infecting humans regularly.\nFigure 1 Multiple sequence alignment for the S2 subunit fragment of SARS-CoV-1 spike glycoprotein with other relevant coronaviruses\nMERS: Middle East respiratory syndrome; SARS: severe acute respiratory coronavirus 1; SARS-CoV-2: severe acute respiratory coronavirus 2.\nThe 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.\nColour 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.\nTable 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\nQuery/reference Pairwise amino-acid identity (%)\nSARS-CoV-2 BatRaTG13 SARS-CoV-1 MERS OC43 HKU1 229E NL63\nFragment region of spike S2\nSARS-Co-V2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 99.40 100.00 SB SB SB SB SB SB\nSARS 93.10 92.50 100.00 SB SB SB SB SB\nMERS 39.00 39.00 39.00 100.00 SB SB SB SB\nOC43 39.00 39.00 38.40 51.20 100.00 SB SB SB\nHKU1 32.70 32.70 30.80 50.60 68.40 100.00 SB SB\n229E 30.80 30.20 32.10 31.50 29.70 30.40 100.00 SB\nNL63 30.80 30.20 30.20 32.10 31.60 33.50 64.20 100.00\nFull spike protein\nSARS-CoV-2 100.00 SB SB SB SB SB SB SB\nBatRaTG13 97.70 100.00 SB SB SB SB SB SB\nSARS-CoV-1 77.80 78.20 100.00 SB SB SB SB SB\nMERS 35.40 35.40 35.20 100.00 SB SB SB SB\nOC43 37.30 37.10 36.90 39.50 100.00 SB SB SB\nHKU1 35.20 35.30 35.00 39.00 67.00 100.00 SB SB\n229E 41.70 41.50 41.80 41.80 43.50 43.50 100.00 SB\nNL63 36.30 36.20 36.20 35.40 39.70 37.80 64.70 100.00\nMERS: Middle East respiratory syndrome; SARS-CoV-1: severe acute respiratory coronavirus; SARS-CoV-2: severe acute respiratory coronavirus; SB: shown below. High to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds.\nThe 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. We also studied the sequence diversity across 174 SARS-CoV-2 S proteins derived from nt sequences shared via the GISAID platform [27]. 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). 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.\n\nFour murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2\nFour 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. 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. 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. Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead. 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). 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.\nFigure 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\naa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\n\nFour murine monoclonal antibodies bind to the full-length S protein of SARS-CoV-2\nNext, 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. As shown in Figure 3, all four mAbs bound to the full-length S protein of SARS-CoV-2 (Figure 3A).\nFigure 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\nDAPI: 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.\nA. Each photo depicts a western blot using the primary antibody indicated below it (2B2, 1A9, 4B12, or 1G10). 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’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. 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.\nB. The abilities of 2B2, 1A9, 4B12 and 1G10 monoclonal antibodies to bind to SARS-CoV-2 S protein was determined by ELISA. Individual wells were coated with 20 ng of SARS-CoV-2 S protein and incubated with serially diluted mAbs as indicated. 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.\nC. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). 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). The indicated primary antibodies were used followed by Alexa Fluor 488-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. 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. Binding to S protein was observed for all four mAbs with 1A9 showing the strongest binding (Figure 3B). Similarly, all four mAbs bound to the full-length S protein of SARS-CoV-2 when tested via IF (Figure 3C). Collectively our data demonstrates the ability of all four mAbs to bind full-length S protein in both its native and denatured forms.\n\nUtility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells\nBased on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs. 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. 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. 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. 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.\nFigure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells\nHA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.\nA. 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. 1A9 and CR3022 were used as capture and detector antibodies respectively. His-tagged HA protein of influenza A (H7N7) virus was used as a negative control. Averaged readings across three replicate experiments are presented. Error bars represent standard deviations across the three replicate experiments. Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p \u003c 0.05.\nB. Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1). At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells. 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."}

PMC:7376845 / 15559-27108 JSONTXT

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PMC:7376845 / 15559-27108 JSON TXT