Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: HuH7 cells were left untreated or were infected with HCoV-229E or MERS-CoV (MOI=1) for 3, 6, 12, or 24 h. Transcriptomic data (by RNA-seq, n=2) and proteomic data (by LC-MS/MS, n=2, three technical replicates per sample) were derived from samples obtained at the indicated time points p.i. and subsequently used to extract expression values for the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. (A) Scatter plots show mean normalized protein / mRNA expression values for each component, fitted linear regression lines, confidence intervals and correlation coefficients for mock-infected HuH7 cells and HuH7 cells infected for 24 h. (B) Correlation matrix of Pearson’s r across all conditions. All p values are given in supplementary Table S1. (C) The heatmap shows mean ratio values of differentially expressed mRNAs or proteins based on significant differences (fold change ≥ 2, p ≤ 0.01) calculated from the two biological replicates. See also and Table S1.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: Projection of normalized ratio values for transcriptomic (by RNA-seq) and proteomic (by LC-MS/MS) data derived in parallel from HuH7 cells infected for 24 h with HCoV-229E or MERS-CoV with a MOI=1 on the components of the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. The left side of the boxes show mRNA values, right sides show protein values.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Infection

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Schematic overview of parameters used to monitor virus- and thapsigargin-mediated ER stress. (B) Schematic presentation of HCoV-229E infection of cells and/or treatment with thapsigargin for different periods of time as applied in this study. (C) HuH7 cells were left untreated or infected with HCoV-229E (MOI=1) for 24 h and treated with thapsigargin (1 μM) according to (B). Supernatants and RNA-isolated from the cell pellets were used to determine viral titers (upper graphs) and expression of HCoV-229E S gene-containing RNA (lower graphs). (D) Representative fluorescence microscopy images showing subcellular HCoV-229E replication sites (at 24 h p.i.) identified by nsp8- or double-strand RNA-specific antibodies in the presence or absence of thapsigargin. Cells were infected with virus and treated with thapsigargin (1 μM) for 24 h. (E) Total cell extracts from HuH7 cells infected with HCoV-229E (MOI=1) and treated with thapsigargin (1 μM) as shown in (B) were analyzed for the expression/modification of the indicated host cell and viral proteins by immunoblotting. (F) Quantification of immunoblot data from multiple experiments. (G) HuH7 cells were left untreated, or were infected with HCoV-229E (MOI=1) in the presence / absence of increasing concentrations of thapsigargin or DMSO as solvent control. After 24 h, cell viability was assessed by MTS assay. (H) Data from (G) were used to compute half maximal cellular cytotoxicity (CC 50 ) concentrations of thapsigargin. (I) HuH7 cells were treated and infected according to (B). 30 min before harvesting, puromycin (3 μM) was added to the cells as indicated. Total cell extracts were analyzed by immunoblotting. The blot membrane was stained with CBB to assess the steady state proteomes and then hybridized with anti-puromycin antibodies to detect de novo synthesized nascent polypeptides. The upper graphs show representative images and the lower graph shows the quantification of multiple replicates. Puromycin signals of each lane were normalized to the corresponding CBB staining and were background corrected by subtracting signals of samples in which puromycin had been omitted. Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, Isolation, Expressing, Fluorescence, Microscopy, Modification, Western Blot, MTS Assay, Staining, Synthesized, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A-D) Human embryonic MRC-5 lung fibroblasts were infected with HCoV-229E according to the scheme shown in . Viral titers (A, upper graph), and expression of viral S gene-containing RNAs (A, lower graph), viral and host cell proteins (B, C) and cell viability (D) were analyzed and quantified as described in the legend of . (E-K) Similarly, HuH7 cells or Vero E6 African green monkey kidney epithelial cells were infected with MERS-CoV (MOI=0.5) or SARS-CoV-2 (MOI=0.5) for 12 h or 24 in the presence / absence of 0.4 μM or 1 μM thapsigargin. (E, F) show viral titers and (G, H) the corresponding expression of MERS-CoV / SARS-CoV-2 nucleocapsid (N) and host cell proteins, respectively. (I) Dose-dependent suppression of MERS-CoV-2 replication by thapsigargin (upper graph) and the estimated effective inhibitory concentration (EC 50 ) in HuH7 cells infected with an MOI of 0.5 (lower graph). (J) Dose-dependent suppression of SARS-CoV-2 replication by thapsigargin (left graph) and the calculated effective inhibitory concentration (EC 50 ) in Vero E6 cells infected with an MOI of 0.5 (right graph). (K) The CC 50 of thapsigargin in Vero E6 cells was calculated by MTS assays as described in the legend of . Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests. See for quantifications from replicates for MERS-CoV / SARS-CoV-2 immunoblot experiments.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, Expressing, Concentration Assay, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Venn diagram demonstrating the overlap of orthologues proteins expressed in HuH7 and Vero cells based on the NCBI gene IDs corresponding to majority protein IDs. (B) The overlap of virus- and thapsigargin-regulated proteins common to HuH7 and Vero E6 cells was calculated based on gene IDs. This analysis identifies 180 proteins with higher and 61 proteins with lower expression in thapsigargin-treated infected cells compared to virus infection alone (ratio > 0, p ≥ −log 10 1.3). (C) Heatmaps showing individual mean ratio values of log 2 -transfomed normalized protein intensities for the top 50 up- or downregulated proteins in virus-infected and thapsigargin-treated cells. Ratio values of infected or thapsigargin-treated conditions compared to untreated cells (-) cells are shown for comparison. Note that ratios are sorted and color coded according to the virus plus thapsigargin conditions with infection alone conditions set as denominator. Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2-regulated protein in an independent study ( Stukalov et al ., 2020 ). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone. Gene ID lists were analyzed using Metascape software ( Zhou et al ., 2019 ). (E) Experimental evidence, co-occurrence, co-expression and confidence scores from the STRING database ( Szklarczyk et al , 2019 ) were used to identify protein:protein interactions (PPI) amongst the 180 up- and 61 downregulated thapsigargin-sensitive proteins. As shown, based on experimental evidence and combined STRING score criteria, 59 and 26 coregulated proteins are engaged in defined PPI networks; the remaining proteins are not known to interact. (F) Validation of thapsigargin-induced HERPUD1 and CTH upregulation by immunoblotting of HuH7 or Vero E6 whole cell extracts from cells treated as described above. BiP and IRE1α levels are shown for comparison. (G) Quantification of thapsigargin-mediated re-expression of HERPUD1 and CTH in cells infected with HCoV-229E, MERS-CoV or SARS-CoV-2 from two independent immunoblot experiments. Error bars show s.d.. (H) Heatmap showing thapsigargin-reprogrammed proteins of KEGG 04141 (mean ratio ≥ 1.5 fold) along with p values. See also for projection of thapsigargin-mediated protein changes on the KEGG pathway map. (I) Venn diagram showing the intersection of thapsigargin- / virus-regulated proteins with all novel ERAD components (FDR of 1 %) identified by ( Leto et al ., 2019 ). The regulation of 31 overlapping components is shown as a heatmap displaying mean ratio values in thapsigargin-treated or infected cells. Red colors highlight UBA5 and ZNF622 as discussed in the text. (J) Summary of the main findings of our study. Abbreviations: M, MERS-CoV; S, SARS-CoV-2; T, thapsigargin.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Expressing, Infection, Software, Western Blot

Journal: Protein science : a publication of the Protein Society

Article Title: Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

doi: 10.1002/pro.5103

Figure Lengend Snippet: FIGURE 1 Frequency of amino acid substitutions within the RdRp complex protein subunits. The graphs illustrate the sums of all different mutations at each position within the amino acid sequences of the proteins comprising RdRp complex. The selected amino acid substitutions are depicted in red: (a) amino acid exchanges in the nsp7 sequence, (b) amino acid substitutions within the nsp8 sequences and (c) identified amino acid substitutions in nsp12. The source data were retrieved from (covidcg.org) over the period from January 2020 to June 30, 2022. The entire dataset subjected to analysis comprised 15,243,203 sequences.

Article Snippet: Expression vectors encoding 6 His-TEV-nsp7 (#154757), 6 His-TEV-nsp8 (#154758) and 6 HisMBP-TEV-nsp12 (#154759) were obtained from AddGene (Hillen et al., 2020).

Techniques: Sequencing

Journal: Protein science : a publication of the Protein Society

Article Title: Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

doi: 10.1002/pro.5103

Figure Lengend Snippet: FIGURE 2 Schematic representation of SARS-CoV-2 RdRp complex and selected amino acid residues in RdRp protein subunits. (a) A model structure (PDB id: 6yyt (Hillen et al., 2020)) and domain description of the proteins comprising the SARS-CoV-2 minimal RdRp complex: A single nsp7 (blue) and two nsp8 subunits are bound to nsp12; nsp8a directly and nsp8b through nsp7. Nsp8 consists of two subdomains—a head domain (magenta) and a rod-like helical extension (pink) that stabilizes the nascent RNA duplex (green). The multidomain nsp12 is composed of the polymerase (wheat), interface (orange), and NiRAN (yellow) domains. The localization of amino acid residues selected for this study are visualized in close-up views at panels b, c, d, e, and f: (b) view of the nsp8a interaction with nsp12 interface and RdRp domains, (c) close-up view of the nsp8 extension domains, (d) selected amino acid residues in nsp8b, (e) nsp7 loop interacting with nsp8b and (f) the P323 residue in nsp12 interface domain and its interaction with nsp8b.

Article Snippet: Expression vectors encoding 6 His-TEV-nsp7 (#154757), 6 His-TEV-nsp8 (#154758) and 6 HisMBP-TEV-nsp12 (#154759) were obtained from AddGene (Hillen et al., 2020).

Techniques: Residue

Journal: Protein science : a publication of the Protein Society

Article Title: Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

doi: 10.1002/pro.5103

Figure Lengend Snippet: FIGURE 3 Assessment of protein stability of individual RdRp proteins and their complexes. (a) SDS-PAGE analysis of purified nsp7, nsp8, and nsp12 variants. The thermal stability (TS) of individual RdRp proteins is shown in panels b, c, d: (b) TS of S25L and S26F of nsp7 mutants, (c) TS of nsp8 mutants, (d) TS of nsp12 wt and P323L. (e) TS of heterodimeric nsp7-nsp8 complexes formed by wt and indicated nsp7 and nsp8 mutants was assessed. (f) TS of nsp12 mixtures with nsp7 and nsp8 variants in a 1:1:2 ratio (nsp12:nsp7:nsp8). The tested complexes were formed by mixing two wt components with one mutant component, for example, the RdRp nsp7 S25L complex was formed by wt nsp12, wt nsp8, and S25L variant of nsp7. The melting temperature (TM), shown in blue, determined using nanoDSF is represented by the inflection point in the 330/350 nm ratio curve. The turbidimetrically assessed aggregation temperature (TAgg), shown in red, represents the initial temperature at which the aggregation starts. The displayed results represent data from three independent measurements for each sample processed with the Panta analysis software.

Article Snippet: Expression vectors encoding 6 His-TEV-nsp7 (#154757), 6 His-TEV-nsp8 (#154758) and 6 HisMBP-TEV-nsp12 (#154759) were obtained from AddGene (Hillen et al., 2020).

Techniques: SDS Page, Purification, Mutagenesis, Variant Assay, Nano Differential Scanning Fluorimetry, Software

Journal: Protein science : a publication of the Protein Society

Article Title: Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.

doi: 10.1002/pro.5103

Figure Lengend Snippet: FIGURE 5 Amino acid substitutions of individual RdRp proteins alter the polymerase activity. (a) A Primed polyU RNA template used to analyze the RdRp activity. (b) Comparison of the fluorescence curves for RdRp complex of wt nsp7, wt nsp8 and wt nsp12 (labeled “wt RdRp”) and nsp7 S25L, wt nsp8 and wt nsp12 (labeled “nsp7 S25L”). (c) RNA polymerase activity of different RdRp variants compared with a combination of wt nsp7, wt nsp8, and nsp12. Each variant comprised one subunit with an amino acid substitution, for example, nsp7 S25L represents a combination of nsp7 S25L, wt nsp8, and wt nsp12. (d) The most significant increase in RdRp activity was observed for a complex of wt nsp7, nsp8 A21V, and nsp12 P323L. Results represent statistically processed data from at least three independent measurements. Outliers were excluded using a Q-test at a 90% significance level. A paired two-tailed t test was utilized to compare results with wt RdRp with indicated p-values (*p ≤0.05; **p ≤0.01; ***p ≤0.001). The percentage of RdRp activity was calculated by comparing the area under the curve (AUC) with the AUC of wt RdRp. RFU—relative fluorescence units.

Article Snippet: Expression vectors encoding 6 His-TEV-nsp7 (#154757), 6 His-TEV-nsp8 (#154758) and 6 HisMBP-TEV-nsp12 (#154759) were obtained from AddGene (Hillen et al., 2020).

Techniques: Activity Assay, Comparison, Fluorescence, Labeling, Variant Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity.

doi: 10.1038/s41598-025-03813-y

Figure Lengend Snippet: Fig. 2. Confirmation of reporter activity through expression of SARS-CoV-2 RdRp. (a) HEK 293 cells were co-transfected with our constructed reporter vector and RdRp at various ratios (1:1, 1:2, and 1:4). Reporter activity was evaluated after 48 h of incubation. (b) Reporter activity was compared between the single expression of RdRp and the co-expression of Nsp7, Nsp8, and RdRp in reporter-transfected HEK 293 cells. (c) The expression of the reporter vector was assessed at various time intervals following the transfection of HEK 293 cells with RdRp. The reporter activity value of the reporter vector and RdRp co-expression group was divided by the reporter activity value of the reporter vector expression group and expressed as a ratio. (d) Reporter activity was measured following the mutation of essential residues Lys73 and Asp218 (depicted as K73 and D218) in the NiRAN domain of RdRp, or the mutation of key residues Asp618, Asp760, and Asp761 (depicted as D618, D760, and D761) in the RdRp domain of RdRp. Each key residue that regulates the activity of SARS-CoV-2 was replaced with alanine, indicated as ‘A’ in the graph. All data on reporter activity are expressed as a percentage compared to the reporter expression group. The data represent the results of three independent experiments. Statistical significance is indicated as follows: ap < 0.05, bp < 0.01, and cp < 0.001 compared to the reporter vector expressed group; **p < 0.01 and ***p < 0.001 compared to the reporter vector and RdRp co-expressed group; n.s., non-significant.

Article Snippet: Strep-tagged plasmids including SARS-CoV-2 RdRp (plasmid #141378), Nsp7 (plasmid #141373), and Nsp8 (plasmid #141374) were obtained from Addgene (MA, USA).

Techniques: Activity Assay, Expressing, Transfection, Construct, Plasmid Preparation, Incubation, Mutagenesis, Residue

Journal: Frontiers in Immunology

Article Title: Immune responses during COVID-19 breakthrough cases in vaccinated children and adolescents

doi: 10.3389/fimmu.2024.1372193

Figure Lengend Snippet: Specific IgG levels against different SARS-CoV-2 proteins in plasma of breakthrough cases of children and adolescents. Titers of specific IgG levels against different SARS-CoV-2 in twelve breakthrough cases. (A) Antibody titer against S1 protein. (B) Antibody titer against N protein. (C) Antibody titer against M protein. (D) Antibody titer against E protein. (E) Antibody titer against NSP8 protein. An in-house indirect ELISA assay was used to assess plasma IgG specific antibody titers against these proteins. Data were transformed, plotted as reciprocal dilution in log base 2 on a linear scale. Circles, triangles and squares correspond to subjects 3-5 yo, 6-11 yo and 12-17 yo, respectively. On the other hand, the black, blue and red colors represent the samples evaluated at pre-immune, 2 nd dose + 4 weeks and 2 to 8 weeks post-infection, respectively, by before-after graph. Black values below the significance line indicate geometric mean titers (GMT), blue values above the significance line indicate an increase in GMT of the two time points compared, and red values above the significance line indicate a decrease in GMT of the two time points compared. The data was analyzed using a mixed effects model, ***p<0.001; **p<0.01; *p<0.05, not significant (ns).

Article Snippet: Total IgG titers for the structural proteins Spike (S1: subunit 1) (SinoBiological, #40591-V08B1), Nucleocapsid (N) (R&D systems, #10474-CV), Membrane (M) (R&D systems, #10690-CV), and Envelope (E) (SinoBiological, #40609-V10E3), as well as the Non-Structural Protein 8 (NSP8) (R&D systems, #10633-CV) of SARS-CoV-2, were tested by in-house indirect ELISA assays, as previously described ( ).

Techniques: Clinical Proteomics, Indirect ELISA, Transformation Assay, Infection