Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: (A-B) The tryptic peptides identified via LC-MS/MS derived from (A) EBOV and (B) SUDV recombinant VP40 protein. The precursor MS1 intensities are calculated in Maxquant software as the summed up extracted ion current of all isotopic clusters associated with the identified peptide sequence. Peptides are numbered according to their order in the linear sequences. (C) Map of EBOV VP40 tryptic peptides ≥ 7 amino acids in length. Peptide 2 (p2, blue underlines) is not aligned between the two species. The labels below indicate amino acid residues that are prone to be oxidized (●); identical between EBOV and SUDV VP40 (=); non-conservative substitution (x); conserved substitution between groups of strongly similar properties, i.e., scoring > 0.5 in the Gonnet PAM 250 matrix (:); conserved substitution between groups of weakly similar properties, i.e., scoring = < 0.5 in the Gonnet PAM 250 matrix (.); and arginine or lysine followed by proline motif that are prone to be miss cleaved by trypsin (○○). Red residues indicate potential tryptic cleavage sites. The peptides are numbered identically as shown in (A-B). (D) Candidate peptide antigenicity determined by predictive algorithms from (left) Genscript or (right) Thermo Scientific. Blue and purple dots respectively indicate data for the EBOV and SUDV species, while the closed or open dots indicate the antigenicity scores for a given peptide sequence that are above (closed) or below (open) the antigenicity score threshold (dotted red line) of a good antigen peptide defined by each algorithm. (E) Sequence alignment and molecular weights of VP40 peptide 8 and 12 regions for the four species known to causing human infections. Conservative (: or.) and divergent (x) substitutions are marked in red. Asterisks indicate the sequences serving as antigens to generate peptide antibodies.

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Recombinant, Software, Sequencing

Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: Both assays employ four steps for sample analysis. ( A ) In the IP-MS assay for VP40 peptide variants, samples are inactivated using a method described by 2005 CDC Interim Guidance and digested to release the target VP40 peptides, which are then immunoprecipitated, and analyzed by mass spectrometry to detect mass differences among the peptide 8 and 12 sequences encoded by different ebolavirus species. The VP40 p8 and p12 target peptides and internal standard (IS) peptide are captured during immunoprecipitation (IP) by target-specific antibody-coupled magnetic beads. The peak area ratio between known amounts of VP40 p8 (blue peak) and its corresponding IS peptide (purple peak) is used to generate a standard curve to permit quantification of endogenous VP40 in clinical samples. ( B ) In the RT-PCR assay for the GP gene, a carrier RNA and a high concentration internal control are spiked into the sample before extraction. Each extracted RNA sample is tested with the internal control RT-PCR RNA Assay to evaluate the yield of the spiked-in internal control. If the internal control amplified within manufacturer-designated ranges, further quantitative analysis of the viral target is performed using standard procedures.

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Immunoprecipitation, Mass Spectrometry, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Amplification

Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: (A-B) The tandem MS spectra of (A) EBOV and (B) SUDV VP40 p12 identified in recombinant protein tryptic digests. All matched ion peaks are annotated, with the six selected p12 MRM transitions colored. (C-D) Extracted ion chromatograms of six MRM transitions in (C) EBOV and (D) SUDV recombinant VP40 spiked-in PBS samples at a concentration of 70 nM. (E-F) Curve fitting between p12 MRM transition summed peak area and VP40 concentration in (E) EBOV and (F) SUDV VP40 spiked-in PBS samples.

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Recombinant, Concentration Assay

Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: (A) Generation of VLPs by co-transfecting 293T cells with pWRG7077 plasmid containing VP40 (orange) and the plasmid containing GP genes (green) from ebolavirus. (B) Western blot quantification of VP40 content in VLPs. Wells 1–5 and 6–10 respectively indicate two-fold serial dilutions of recombinant VP40 protein (starting from 0.87 μg of VP40) and VLP protein extract (starting from 2.5 μg total VLP protein). The position of a 38 kDa protein marker that migrates close to VP40 is indicated next to each Western blot. Data is shown as mean with standard deviation in the bar plot (n = 2). (C-D) Extracted ion chromatograms of VP40 peptide 8 (C) and peptide 12 (D) in human plasma spiked with 50 ng of EBOV or SUDV virus-like particles expressing VP40. The measured retention time and peak area of each peptide are shown in the left and right upper corners respectively in the upper panels. (E-F) Quantification curve for (E) EBOV and (F) SUDV VP40 peptide 8 in human plasma spiked with the corresponding VLPs. The extracted peptide peaks were used to calculate the peak area ratio for (left) the standard curve of VP40 peptide signal to VLP protein input, and (right) the LOD (red arrow). Data is shown as mean with standard deviation (n = 3).

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Plasmid Preparation, Western Blot, Recombinant, Marker, Standard Deviation, Expressing

Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: (A) MALDI-TOF MS spectra of IP enriched peptides from (upper) NHP plasma spiked with 10 μg EBOV VLPs protein and (lower) authentic EBOV infected NHP plasma at day 7 post-infection (Animal ID 6311, viral titer 7.91×10 9 GEq/mL). For simplicity, only the two peptide target peaks were indicated with their mass-to-charge ratio (m/z). (B-C) Internal standard (IS) of EBOV VP40 peptide 8 was spiked into the plasma after digestion for VP40 quantitation. Quantitative standard curves of MS peak intensity ratios between the light (endogenous peptide) and heavy (internal standard peptide) VP40 peptide 8 and the amount of (B) recombinant protein and (C) VLP proteins spiked into NHP plasma. The data is shown as mean with standard deviation (n = 3). Red arrows indicate the assay LOD as 240 or 930 ng/mL in the absence or existence of LDS buffer, respectively.

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Infection, Quantitation Assay, Recombinant, Standard Deviation

Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: (A-B) A concentration gradient of recombinant VP40 is used as standard, and serial dilution of NHP plasma post-infection (PI) is measured on the same membrane. Well 1–5 represent for a two-fold decrease of VP40 loading starting from 870 nanogram. Well 6 represents for the undiluted NHP plasma infected with EBOV, and well 7–10 represent for a two-fold serial dilution of the same sample. Two microliters of both diluted and undiluted plasma were loaded into each well. The band of VP40 is indicated by a black arrowhead on each membrane. (C-D) The standard curves of densitometry of VP40 band in each sampling time point for (C) ID 436 and (D) ID 7284 (died at Day 7 PI). The correlation co-efficiency (R 2 ) of each curve is indicated. (E-F) Densitometry values of detected bands in the expected VP40 region in infected plasma dilutions. (G-H) Changes of NHP plasma VP40 concentration (grey bars, left axis) measured by western blot and viral titer (red bars, right axis) measured by RT-PCR during the infection course. The data is shown as mean+SD (n = 3).

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Concentration Assay, Recombinant, Serial Dilution, Infection, Sampling, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

doi: 10.1371/journal.ppat.1010039

Figure Lengend Snippet: ( A-B ) Full MS scan of IP enriched peptides from (A) 10 μg/mL EBOV VLP-spiked human plasma sample and (B) from 10 μg/mL SUDV VLP-spiked human plasma sample in the mass range of 660–1,310 m/z. ( C-D ) The tandem MS spectrum of (C) EBOV VP40 peptide 8 with the sequence LGPGIPDHPLR and (D) SUDV VP40 peptide 8 with the sequence LGQGIPDHPLR in the mass range of 330–1,140 m/z.

Article Snippet: Recombinant BDBV VP40 protein was purchased from Sino Biological (Beijing, China; Cat. # 40448-V07E).

Techniques: Sequencing

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Mutations on the spike glycoprotein of SARS-CoV-2 Omicron subvariants. Residues that are not identical among Omicron subvariants are colored red. b , Workflow to generate cryo-EM structure of BA.2, BA.3, BA.2.13, BA.2.12.1, BA.4/5 spike glycoprotein trimer with S6P and R683A, R685A substitutions. c , Binding affinities of Omicron variants spike trimers to hACE2 measured by SPR. SPR analyses were conducted in biological duplicates. d , MD simulated interactions between hACE2 and RBD of Omicron variants. Structures of the RBD from Omicron variants and hACE2 are shown as ribbons.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , FACS analysis of pooled memory B cells (IgM − CD27 + ) from plasma of individuals who have recovered from BA.1 breakthrough infection after vaccination, vaccinated individuals and unvaccinated individuals who have recovered from BA.1 breakthrough infection. The percentage of cells recognizing WT or BA.1 RBD are shown. b , The heavy chain V domain somatic hypermutation (SHM) rate of BA.1-specific ( n = 968) and BA.1–WT cross-reactive ( n = 4,782) BCRs obtained from 10X scVDJ-seq from individuals who have recovered from BA.1 breakthrough infection after vaccination. Two-tailed Wilcoxon rank-sum test. Boxes show 25th percentile, median and 75th percentile, and violin plots show kernel density estimation curves of the distribution. c , t -SNE and unsupervised clustering of antibodies that bind WT SARS-CoV-2 RBD. Twelve epitope groups were identified on the basis of DMS of 1,538 antibodies. d , e , Epitope distribution and projection of antibodies from plasma of individuals who had recovered from infection with the WT virus, individuals who have recovered from BA.1 breakthrough infection after vaccination, and vaccinated individuals who had recovered from SARS. f , ACE2 competition level determined by competition ELISA ( n = 1,286) were projected onto the t -SNE. g , Neutralizing activity against SARS-CoV-2 D614G ( n = 1,509) and SARS-CoV-1 (HKU-39849; n = 1,457). h , Average mutational escape score projection of each epitope group on SARS-CoV-2 RBD (Protein Data Bank (PDB): 6M0J). All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Infection, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – c , Neutralizing activity against SARS-CoV-1 and SARS-CoV-2 subvariants by NAbs in group E1 ( a ; n = 70), F2 ( b ; n = 171) and F3 ( c ; n = 69). The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. P -values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, compared with the IC 50 for BA.2. d , The epitope of Group E1 antibody BD55-3152 on the BA.1 RBD. e , Overlay of BD55-5840 in the complex with BA.1 or BA.2 RBD. f , g , The epitope and interactions on the binding interface of BD55-1239 (group F2) ( f ) and BD55-3372 (group F3) ( g ). Antibody residues are shown in blue, and RBD residues are in black or red. Residues highlighted in red indicate sites that are mutated in Omicron variants. h , Average escape maps of antibodies in epitope groups E1, F2 and F3, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Activity Assay, Two Tailed Test, Binding Assay, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Cartoon models of Cryo-EM structures of BD55-3152 in complex of BA.1 RBD, BD55-1239 in complex of BA.1 RBD, and BD55-3372 in complex of Delta RBD. b , Workflow to generate refined structural model of BD55-3152 and BD55-1239 in complex of BA.1 RBD, BD55-3372 in complex of Delta RBD, and BD55-5840 in complex of BA.2 RBD. c , Neutralizing activity of representative NAbs in group E1 (n = 68), F2 (n = 139) and F3 (n = 61) against SARS-CoV-2 D614G, in addition to D614G+D405N and D614G+R408S. Geometric mean of IC50 fold changes compared to IC50 against D614G are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. d , Conformational comparison between BA.1 and BA.2 RBD regarding the 366-377 hairpin. e , Biolayer interferometry analysis of Group E1 antibodies S309 and BD55-5840 binding to Omicron BA.1 and BA.2 Spike trimer. Biolayer interferometry analyses were conducted in biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Cryo-EM Sample Prep, Activity Assay, Two Tailed Test, Neutralization, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Four epitope groups were identified among 102 BA.1-specific NAbs via k -means clustering and t -SNE of BA.1 RBD-based DMS profiles. b , c , Distribution of ACE2 competition level ( b ) and neutralizing activities ( c ) against BA.1. d , Neutralizing activities of BA.1-specific antibodies against pseudovirus with SARS-CoV-1 and SARS-CoV-2 spike variants (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. e , Average mutational escape score projection of each BA.1-specific epitope group on SARS-CoV-2 RBD (PDB: 7WPB). f , Averaged escape maps at escape hotspots of the 102 NAbs in the four epitope groups, and corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron variants are marked in bold. WT-related escaping mutations are highlighted. g , Neutralizing activities of BA.1-specific NAbs against BA.1- or BA.2-based pseudoviruses carrying single substitutions (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. Wilcoxon signed-rank test of paired samples, compared with IC 50 for BA.1. All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Sequencing, Mutagenesis, Neutralization

Journal: Journal of Virology

Article Title: Transmission and Protection against Reinfection in the Ferret Model with the SARS-CoV-2 USA-WA1/2020 Reference Isolate

doi: 10.1128/JVI.02232-20

Figure Lengend Snippet: Antibody and viral titers in SARS-CoV-2-infected mock- and RBD-vaccinated ferrets. (A) Displays binding antibody titers against the S protein RBD determined by ELISA on days 0, 14, 28, 42, and 56 postprimary vaccination. Red open symbols represent RBD-vaccinated ferrets. Closed black symbols represent mock-vaccinated animals. Animals were given a secondary vaccination on day 28. (B) Displays neutralizing antibody titers on day 56. (C and D) Display nasal wash titers in mock- and RBD-vaccinated animals challenged with SARS-CoV-2, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left y axis), and bar graphs indicate infectious titers (right y axis) determined via TCID50 on Vero cells. Horizontal dashed lines indicate limit of detection.

Article Snippet: Ferrets were mock vaccinated ( n = 4) with Sigma adjuvant system adjuvant (SAS) alone or receptor-binding domain vaccinated ( n = 4) with 50 μg of recombinant SARS-CoV-2 S-protein RBD (SinoBiological) mixed with SAS.

Techniques: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: iScience

Article Title: Compartment-specific antibody correlates of protection to SARS-CoV-2 Omicron in macaques

doi: 10.1016/j.isci.2024.110174

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 Ebola Glycoprotein , Sino Biological , 40459-V08H.

Techniques: Recombinant, Isolation, Cell Isolation, Chromatography, Software, Luminex