cchfv gp38  (ATCC)

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Matrix of competition-binning experiments. For on-yeast competition experiments (top left quadrant), results are displayed with surface-presented IgGs on the y axis and competitive pre-complexed Fabs on the x axis. For BLI competition assays (the other three quadrants), binning was performed in an IgG vs. IgG format. (B) Binning analysis of on-yeast competition assays of all 188 antibodies; each color represents 1 of 11 overlapping bins and the Unknown/Weak Affinity mAbs are shown in gray . Distribution of overlapping bins of the antibody panel (left) and by each donor (right). Total number of mAbs is indicated in the circular diagram and total mAbs from each donor are indicated above each bar graph.

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques:

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Matrix of percent sequence identity of GP38 amino acid residues (AA) across six CCHFV isolates. (B) Single concentration BLI binding analysis of 188 antibodies to the six rGP38 proteins as a whole panel (top) and broken down by bin (bottom). Shades of green represent the number of rGP38 proteins bound by a single antibody (from 0 in gray to 6 in darkest green). Total number of mAbs is indicated in the circular diagram and total mAbs from each bin are indicated above the bar graph. (C) Carterra system HT-SPR binding analysis of six lead antibody candidates binding to six rGP38 proteins. The highest binding affinities are in dark green and the lowest binding affinities are in white. Calculated K D values appear in each rectangle of the heatmap; for samples that were off-rate limited, K D values are denoted as < the calculated K D . The one interaction for which a curve could not be fit is denoted as P.F.

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques: Sequencing, Concentration Assay, Binding Assay

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Neutralization curves for CCHFV IbAr10200 tecVLPs, as measured by the reduction in luciferase activity compared with no-antibody treatment on Vero cells. (B–E) Neutralization curves of the indicated mAbs against authentic (B) CCHFV IbAr10200, (C) CCHFV Afg09, (D) CCHFV Turkey2004, and (E) CCHFV Oman as measured by the reduction in infection compared with no-antibody treatment on SW-13 cells. The average of n = 3 replicates each from two independent experiments ( n = 6 total) is shown for all neutralization curves.

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques: Neutralization, Luciferase, Activity Assay, Infection

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A–C) IFNAR1 −/− mice were treated with the indicated mAbs at 1 mg/mouse 1 and 4 days post-challenge (2 mg total; n = 10 mice per group) with IbAr10200. (A) Survival curves (vehicle vs. test mAb), (B) associated mean weight loss, and (C) clinical score data are shown. (D–L) STAT1 −/− mice were challenged with (D–F) CCHFV-Afg09, (G–I) CCHFV-Turkey2004, or (J–L) CCHFV-Oman and then treated with 0.2 mg/mouse of mAb or vehicle 30 min post-exposure (n = 5–6 mice per study; represented by 2 replicate studies). (D, G, and J) Survival curves. (E, H, and K) Associated mean weight loss. (F, I, and L) Clinical scores are defined as: 1 = decreased grooming and/or ruffled fur; 2 = subdued behavior when un-stimulated; 3 = lethargy, hunched posture, and/or subdued behavior even when stimulated; 4 = bleeding, unresponsiveness, severe weakness, or inability to walk. Mice scoring a 4 were considered moribund and were humanely euthanized based on IACUC-approved criteria (denoted as X over white).

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques:

Journal: NPJ Vaccines

Article Title: GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41541-023-00663-5

Figure Lengend Snippet: Characterization of rhabdoviral-vectored CCHFV vaccines through Immunofluorescence ( a , b ), flow cytometry ( c , d ), SDS PAGE protein gel ( e ), Western Blot ( f ), and Growth Curves ( g – i ). Vero E6 cells were infected at MOI 0.01 and fixed after 72 or 24 h for RABVs and VSVs, respectively. Cells were stained with α-RABV-G 4C12 (purple) and α-CCHFV-G C 11E7 ( a ) or α-CCHFV-GP38 13G8 ( b ) (red) and mounted with mounting media containing a nuclear DAPI stain (blue). In the merged images, GFP from VSV GFP is green, and areas where there is overlapping expression of RABV-G and the CCHFV glycoproteins are pink. Images were taken at 40X magnification with a 2X zoom. Scale bars represent 10 µm. c Vero E6 cells were infected at MOI 10 and fixed after 48 h for RABVs or infected at MOI 5 and fixed after 8 h for VSVs. Cells were probed for α-RABV-G 4C12 and α-CCHFV-G C 11E7 ( c ) or α-CCHFV-GP38 13G8 ( d ) and analyzed by flow cytometry. Assay was performed multiple times, and the graph is one representative experiment. e SDS PAGE protein gel of sucrose purified virions. 1 µg of each virus was loaded onto the gel and all native rhabdoviral proteins and foreign proteins are indicated by the arrows next to each gel. f Western blot of sucrose purified virions. 1 µg of each virus was loaded onto the gel and transferred to a nitrocellulose membrane for western blotting. Blots were either probed with α-CCHFV-GP38 13G8 (top panel), α-CCHFV-G C 11E7 (middle panel) or α-RABV-G 4C12 (bottom panel). g-i Multi-step and one-step growth curves. Cells were infected at MOI 0.01 for multi-step ( g ) or MOI 10 for one-step ( h , i ) growth curves and samples were titered in triplicate. Error bars represent standard deviation. Statistics are differences in titer compared to the parental vector for each growth curve (**** P < 0.0001; *** P < 0.0002; ** P < 0.0021; * P < 0.0332).

Article Snippet: Positive controls (when available) for each assay were as follows: α-CCHFV-GP38 13G8 at a starting concentration of 1 μg/mL for IgG Fc and IgG2b GP38 ELISAs; α-CCHFV-GP38 10E11 (BEI Resources, NR-40276) at a starting concentration of 1 μg/mL for IgG1 GP38 ELISAs; α-CCHFV-G C 11E7 at a starting concentration of 2 μg/mL for IgG Fc Gc ELISAs; α-RABV-G 1C5 (Abcam, Ab82460) at a starting concentration of 1 μg/mL for IgG Fc RABV-G ELISAs.

Techniques: Vaccines, Immunofluorescence, Flow Cytometry, SDS Page, Western Blot, Infection, Staining, Expressing, Purification, Virus, Membrane, Standard Deviation, Plasmid Preparation

Journal: NPJ Vaccines

Article Title: GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41541-023-00663-5

Figure Lengend Snippet: Immunogenicity study to look at antibody responses induced by each CCHFV vaccine. a Immunization and blood draw schedule for mouse studies. Groups of 5 mice were immunized with 10 µg/dose of BPL inactivated vaccines adjuvanted with 5 µg of PHAD in 2% SE per dose. Syringes represent immunizations, red blood drops indicate the days blood was taken and the skull denotes the conclusion of the study when the mice were sacrificed. Created with Biorender.com. b Table showing the vaccine groups used in this study and the symbols and colors used to denote each group and assay controls. c , e , g Group average ELISA curves for each antigen at the peak of the antibody response. Error bars represent standard deviation (SD). d , f , h EC 50 ELISA titers over time for each antigen. Error bars indicate the mean with SD for groups of 5 mice with samples run in duplicate. An ordinary one-way ANOVA with Tukey’s Multiple Comparison Test was used to determine statistical differences between groups at each time point. All groups with detectable antibody titers have 4-star significance compared to groups where no antibody titers were detected (**** P < 0.0001; *** P < 0.0002; ** P < 0.0021; * P < 0.0332; ns not significant). c , d α-CCHFV-GP38 ELISAs, e , f α-CCHFV-G C ELISAs, and g , h α-RABV-G ELISAs. ●, mouse 1; ■, mouse 2; ▲, mouse 3; ▼, mouse 4; ◆, mouse 5.

Article Snippet: Positive controls (when available) for each assay were as follows: α-CCHFV-GP38 13G8 at a starting concentration of 1 μg/mL for IgG Fc and IgG2b GP38 ELISAs; α-CCHFV-GP38 10E11 (BEI Resources, NR-40276) at a starting concentration of 1 μg/mL for IgG1 GP38 ELISAs; α-CCHFV-G C 11E7 at a starting concentration of 2 μg/mL for IgG Fc Gc ELISAs; α-RABV-G 1C5 (Abcam, Ab82460) at a starting concentration of 1 μg/mL for IgG Fc RABV-G ELISAs.

Techniques: Immunopeptidomics, Vaccines, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: NPJ Vaccines

Article Title: GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41541-023-00663-5

Figure Lengend Snippet: Isotype subclass ELISAs for each vaccine that had detectable antibodies in the CCHFV glycoprotein IgG Fc ELISAs. a , c EC 50 antibody titers for each isotype subclass. Error bars indicate the mean with standard deviation (SD) for groups of 5 mice with samples run in duplicate. b , d Isotype ratios comparing EC 50 titers of IgG2c or IgG2b to IgG1. Any animals with undetectable IgG1 were excluded from isotype ratio calculations. Lines represent median values. a , b GP38 isotype subclass ELISAs. c , d G C isotype subclass ELISAs. Mann–Whitney test was used to determine statistical differences between groups for each isotype. (**** P < 0.0001; *** P < 0.0002; ** P < 0.0021; * P < 0.0332; ns not significant).

Article Snippet: Positive controls (when available) for each assay were as follows: α-CCHFV-GP38 13G8 at a starting concentration of 1 μg/mL for IgG Fc and IgG2b GP38 ELISAs; α-CCHFV-GP38 10E11 (BEI Resources, NR-40276) at a starting concentration of 1 μg/mL for IgG1 GP38 ELISAs; α-CCHFV-G C 11E7 at a starting concentration of 2 μg/mL for IgG Fc Gc ELISAs; α-RABV-G 1C5 (Abcam, Ab82460) at a starting concentration of 1 μg/mL for IgG Fc RABV-G ELISAs.

Techniques: Standard Deviation, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41541-023-00663-5

Figure Lengend Snippet: Challenge study to determine the utility of a VSV-based surrogate challenge virus when looking at vaccine protective efficacy. a Experimental timeline. Groups of 10 mice, 5 male and 5 female, were immunized with 10 µg/dose of BPL inactivated vaccines adjuvated with 5 µg of PHAD in 2% SE per dose as indicated by the syringe with the rhabdovirus containing multiple glycoproteins. Challenge of 5E5pfu of surrogate virus is indicated by the syringe with a VSV with a singular set of glycoproteins. Red blood drops indicate the days blood was taken, and the skull denotes the conclusion of the study when any surviving mice were sacrificed. Created with Biorender.com. b Table of vaccine groups and representative colors. GP38 EC 50 titers pre-challenge ( c ) and post-challenge ( f ). Error bars indicate the mean with standard deviation (SD) for groups of 5 mice with samples run in triplicate. d Average group weight curves. Error bars indicate SD. e Viral RNA copies in the blood as determined by VSV-N qPCR. LOD, limit of detection. Error bars indicate the mean with SD. Results show the combination of two independent experiments; hollow symbols represent the first experiment and symbols with a black outline represent the second experiment. An ordinary one-way ANOVA with Tukey’s Multiple Comparison Test was used to determine statistical differences between groups at each time point for EC 50 titers and viremia ( c , e , f ). Two-way ANOVA with Tukey’s Multiple Comparison Test was used to determine statistical differences between groups for the weight curves ( d ). All comparisons between groups not listed on the EC 50 or weight change graphs had 4-star significant difference. (**** P < 0.0001; *** P < 0.0002; ** P < 0.0021; * P < 0.0332; ns not significant).

Article Snippet: Positive controls (when available) for each assay were as follows: α-CCHFV-GP38 13G8 at a starting concentration of 1 μg/mL for IgG Fc and IgG2b GP38 ELISAs; α-CCHFV-GP38 10E11 (BEI Resources, NR-40276) at a starting concentration of 1 μg/mL for IgG1 GP38 ELISAs; α-CCHFV-G C 11E7 at a starting concentration of 2 μg/mL for IgG Fc Gc ELISAs; α-RABV-G 1C5 (Abcam, Ab82460) at a starting concentration of 1 μg/mL for IgG Fc RABV-G ELISAs.

Techniques: Virus, Vaccines, Standard Deviation, Comparison