Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38

Abstract

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, unique to Nairoviridae, is a target of protective antibodies, but extensive mapping of the human antibody response to GP38 has not been previously performed. Here, we isolated 188 GP38-specific antibodies from human survivors of infection. Competition experiments showed that these antibodies bind across five distinct antigenic sites, encompassing eleven overlapping regions. Additionally, we reveal structures of GP38 bound with nine of these antibodies targeting different antigenic sites. Although GP38-specific antibodies were non-neutralizing, several antibodies were found to have protection equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and inform the development of broadly effective CCHFV antibody therapeutics.

Keywords: CCHFV; Crimean-Congo hemorrhagic fever virus; GP38; Nairoviridae; antibody therapeutics; human monoclonal antibody; tickborne; viral glycoprotein.

Figure 1.. Isolation of monoclonal antibodies and genetic signatures of the B cell repertoire.

(A) Flow cytometric analysis of avid-rGP38 binding of B cells (top panel) and IgM and IgD expression on the surface of rGP38-reactive B cells (bottom panel). Donor 1 PBMCs were gated on CD3⁻CD8⁻CD14⁻CD16⁻PI⁻CD19⁺ lymphocytes; Donor 5 and 6 PMBCs were gated on CD3⁻CD8⁻CD14⁻CD16⁻PI⁻CD19⁺CD20⁺ lymphocytes. (B) Single concentration BLI binding analysis of 188 antibodies to IbAr10200 rGP38 protein. Dotted horizontal lines indicate antibodies for which no binding (N.B.) was detected or for which poor fits (P.F.) to the binding model were obtained (C) Analysis of VH nucleotide substitutions of each of the mAbs. Statistical comparison was performed using the Mann-Whitney Test (*p<0.05). (D) Clonal lineage analysis of B cells from Donors 1, 5, and 6. B cells with antibody sequences that had the same V heavy and V light germline gene usage and CDRH3s of the same length with >80% nucleotide sequence identity were considered to be clonally related. Colored slices represent the percentage of clones from each donor that are related. Total number of isolated mAbs from each donor are indicated in each corresponding circular diagram. (E) Analysis of CDRH3 lengths of mAbs from the three donors. (F) Heatmap of VH and VL germline gene usage across mAbs from the three donors; shades of green represent the number of B cells that used a certain germline gene pairing. (G) Analysis of VK1–29, VK3–20, and VL3–21 germline gene usage broken down by donor. (H) Analysis of VH4–4/VK3–20 and VH3–48/VL3–21 germline gene usage broken down by donor.

Figure 2.. Competition-binning profile of GP38 antibodies.

(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 one of 11 overlapping bins and the Unknown/Weak Affinity mAbs are shown in gray (Supplementary Table 1). Distribution of overlapping bins of the antibody panel (left) and broken down by donor (right). Total number of mAbs is indicated in the circular diagram and total mAbs from each donor are indicated above the bar graph.

Figure 3.. Analysis of antibody binding to GP38 proteins derived from six CCHFV isolates.

(A) Matrix of percent sequence identity of GP38 amino acid residues across six CCHFV isolates. (B) Single concentration BLI binding analysis of 188 antibodies to the six rGP38 proteins as a whole panel (left) and broken down by bin (right). Shades of green represent the number of rGP38 proteins bound by a single antibody. Total number of mAbs is indicated in the circular diagram and total mAbs from each donor 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 apparent binding affinities are in dark green and the lowest apparent binding affinities are in white. Calculated K_Ds appear in each rectangle of the heat map; for samples that were off-rate limited, K_Ds are denoted as < the calculated K_D. The one interaction for which a curve could not be fit is denoted as P.F.

Figure 4.. CCHFV tecVLPs and authentic virus neutralization assays of GP38 antibodies.

(A) Individual neutralization curves for CCHFV IbAr10200 tecVLPs, as measured by the reduction in luciferase activity compared to 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 to 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.

Figure 5.. Structural characterization of GP38 antibodies.

(A) Yeast-based mapping strategy of select antibodies to identify critical binding residues on GP38. The percentage of antibody binding retained by each GP38 variant is colored according to the key. Critical residues are defined as mutations that led to a binding disruption of 75% or more and are colored by the assigned antigenic site. (B) Yeast-based critical residues mapped on the surface of GP38 (PDB ID: 6VKF): bin I (blue, residues Val385, Pro388), bin II (green, residues Gly371, Leu374, Ile375, Lys404, Lys488, Leu499), bin III (yellow, residues Ser428–Ala429, Asp444–Asp446, Lys474–Leu475, Asp477), bin IV (orange, residues Ile253–Leu255, Leu257, Lys262, Gly266, Glu277, and Glu281), bin V (red, residues Glu285, Arg289, and Gly292). (C) Composite structure of GP38 (PDB ID: 6VKF) bound with representative antibodies. GP38 is shown as a rainbow ribbon, and Fabs as molecular surfaces. Heavy chains are colored to represent the 5 non-overlapping bins, and light chains are white. Black dashed lines highlight the vertical alignment of Fabs along one plane (left) and the opposing binding directions to another plane (right).

Figure 6.. High-resolution structures of GP38–antibody complexes.

(A) Crystal structure of GP38 bound with ADI-46143 (bin I, blue) with heavy chain interactions (top) and light chain interactions (bottom). (B) Cryo-EM structure of GP38 bound with ADI-58048 (bin II, green, left) and ADI-46152 (bin IV+V, red, right). Heavy chain interactions (top left, top right) and light chain interactions (bottom left, bottom right) are shown in the insets. (C) Crystal structure of GP38 bound with c13G8 (bin IV+V, red) with heavy chain interactions (top) and light chain interactions (bottom). For all panels, hydrogen bonds are indicated by black dashed lines. GP38 residues are labeled in white text with a black outline.

Figure 7.. Protective efficacy of lead mAbs in two murine models of lethal CCHFV challenge.

(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). (A) Survival curves (vehicle versus 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 minutes post-exposure (n=5–6 mice per study; represented by 2 replicate studies). (D, G, J) Survival curves, (E, H, K) associated mean weight loss, and (F, I, L) clinical scores.