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

Abstract

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, exclusive to Nairoviridae, is a target of protective antibodies and is a key antigen in preclinical vaccine candidates. Here, we isolate 188 GP38-specific antibodies from human survivors of infection. Competition experiments show that these antibodies bind across 5 distinct antigenic sites, encompassing 11 overlapping regions. Additionally, we show structures of GP38 bound with 9 of these antibodies targeting different antigenic sites. Although these GP38-specific antibodies are non-neutralizing, several display protective efficacy 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 may inform the development of broadly effective CCHFV antibody therapeutics.

Keywords: CCHFV; CP: Immunology; 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) and IgM and IgD expression on the surface of rGP38-reactive B cells (bottom). Donor 1 PBMCs were gated on CD3⁻CD8⁻CD14⁻CD16⁻PI⁻CD19⁺ lymphocytes; donors 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. The total number of isolated mAbs from each donor is indicated in each corresponding circular diagram.

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 1 of 11 overlapping bins and the Unknown/Weak Affinity mAbs are shown in gray (Table S2). 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.

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 (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.

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

(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.

Figure 5.. Structural characterization of GP38-specific 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: 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, Glu281), bin V (red, residues Glu285, Arg289, Gly292). (C) Composite structure of GP38 bound with representative antibodies. GP38 is shown as a rainbow ribbon and Fabs as molecular surfaces. Heavy chains are colored to represent the five 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, heavy chains are colored, light chains are gray, polar interactions are indicated by black dashed lines, and 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) 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).