Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. Structural information is lacking in regard to the CCHFV membrane fusion glycoprotein Gc—the main target of the host neutralizing antibody response—as well as antibody–mediated neutralization mechanisms. We describe the structure of prefusion Gc bound to the antigen-binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show the two Fabs acting in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The structures also revealed the neutralization mechanism of previously reported antibodies against CCHFV, providing the molecular underpinnings essential for developing CCHFV–specific medical countermeasures for epidemic preparedness.

Fig. 1.. Structures of CCHFV Gc.

A) Organization of the CCHFV glycoprotein precursor B) Mechanism of bunyavirus class II membrane fusion proteins. C) X-ray structure of the CCHFV Gc ectodomain in post-fusion conformation. The front protomer is colored by domains and the trimer axis is shown in light blue. Secondary structure elements and disulfide bonds (green numbers) are labeled. An orthonairovirus-specific insertions cluster (IC) is depicted in brown. D) X-ray structure of the CCHFV Gc monomer in complex with the ADI-37801 and ADI-36121 Fabs.

Fig. 2.. ADI-37801 binds HMIS residues required for Gc driven syncytia formation.

A) The CCHFV HMIS of the post-fusion trimer (left) and in complex with ADI-37801 (right). In the left panel, W1191, W1197 and W1199, mutated to obtain the crystals, have been modeled for clarity. B) The hantavirus fusion loops in the post-fusion trimer forming the HMIS (left, PDB:6y68, MPRLV structure) and in the pre-fusion Gn/Gc heterodimer, where the HMIS is not formed (right, PDB:6y62) (15). C) Fusion loop sequences of CCHFV Gc with consensus sequence logo for the Orthonairovirus (top) and Orthohantavirus (bottom) genera. The bar chart shows the exposed surface area per residue in pre- (hantavirus Gc) and post-fusion (CCHFV and hantavirus Gc) structures. The accessible and buried surface per residue are represented in grey and black, respectively. Non-polar residues are black, acidic red, basic blue and cysteines green. D) CCHFV Gc-induced syncytia formation by wild-type and indicated mutant Gc at neutral and acidic pH. The transfected cell surface expression is shown for each mutant below. E) Details of two alternative conformations of the N-tail and a pH-sensitive salt bridge between domains I and III. The helical conformation (top) is dominant, whereas the β-hairpin (bottom) is well defined in only two of the six polypeptide chains in the asymmetric unit of the monoclinic crystals obtained at pH 7.5. The view is as in Fig. 1C. F) Interface between the ADI-37801 CDRs and the Gc fusion loops. The antibody heavy and light chain CDRs are respectively colored blue and gray. CCHFV Gc is colored orange (cd loop) and yellow (bc loop). Polar interactions denoted as dashed lines. G) BLI sensorgrams showing binding kinetics of CCHFV Gc¹⁵⁷⁹ to ADI-37801 at pH 7.5 (top) or pH 5.5 (bottom).

Fig. 3.. ADI-36121 epitope is buried at the trimer interface of the post-fusion hairpin.

A) The CCHFV Gc monomer in complex with the ADI-36121 Fab. B) The CDRs interacting with the Gc domain II base. Green and gray indicate heavy and light chains, respectively, and yellow indicates Gc domain II. Polar interactions are shown by dashes. C) Superposition of the ADI-36121 complex with the Gc post-fusion trimer. The trimer’s front protomer is shown in ribbons colored by domains and the flanking protomers as a white surface. D) One trimer protomer shown as surface colored by domains with the trimer interface outlined and the ADI-36121 footprint superposed in green, illustrating that the epitope is occluded in the trimer. E) BLI sensograms showing binding kinetics of the monomeric fraction (top) or the trimeric fraction (bottom) of CCHFV Gc¹⁵⁷² W3 to ADI-36121 at pH 7.5. F) BLI sensograms showing binding kinetics of CCHFV Gc¹⁵⁷⁹ to ADI-36121 at pH 7.5 (top) or pH 5.5 (bottom). See Materials and Methods for details of the constructs used.

Fig. 4.. The epitopes of CCHFV-neutralizing human antibodies map to Gc surfaces involved in driving membrane fusion.

A) Antigenic sites mapped on the surface of one CCHFV Gc protomer within the post-fusion trimer. The trimer axis is shown in light blue. Only the front Gc subunit is shown in the right panel, after a 180° rotation about the trimer axis. The trimer interface is outlined in black. B) Sequence variability across 15 representative CCHFV strains (Fig. S3) color-plotted on the Gc surface. C) Sequence variability across 14 species in the Orthonairovirus genus (Fig. S4).