Neutralization of Crimean-Congo hemorrhagic fever pseudotyped virions with heavy chain antibodies.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus that causes severe hemorrhagic fever with high mortality rates in humans. No licensed vaccines or efficacious antiviral therapies are currently available. Here, we identified seven heavy chain antibodies targeting CCHFV Gc, which consist of heavy-chain variable domain (VHH) fused to human IgG1 Fc region (VHH-Fc). These VHH-Fc antibodies exhibited neutralizing activity against both recombinant vesicular stomatitis virus (VSV)-vectored CCHFV pseudoviruses and CCHFV transcription- and entry-competent virus-like particles (tecVLPs). Among these, N025 achieved the most potent pseudovirus neutralization, while N013 showed remarkable efficacy in tecVLP systems, with IC(50) values of 22.7 ng/mL and 33.0 ng/mL, respectively. AlphaFold3 structural predictions revealed that all characterized VHH-Fc antibodies target epitopes within Domain II of the Gc protein, with partial or complete overlap with the fusion loop region. Alanine scanning mutagenesis confirmed the functional significance of these epitopes, with N013 showing the highest binding energy change (ΔΔG = 25.36 kcal/mol) and moderate competition with a known fusion loop-targeting antibody. Sequence conservation analysis across representative CCHFV strains from different genetic lineages demonstrated complete conservation of the N013 and N025 epitopes, suggesting potential for broad-spectrum neutralizing activity. Together, our findings provide a novel strategy for developing CCHFV therapeutics and identify promising antibody candidates that could inform future broad-spectrum antiviral development efforts.