Llama-derived nanobody VHH21 destroys the spike-trimer of SARS-CoV-2

Neutralizing antibodies that merely block receptor binding are losing ground against heavily mutated SARS-CoV-2 Omicron sub-variants. A new approach now exploits a llama-derived nanobody—VHH21—that does not just bind the spike (S) protein but actively tears the trimer apart within seconds. Bactrian camels were immunized with a cocktail of recombinant S proteins from ancestral and VOC strains, yielding a high-diversity VHH phage library. Multi-round biopanning and BLI screening singled out six nanomolar-affinity binders; VHH21, which spontaneously dimerizes, stood out by destroying 68 % of surface-immobilized S-trimers in 20 min, far outperforming ACE2 or conventional nanobodies.

Functional validation combined fluorescence super-resolution, SEC, negative-stain EM and real-time TIRF imaging. Within milliseconds after contact, VHH21 triggered release of fluorescent S1 subunits and left dissociated S2 stalks, effectively converting the metastable prefusion trimer into post-fusion debris. Cryo-EM at 3.3 Å revealed two VHH21 dimers wedged between neighboring RBDs in a half-up/closed configuration, locking the spike in an open state and preventing refolding. The epitope—loops 345-346 and 440-450 on the outer face of the RBD—lies outside the ACE2 receptor-binding motif, explaining why VHH21 retains nanomolar affinity against Alpha, Beta, Gamma, Delta and Omicron BA.1-BA.5 S1 proteins and neutralizes corresponding lentiviral pseudoviruses with EC50 values in the low nanomolar range.

Sequence alignment and structural modelling show the epitope is conserved across SARS-CoV-2 lineages and largely preserved in SARS-CoV and bat CoV RaTG13, although a few substitutions weaken binding to MERS-CoV. Because mutations that underlie immune escape rarely alter this ridge, VHH21 offers a breadth unmatched by current monoclonals. The spontaneous dimerization mimics engineered bivalent constructs, delivering avidity without chemical conjugation and reducing the risk of viral escape. Small size, high stability and simple bacterial production make VHH21 an attractive candidate for intranasal prophylaxis or inclusion in multivalent therapeutics.