Llama-derived Nanobody Vhh21 Destroys The Spike-trimer Of Sars-cov-2

Neutralizing antibodies that simply artifact receptor binding are losing crushed against heavy mutated SARS-CoV-2 Omicron sub-variants. A caller attack now exploits a llama-derived nanobody—VHH21—that does not conscionable hindrance nan spike (S) macromolecule but actively tears nan trimer isolated wrong seconds. Bactrian camels were immunized pinch 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 retired six nanomolar-affinity binders; VHH21, which spontaneously dimerizes, stood retired by destroying 68 % of surface-immobilized S-trimers successful 20 min, acold outperforming ACE2 aliases accepted nanobodies.

Functional validation mixed fluorescence super-resolution, SEC, negative-stain EM and real-time TIRF imaging. Within milliseconds aft contact, VHH21 triggered merchandise of fluorescent S1 subunits and near dissociated S2 stalks, efficaciously converting nan metastable prefusion trimer into post-fusion debris. Cryo-EM astatine 3.3 Å revealed 2 VHH21 dimers wedged betwixt neighboring RBDs successful a half-up/closed configuration, locking nan spike successful an unfastened authorities and preventing refolding. The epitope—loops 345-346 and 440-450 connected nan outer look of nan RBD—lies extracurricular nan 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 pinch EC50 values successful nan debased nanomolar range.

Sequence alignment and structural modelling show nan epitope is conserved crossed SARS-CoV-2 lineages and mostly preserved successful SARS-CoV and bat CoV RaTG13, though a fewer substitutions weaken binding to MERS-CoV. Because mutations that underlie immune flight seldom change this ridge, VHH21 offers a breadth unmatched by existent monoclonals. The spontaneous dimerization mimics engineered bivalent constructs, delivering avidity without chemic conjugation and reducing nan consequence of viral escape. Small size, precocious stableness and elemental bacterial accumulation make VHH21 an charismatic campaigner for intranasal prophylaxis aliases inclusion successful multivalent therapeutics.