VHH DIMER P559-R45L NEUTRALIZES SARS-CoV-2 BY STABILIZING SPIKE IN UP CONFORMATION

ABSTRACT

Background: SARS-CoV-2 Omicron subvariants are highly resistant to vaccineinduced immunity and therapeutic monoclonal antibodies. We previously reported anti-SARS-CoV-2 spike alpaca nanobodies (VHHs) P86 and P17 that potently neutralize the wild type and VOCs from Alpha to Omicron BA.1 and BA.2, but not Omicron subvariants after that such as BA.4/5. Thus, we tried to establish a new VHH that can neutralize all the variants including BA.4/5. Method(s) We developed VHH trimers and heterodimers based on the structural and computational analysis of Delta spike-immunized alpaca VHH library. We tested representative VHHs against SARS-CoV-2 spike by pseudovirus assays and generated VHH heterodimers. We further obtained Cryo-EM structure of Spike trimer and VHH monomer or heterodimer. Result(s) First, we generated series of P86 mutants to counteract L452R mutation in Delta or Omicron BA.5 subvariants and found that P86 R45L was most potent against D614G with an IC50 of 0.03 mug/mL. From the Delta spike-immunized VHH library, we also identified that homo-trimer of a new clone P559 neutralized SARS-CoV-2 Delta and Omicron BA.5 variants with an IC50 of 0.077 and 0.54 mug/mL, respectively. We finally generated P559-R45L heterodimer that neutralized all the variants so far including Omicron BA.5 with an IC50 of 0.39 mug/mL. Cryo-EM structure revealed that three molecules of P559- R45L heterodimer bridged two RBD molecules in the spike trimer and stabilized spike timers with RBD in the up conformation. Conclusion(s) We developed VHH P559-R45L heterodimer that potently neutralized all the variants including Omicron subvariants through unique structural interaction.