The rapid emergence of the COVID-19 pandemic, and the SARS and MERS epidemics within the past two decades highlight coronaviruses as critically important human pathogenic viruses with pandemic potential. Our strategy is to utilise nanobodies to disrupt coronavirus entry. Nanobodies are modular, easily adaptable constructs with unique attributes: small size, high stability, quick clearance and ease of production making them a desirable tool in therapeutic development
To develop more potent neutralising nanobodies, we have immunised an alpaca with the SARS-CoV-2 Omicron Ba1 spike in a prime-boost immunisation strategy to analyse the expansion of nanobody clonal groups over time. Due to the dynamic and ever-changing mutational landscape resulting in immune evasion against COVID-19, omicron variants of SARS-CoV-2 pose as an ideal candidate for investigating this hyperimmunization strategy.
Here we have completed several phage display campaigns against a panel of Omicron variants using nanobody libraries derived from alpacas immunised with recombinant Omicron Ba1 spike. Using next-generation sequencing, we identify collections of nanobodies that recognize multiple Omicron variants. We have identified a collection of nanobodies from these libraries and characterised their ability to bind to SARS-CoV-2 Ba5 spike subunits.
Of 94 nanobodies, we found 22 RBD, 20 NTD and 27 S2 binders, giving us coverage of all spike domains. Using biolayer interferometry, 12 nanobodies were shortlisted from the cohort comprising of 3 nanobodies selected as high affinity binders for each domain. Structural characterisation of these nanobodies will allow us to determine the recognised epitopes and assess their resilience in the face of antigenic variation. Finally, we will compare nanobodies from the first and second immunisations using MNV and BLI to determine whether this immunisation strategy improves nanobody affinity and breadth.