Characterisation of nanobodies directed against emerging viruses

Sammanfattning: The emergence of new viral pathogens, such as SARS-CoV-2, or re-emergence of known pathogens, like CHIKV, point out the need for further understanding of the biology behind viruses, as well as the urgent need for the development of therapeutic and diagnostic tools. Nanobodies, small antigen-binding fragments derived from camelid heavy-chain antibodies, have gained attention for their use in viral research due to their wide range of applications: from the study of protein-protein interactions, uncovering of new viral targets, to the generation of new diagnostic tools or therapeutics. In paper I, we isolated a nanobody, Ty1, targeting the receptor binding domain (RBD) of SARS-CoV-2. We showed the ability of Ty1 to neutralise SARS-CoV-2 pseudotyped lentivirus potently (IC50 of 0.77 μg mL-1). The highly neutralising ability of Ty1 was likely due to its ability to bind the RBD in the ‘up’ and ‘down’ conformations, causing direct blocking to the cellular receptor and steric hindrance, respectively. Moreover, staining of SARS-CoV-2-infected cells with Ty1 confirmed its high specificity. In paper II we made use of a novel and rapid strategy to create nanobody multimers. We first functionalised the nanobodies using sortase A ligation to attach click chemistry functional groups. Then, the functionalised nanobodies were used to create C-to-C terminal bi- and tetravalent nanobody constructs by Cu-free strain-promoted azide-alkyne click chemistry (SPAAC). The bivalent and tetrameric nanobody constructs showed an increased potency with respect to the monomeric Ty1 of 150-fold and 4000-fold, respectively. This was true both for SARS-CoV-2 spike pseudotyped lentivirus and infectious SARS-CoV-2. In paper III, we generated nanobodies targeting the spike complex of CHIKV. We used a combinatory immunisation strategy with a cDNA prime followed by a protein boost. The CHIKV spike complex is formed by homotrimers of heterodimers of the E1 and E2 proteins. While E2 binds to the cellular receptor, E1 is responsible for the fusion of viral and cellular membranes, both essential steps of viral entry. We made use of a bivariate mining approach coupled to NGS and calculation of enrichment (fold difference in frequency between basal and enriched libraries) for the quick selection of nanobodies targeting either protein. We identified 12 nanobodies that detected cells infected with the 3 CHIKV lineages (ECSA, Asian and WA). Surprisingly, neutralisation of the ECSA and Asian genotypes was below 50% for all tested nanobodies, while 2 nanobodies, Dy010 and Dy059 could neutralise the WA lineage above 50% with PRNT50 values of 563 and 722 nM, respectively. Fusion to an Fc fragment produced an increase in potency of 130- and 63-fold for Dy010 and Dy059. Moreover, 4 of the nanobodies, Dy009, Dy025, Dy027 and Dy201 cross-reacted with other alphaviruses including ONNV, RRV and SFV, while one nanobody, Dy007, showed great specificity for CHIKV. These nanobodies expand the toolbox for research of this important human pathogen and could form a basis for the development of therapeutic or diagnostic tools.