Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

Abstract

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

Nanobodies in the limelight: Multifunctional tools in the fight against viruses

“…To isolate nanobodies with specific functions, such as antiviral activity, phenotypic screens based on cell survival of viral infections have been described [11,30] and have the advantage to rapidly test a larger selection of nanobodies, significantly increasing the chance of identifying exceptionally potent candidates. We and our colleagues have recently bridged the gap between phage display and phenotypic testing by using a multivariate phage selection combined with next-generation sequencing and sequence enrichment analysis, followed by high-throughput neutralization testing [31]. In an immune library, nanobodies will have varying frequencies, in many cases independent on their binding affinity.…”

“…Although this might be beneficial for many applications, including diagnostic imaging and cancer treatment (reviewed in [80,81]), an increased half-life may be beneficial and preferred for prophylactic therapy or treatment of viral (and other) diseases. Methods to increase the half-life of nanobodies include multimerization, PEGylation [82], serum albumin fusion, or fusion to another nanobody specific for serum albumin [14,31,83], and genetic fusion to an Fc domain [84].…”

“…In our approach, phage pools are sequenced prior to and after each round of panning and the increase in frequency is used for the selection of nanobody candidates. Interesting nanobodies can then be synthesized based on sequence information without relying on physical isolation of the clones from the libraries [31]. This approach enabled testing >70 nanobodies from diverse lineages to ultimately identify the most potently SARS-CoV-2 neutralizing candidates from the entire repertoire.…”

“…Epitope mapping can also be achieved by hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS), which measures the difference of deuterium uptake between ligand bound and unbound antigens. This approach appears relatively fast compared to other structural approaches and permits epitope characterization for a larger selection of nanobodies, as recently demonstrated for numerous SARS-CoV-2 RBD-specific nanobodies [31, 68].…”