RASP: rapid antibody functional screening by pentavalent phage display

The heavy-chain antibody variable domain (VHH) is the smallest antigen-binding domain of such antibodies, which are derived from camelids. In the past three decades, VHHs, which are also called single-domain antibodies, have been extensively used to target pathogens and/or toxins. Conventional screening methods, such as phage display, rely only on antibody-antigen binding as the sole criterion for selection. Despite being robust and high-throughput, such methods often require additional downstream experiments to identify VHH that neutralize their target. Here, we describe an innovative, high-throughput functional screening method, Rapid Antibody functional Screening by Pentavalent phage display (RASP), that incorporates purified antibody-displaying phages for virus neutralization assays, and thus can be used to directly identify neutralizing VHHs. As a proof-of-concept, we first displayed previously identified neutralizing VHHs specific for the spike proteins of SARS-CoV-2 and respiratory syncytial virus on phages and demonstrated a dose-dependent blockade of viral infection. We further improved our method by utilizing the pentavalent display feature of hyperphages. We showed that hyperphage-derived VHH phages were superior to helper phage-derived VHH phages in assaying viral neutralization potential. Thereafter, we applied RASP to identify multiple candidates by screening a semi-synthetic VHH library against recombinant vesicular stomatitis viruses pseudotyped with spike glycoproteins from SARS-CoV-2, Junin virus, and Ebola virus, featuring as case studies in antiviral antibody discovery. Further, we benchmarked RASP against established phage ELISA and next-generation sequencing methods. Overall, we successfully used RASP in the context of the discovery of antiviral VHHs, highlighting its broader applicability as a platform that can be used either in isolation or in conjunction with other functional screening methods to accelerate the discovery of antiviral VHHs.