Rapid Antiviral Screening using hyperPhage display
The variable domain of heavy chain antibodies (VHH) or Single-domain antibodies (SdAbs) are the smallest antigen-binding domain derived from camelid HCAbs (heavy chain antibodies). In the last three decades, they have been extensively utilized 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 their robustness and high throughput, such methods often require additional downstream experiments to identify virus-neutralizing VHHs. Here, we describe an innovative, high-throughput functional screening method, RASP, that directly identifies neutralizing VHHs. We select antiviral VHHs using purified antibody-displaying phages in virus-neutralization assays. 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. We demonstrated a dose-dependent blockade of viral infection. We further improved our method by utilizing the pentavalent display feature of hyperphages. We showed that hyperphages were superior to helper 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. Further, we benchmarked RASP against established phage ELISA and next-generation sequencing methods. Overall, we conclude that RASP can be used either as a standalone platform or in conjunction with conventional screening methods to accelerate the discovery of antiviral VHHs.
The variable domain of heavy chain antibodies (VHH) or Single-domain antibodies (SdAbs) are the smallest antigen-binding domain derived from camelid HCAbs (heavy chain antibodies). In the last three decades, they have been extensively utilized 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 their robustness and high throughput, such methods often require additional downstream experiments to identify virus-neutralizing VHHs. Here, we describe an innovative, high-throughput functional screening method, RASP, that directly identifies neutralizing VHHs. We select antiviral VHHs using purified antibody-displaying phages in virus-neutralization assays. 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. We demonstrated a dose-dependent blockade of viral infection. We further improved our method by utilizing the pentavalent display feature of hyperphages. We showed that hyperphages were superior to helper 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. Further, we benchmarked RASP against established phage ELISA and next-generation sequencing methods. Overall, we conclude that RASP can be used either as a standalone platform or in conjunction with conventional screening methods to accelerate the discovery of antiviral VHHs.