Generation of synthetic Acinetobacter baumannii specific nanobodies

The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development. Sequencing of phagemid VHH inserts derived from phage display selection after the second round of phage display. Phage displayed VHHs were selected for binding to either control targets (GFP and V. cholerae) or A. baumannii. A. baumannii binding dataset was filtered by each of the three CDRs (peptide1,2,3) to identify VHHs completely unique to the A. baumannii dataset compared to the control GFP and V. cholerae datasets. See supplementary file "Filtered A.baumannii Binding VHH.csv" linked to the Series record.