Generation of Synthetic Acinetobacter baumannii-Specific Nanobodies

Abstract

The bacterial pathogen Acinetobacter baumannii is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that can 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 the selection of lead candidate nanobodies. Using monoclonal phage display, we validated the binding of lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that specifically bound select A. baumannii strains compared to other common drug-resistant pathogens. These findings support the potential for nanobodies to selectively target A. baumannii and the identification of lead candidates for future investigation.

Keywords: Acinetobacter baumannii; drug resistance; nanobodies; phage display; sequencing.

Figure 1

A. Schematic of the VHH scaffold used on top with the amino acid sequence below. Blue represents the framework region and red corresponds to CDR regions. The Xs in the amino acid sequence represent randomized CDR amino acids based on the Moutel et al. design. B. Flow diagram of phage panning approach to identify A. baumannii-specific nanobodies.

Figure 2

Validation of nanobody leads. A. Monoclonal phage whole-cell ELISA results for the 14 lead nanobodies. Nanobody binding to A. baumannii was assessed by comparison with binding to the control antigen GFP. Error bars represent the standard deviation of three biological replicates. Significance in binding to AB5075 over GFP was determined by unpaired t tests (*p < 0.05). B. Coomassie-stained gel of size exclusion purified nanobodies and a protein standard with the 14 kDa band of the ladder labeled. The expected size of nanobodies from our library is ∼14 kDa. C. Relative binding of purified nanobodies R3_3, Ng2, Ng3, and Ng8 at 5 μM to AB5075 by whole-cell ELISA. Each nanobody signal was normalized and compared to to the background binding of secondary antibody alone. Significance of binding was determined by one-way ANOVA with Dunnet’s multiple comparisons (****p < 0.0001). Error bars represent the standard deviation.

Figure 3

A. Relative binding of R3_3 to A. baumannii and non-baumannii strains. Nanobodies signals were normalized to R3_3 binding to AB5075. Significance was assessed via a one-way ANOVA with Tukey test. Each A. baumannii strain designated as significant was determined to be significantly different from each non-baumannii strain (*p < 0.0001). Error bars represent the standard deviation. B. Binding of R3_3 to AB5075 and isogenic itrA1::Tn capsule mutant. Significance was determined by unpaired t test (* p < 0.05). Error bars represent the standard deviation.