Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. KEY POINTS: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells.

Immunoproteomics Methods and Techniques.

The varied landscape of the adaptive immune response is determined by the peptides presented by immune cells, derived from viral or microbial pathogens or cancerous cells. The study of immune biomarkers or antigens is not new, and classical methods such as agglutination, enzyme-linked immunosorbent assay, or Western blotting have been used for many years to study the immune response to vaccination or disease. However, in many of these traditional techniques, protein or peptide identification has often been the bottleneck. Recent progress in genomics and mass spectrometry have led to many of the rapid advances in proteomics approaches. Immunoproteomics describes a rapidly growing collection of approaches that have the common goal of identifying and measuring antigenic peptides or proteins. This includes gel-based, array-based, mass spectrometry-based, DNA-based, or in silico approaches. Immunoproteomics is yielding an understanding of disease and disease progression, vaccine candidates, and biomarkers. This review gives an overview of immunoproteomics and closely related technologies that are used to define the full set of protein antigens targeted by the immune system during disease.