Discovery and characterization of cross-reactive single-domain antibodies targeting key virulence factors of Salmonellaand Shigella

Abstract

Abstract Study Objective: Here, we utilize a nanobody yeast display library to identify nanobodies that target virulence factors in Shigellaspp. and Salmonella enterica typhimuriumand determine if these nanobodies (Nbs) can inhibit host cell invasion in vitro. Brief Statement of Methods: Using a synthetic yeast display library containing camelid Nbs with randomized CDR loops, we selected for binders to select Enterobacteriaceae virulence proteins, including IpaD/SipD, IcsA, and IcsP by MACS and FACS. Lead Nbs were cloned and expressed as monomeric VHH domains or Nb-Fc fusions. ELISA and flow cytometry surface staining was used to estimate affinity, and functional activity was assayed via a host cell invasion assay with Shigella flexneri, Shigella sonnei, and Salmonella enterica typhimurium. Summary of Results: Several high affinity Nbs were isolated with specificity for the targe proteins, and a subset of these could bind whole bacterial cells. Lead Nbs were binned into epitopes by competitive binding assays. The efficacy of Nbs against specific epitopes of specific virulence proteins in invasion assays were directly compared. Statement of Conclusions: A direct comparison of high-affinity Nb binders against various epitopes of IpaD/SipD, IcsA, and IcsP reveals that only a subset of epitopes are accessible on whole bacteria and able to inhibit bacterial invasion. These results will inform the development of virulence factor-based vaccines or other immunotherapies. Wellcome Trust (REISTANT Consortium)