Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Abstract

ABSTRACTOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of theMycobacterium tuberculosisantigens ESAT6, Ag85B, and Rv2660c were targeted to the surface ofEscherichia coliOMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolRΔtolAderivative of attenuatedSalmonella entericaserovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by theM. tuberculosisantigens and epitopes fromChlamydia trachomatismajor outer membrane protein (MOMP). Also, we showed that delivery ofSalmonellaOMVs displaying Ag85B to antigen-presenting cellsin vitroresults in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.