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

Abstract

iOuter 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 the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ⌬tolR ⌬tolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results 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.

Immune modulation by bacterial outer membrane vesicles

“…Furthermore, the ability of OMVs to activate the innate immune system 14,20,22,24,25,69,114 , together with their particulate nature, provides these nano particles with their own adjuvant activity, as they are able to enhance antibody and T cell responses to antigens 76,83,87,115 . Finally, OMVs are very versatile as they can be bioengineered to express any chosen antigen 77,112,[116][117][118] and they can be manipulated to reduce their endotoxicity [119][120][121] . Despite the enormous vaccine potential of OMVs, it has taken more than 20 years for an OMV-based vaccine to be licensed for human use.…”

Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway

“…Although most autodisplay work has been conducted in E. coli , a few research groups have validated the use of autotransporter‐based surface display in alternative organisms (Tozakidis et al ., ). Recent studies have extended the basic concept of autodisplay and shown that polypeptides fused to autotransporters can also be presented on the surface of OM vesicles and bacterial ghosts (Daleke‐Schermerhorn et al ., ; Hjelm et al ., ). Taken together, the numerous examples of autodisplay that have appeared in the literature demonstrate that a remarkable diversity of polypeptides can be secreted effectively by the autotransporter pathway.…”

Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

“…In addition, expressed heterologous proteins that had the ability to anchor to the surface of E. coli cells were also presented on the derived OMVs18. Technologically, fusion with ClyA (a pore-forming hemolytic protein), HBP or AIDA can bring exogenous proteins to the surface of OMVs1920212223. Moreover, exogenous proteins can also be presented in the inner lumen of OMVs by using an appropriate leader protein17.…”