Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

Abstract

The virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live and serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated serovar Typhimurium cells displaying an adapted autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

“…Genetic dissection of the hepatitis B core antigen generated an exposed terminus, which was better able to display fusions on the VLP (SplitCore HBcAg) ( 8 ). However, sometimes compromises in genetically fused VLPs have decreased the chances of long-term success: if full domains cannot be genetically fused, fusion of VLPs to short peptides has often been tried or antigens were genetically dissected and distributed across the carrier ( 23 , 24 ). Such peptide-decorated VLPs can generate antibodies that cross-react with the parent protein.…”

“…SpyTag was first used for OMV display through expression of a SpyTag-OmpA fusion, allowing conjugation of an enzyme for detoxification of nerve agents ( 109 ). SpyTag was subsequently displayed on hemoglobin protease, yielding OMVs (30–200 nm in diameter) from E. coli and Salmonella Typhimurium displaying SpyCatcher-linked pneumococcal antigens ( 24 ). In addition, this platform was amenable to SnoopTag/SnoopCatcher antigen display ( 24 ).…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…In a proof-of-principle experiment, nanobodies were anchored on E. coli by means of the SpyTag/SpyCatcher ligation system. In this case bacteria displayed the SpyTag that was available for binding to SpyCatcher-fused anti-GFP antibodies [96]. Of course, the method can be expanded by selecting nanobodies specific for other antigens and could be for instance used to decorate Virus Like-Particles with nanobodies suitable for improving their targeted delivery [97].…”

Post‐translational Assembly of Protein Parts into Complex Devices by Using SpyTag/SpyCatcher Protein Ligase

“…Briefly reviewed by Banerjee and Howarth, modular vaccine assembly with the SpyTag/SpyCatcher system allowed the rapid production of experimental vaccines. Antigen presentation facilitated by coupling proteins to an outer‐membrane vesicle allowed multiple antigens to be displayed for optimal presentation to the immune system . This system was also previously used to irreversibly decorate virus‐like particles (VLPs) by post‐translational mixing with protein antigen (Figure A) .…”

“…Antigen presentation facilitated by coupling proteins to an outer-membrane vesiclea llowed multiple antigenst ob ed isplayed for optimal presentation to the immune system. [40] This system was also previously used to irreversibly decorate virus-like particles (VLPs) by post-translational mixingw ith protein antigen ( Figure 3A). [41,42] Injectiono f synthetic VLPs that were decorated with am alariala ntigen led to an improved immune response in mice.…”