Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

Take transfection polyplexes containing target plasmids bound to a polymer. The plasmids encode a viral antigen fused to a peptide tag and an affinity tag.

Incubate the polyplexes with mammalian cells, which internalize the polyplexes inside endosomes.

The polymer induces vesicle rupture. The released plasmids express the viral antigen, which is secreted extracellularly.

Harvest the supernatant containing the antigen and contaminants. Add a competing agent at a low concentration.

Load it onto an affinity chromatography column.

The affinity tags bind to the column resin, immobilizing the antigen, while the competing agent decreases non-specific binding.

Flow the competing agent at a low concentration, removing weakly bound contaminants.

Wash with increasing competing agent concentrations to displace the strongly bound antigens, eluting them.

Introduce bacterial outer membrane vesicles, or OMVs, with a surface-bound capturing protein and incubate.

Antigen peptide tags bind to the capturing proteins, forming an OMV vaccine with a multivalent antigen display.

Perform OMV SpyCatcher purification. Start with centrifuging the bacterial solution. Filter the supernatant with a 0.22-micromet