A modular plug-and-play vaccine construction platform using bio-click chemistry and virus-like particles

Virus-like particles (VLPs) are a convenient platform for the presentation of antigens to enhance their immunogenic properties. In the present study, we combined sortase-based labelling of the outer surface protein C (OspC) of Borrelia burgdorferi (as an antigen) with click chemistry to couple it to functionalised VLPs of the hepatitis B core antigen (HBc) expressed in Escherichia coli. The first step involved p-azido-phenylalanine (pAzF) being bioorthogonally incorporated into HBc. The major immunodominant region (MIR) was chosen as the incorporation site because HBc is known to form dimers that assemble into VLPs and expose the MIR on the surface. In a second step, dibenzocyclooctyne-amine (DBCO-amine) was C-terminally attached to the OspC using the sortase reaction. Finally, both of these functionalised proteins were covalently coupled by a strain-promoted azido-alkyne cycloaddition (SPAAC) reaction. The combination of an enzymatic functionalisation followed by a click chemistry-based covalent coupling is referred to as bio-click chemistry. An optimal amount of OspC-DBCO, corresponding to a three- to five-fold molar excess over HBc-pAzF in the reaction, was identified. A reaction yield of over 90% was achieved by incubation at 37 °C for 4 h. Prolonged incubation resulted in similar results at 25 °C. Particle size measurements using different methods showed homogenously increasing diameters for VLPs that were purified after the SPAAC reaction when compared to the initial HBc-pAzF VLPs. The zeta potential was increased by approximately 20 mV, and the VLPs showed an altered electrophoretic mobility, which further indicated an increased surface charge. Heterodimers of HBc-pAzF and the conjugation product were identified in addition to homodimers of either HBc-pAzF or conjugate monomers. No VLPs composed exclusively of HBc-pAzF were detectable. Our approach to generating chimeric VLPs via bio-click chemistry can be adapted to any antigen of choice, independently of the expression host.