Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design

Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find the removal of a signal peptide sequence and alteration of liposome lipid composition improves viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.