SARS‐CoV‐2 Protein Nanoparticle Vaccines Formed In Situ From Lyophilized Lipids

The receptor binding domain (RBD) of the SARS‐CoV‐2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten‐like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile‐filtered freeze‐dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome‐displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween‐80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli‐derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin‐phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome‐bound format prior to injection. Protein particles can readily be formed with sequent‐divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of mice elicits antibodies that neutralize respective viral strains. When K18‐hACE2 transgenic mice are immunized and challenged with ancestral SARS‐CoV‐2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates. A lyophilized lipid cake is developed that can be reconstituted with protein antigen solutions to induce spontaneous nanoparticle formation based on liposome‐display. The resulting nanoparticles can be used for vaccination against SARS‐CoV‐2 variants of concern.