Erythro-VLPs: Anchoring SARS-CoV-2 spike proteins in erythrocyte liposomes.

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/µm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future.

Lyophilized, thermostable Spike or RBD immunogenic liposomes induce protective immunity against SARS-CoV-2 in mice.

The COVID-19 pandemic has spurred interest in potent and thermostable SARS-CoV-2 vaccines. Here, we assess low-dose immunization with lyophilized nanoparticles decorated with recombinant SARS-CoV-2 antigens. The SARS-CoV-2 Spike glycoprotein or its receptor-binding domain (RBD; mouse vaccine dose, 0.1 µg) was displayed on liposomes incorporating a particle-inducing lipid, cobalt porphyrin-phospholipid (dose, 0.4 µg), along with monophosphoryl lipid A (dose, 0.16 µg) and QS-21 (dose, 0.16 µg). Following optimization of lyophilization conditions, Spike or RBD-decorated liposomes were effectively reconstituted and maintained conformational capacity for binding human angiotensin-converting enzyme 2 (hACE2) for at least a week when stored at 60°C in lyophilized but not liquid format. Prime-boost intramuscular vaccination of hACE2-transgenic mice with the reconstituted vaccine formulations induced effective antibody responses that inhibited RBD binding to hACE2 and neutralized pseudotyped and live SARS-CoV-2. Two days following viral challenge, immunized transgenic mice cleared the virus and were fully protected from lethal disease.