Summary Severe respiratory acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19, a world-wide pandemic causing over 525K deaths in the U.S. and over 2.6M deaths world-wide as of this writing. Here, we propose to make phospholipid vesicles and other related nanostructures (droplets and micelles) with recombinant protein motifs that will bind and inactivate the SARS-CoV-2 virion – we call these structures inactosomes. Our inactosomes will be novel hybrid materials into which functional recombinant proteins are reconstituted. The functionalized protein will be variants of oleosin, a naturally occurring surfactant protein. Previously, we have designed and produced novel oleosin variants that assemble into vesicle membranes and micelles and can stabilize droplets due to their triblock-like free-chain amphiphilic structure. We can readily incorporate functional peptide motifs into oleosin recombinantly. SARS-CoV-2 can enter epithelial cells via endocytosis and/or fusion. Binding between the spike protein (S) on SARS-CoV-2 and the ACE2 receptor is essential for the entry of the virus into the epithelium. The ACE2 receptor can mediate endocytosis. Alternatively, after binding to ACE2, a protease (TMPRSS2) can activate a conformational change in the S protein leading to fusogenic entry. We envision a number of chemistries that will be directly useful at interfering with infection by SARS-CoV-2. First, a family of spike protein binding motifs – mini-proteins, single chain antibodies (sybodies), or ACE2 peptide mimetics - will be recombinantly added to the hydrophilic ends of oleosin. These motifs have low dissociation constants with the spike protein receptor binding domain and are much easier to produce than large antibodies. When these virus binding motif-oleosins are reconstituted into nanostructures, the result will be a multivalent particle (inactosomes) that can bind directly to SARS-CoV-2 and competitively blocks its entry. Next, we will incorporate an oleosin that presents a small peptidic fusion inhibitor of the S protein to prevent fusogenic entry of the virus on cell lines expressing TMPRSS2. Peptides that block either binding and fusion can be combined to make multi-functional inhibitory inactosomes. In Aim 1, we will develop and characterize SARS-CoV-2 inactosomes that prevent endocytosis- mediated entry into cells. In Aim 2, we will develop and characterize the SARS-CoV-2 inactosomes that block fusogenic entry, followed by inactosomes which possess both ACE2 blocking peptides and anti- fusogenic peptides. Combinations of SARS-CoV-2 reporter particles (bearing an eGFP gene) and SARS-CoV-2 inactosomes will be incubated in a co-culture to assess the binding and infection into 293T, Vero A6, and Calu- 3 cells. This assay will be used to optimize the chemical composition of inactosomes (type of virus binding motif, nanoparticle structure, total protein density, ratio of virus binding motifs to fusion inhibitory motifs) ...