Document text
Principal Investigator: Daniel A Hammer
Organization: UNIVERSITY OF PENNSYLVANIA
Fiscal Year: 2023
Award: $203,125
Funding agency: National Institute of Allergy and Infectious Diseases
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) to minimize
the entry of SARS-CoV-2. The optimized inactosome formulations will then be tested for inhibiting infection of
live SARS-CoV-2 virus into cells at the Penn Center for Precision Medicine.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><Acute><Amino Acid Motifs><Amino Acids><Antibodies><Assay><BSL-3 facility><BSL3 facility><Bacteria><Binding><Bioassay><Biologic Assays><Biological Assay><Biotech><Biotechnology><COVID-19><COVID-19 infection><COVID-19 virus><COVID-19 virus infection><COVID19><COVID19 infection><COVID19 virus><CV-19><CV19><Cell Body><Cell Line><Cell Membrane Lipids><Cell membrane><CellLine><Cells><Cessation of life><Chemicals><Chemistry><Cholesterol><Choline Glycerophospholipids><Choline Phosphoglycerides><Co-culture><CoV emergence><CoV-2><CoV2><Cocultivation><Coculture><Coculture Techniques><Coronaviridae><Coronavirus><Cytoplasmic Membrane><Death><Development><Disease><Disorder><Dissociation><Endocytosis><Engineering><Epithelial Cells><Epitheliasin Gene><Epithelium><Esteroproteases><Family><Fat Body><Formulation><Future><Genes><Human><Hybrids><Hydrogen Oxide><Hydrophobicity><In Vitro><Incubated><Infection><Infectious Agent><Integral Membrane Protein><Intrinsic Membrane Protein><Lecithin><Length><Lipids><Mediating><Membrane><Membrane Lipids><Micelles><Microfluidic Device><Microfluidic Lab-On-A-Chip><Microfluidic Microchips><Microfluidics><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Nanostructures><Nature><PRSS10><Peptidases><Peptide Domain><Peptide Hydrolases><Peptides><Phosphatides><Phosphatidylcholines><Phospholipids><Plants><Plasma Membrane><Protease Gene><Proteases><Protein Binding Domain><Protein Binding Motif><Protein Domains><Protein Motifs><Protein-Protein Interaction Domain><Proteinases><Proteins><Proteolytic Enzymes><Pulmonary Surfactant-Associated Protein A><Receptor Protein><Recombinant Proteins><Recombinants><Reporter><Respiratory distress><SARS><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV disease><SARS-CoV-1><SARS-CoV-2><SARS-CoV-2 infection><SARS-CoV2><SARS-CoV2 infection><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SP-A Protein><Serine Endopeptidases><Serine Protease><Serine Protein Hydrolases><Serine Proteinases><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome related corona virus 2><Strains Cell Lines><Structure><Surface><Surfactant Protein A><Syndrome><TMPRSS2><TMPRSS2 gene><Tertiary Protein Structure><Testing><Therapeutic><Transmembrane Protein><Transmembrane Protein Gene><Vaccines><Variant><Variation><Vesicle><Viral><Viral Diseases><Virion><Virus><Virus Diseases><Virus Particle><Water><Work><Writing><Wuhan coronavirus><aminoacid><amphiphilicity><angiotensin converting enzyme 2><angiotensin converting enzyme II><biosafety level 3 facility><block viral entry><conformation><conformational><conformational state><conformationally><conformations><corona virus><corona virus disease 2019><corona virus emergence><coronavirus disease 2019><coronavirus disease 2019 infection><coronavirus disease 2019 virus><coronavirus disease-19><coronavirus disease-19 virus><coronavirus emergence><coronavirus infectious disease-19><cultured cell line><density><design><designing><developmental><dimer><emergent CoV><emergent corona virus><emergent coronavirus><emerging CoV><emerging corona virus><emerging coronavirus><hCoV19><hydrophilicity><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><infectious organism><inhibit viral entry><inhibitor><innovate><innovation><innovative><interfacial><membrane structure><microfluidic chip><mimetics><monomer><nCoV><nCoV2><nano particle><nano vesicle><nano-sized particle><nano-sized structures><nano-structures><nanobodies><nanobody><nanoparticle><nanosized particle><nanovesicle><new CoV><new corona virus><new coronavirus><novel><novel CoV><novel corona virus><novel coronavirus><pandemic><pandemic disease><particle><peptide mimetic><peptide mimic><peptidomimetics><plasmalemma><precision medicine><precision-based medicine><prevent><preventing><protein reconstitution><receptor><receptor binding><receptor bound><reconstitute><reconstitution><respiratory><sdAb><self assembly><severe acute respiratory syndrome-CoV><single domain antibodies><success><surfactant><viral entry blocker><viral entry inhibitor><viral infection><virus infection><virus-induced disease><µfluidic>