Fatty Acid Complexes Of Coronavirus Spike Protein And Their Use

Abstract

Complexes of coronavirus spike proteins, as well as fragments or mutants thereof wherein the fragment or mutant thereof at least contains a receptor binding domain of said coronavirus spike protein, with linoleic acid, or a derivative or a salt or a mimetic thereof. Methods for producing the complexes of the invention by incubating coronavirus spike proteins with linoleic acid or a derivative or a salt or a mimetic thereof. . In vitro methods for identifying molecules which have therapeutic potential for diseases caused by coronaviruses by contacting the molecule with a coronavirus spike protein and linoleic acid or a derivative or salt or mimetic thereof. A method of treatment of coronavirus infection by administration of linoleic acid, or a derivative, a salt or a mimetic thereof to a subject in need thereof, by administration of an aerosol formulation or dry powder formulation to the respiratory tract, preferably by nasal administration.

Claims

1.-95. (canceled)

96. An isolated complex of a coronavirus spike protein or a fragment or mutant thereof wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus spike protein, with linoleic acid, or a derivative, salt or mimetic thereof, wherein the derivative or mimetic of linoleic acid is a compound characterised by the following general formula (I): ##STR00002## wherein Q is selected from O, S and NH; R1 is selected from OH, NH.sub.2, and SH; and R2 is a straight hydrocarbyl group having from 13 to 21 C atoms or having 17 C atoms, optionally linked or bound to a detectable label; wherein the mutant has at least 90%, or at least 95%, or has at least 96%, or at least 97%, or at least 98%, or at least 99% sequence homology with the wild-type coronavirus spike protein or fragment thereof; and optionally, wherein the derivative or mimetic of the linoleic acid comprises a detectable label.

97. The complex of claim 96 wherein R2 has at least one unsaturated C—C bond, or two unsaturated C—C bonds, preferably the unsaturated C—C bonds are between C-8 and C-9 and between C-11 and C-12 of the hydrocarbyl group when counted from the carbon bound to the C=Q group in formula (I).

98. The complex of claim 96 wherein the compound is selected from the group consisting of oleic acid, arachidonic acid, elaidic acid, eicosapentaenoic acid, stearic acid, gamma-linoleic acid, calendic acid, arachidic acid, dihomo-gamma-linoleic acid, docosadienoic acid, adrenic acid, palmitic acid and behenic acid, preferably oleic acid.

99. The complex of claim 96 wherein the coronavirus spike protein or fragment or mutant thereof is selected from spike proteins or fragments or mutants thereof of a coronavirus causing respiratory disease, preferably pneumonia, preferably in humans.

100. The complex of claim 99 wherein the coronavirus spike protein or fragment thereof is a spike protein or fragment or mutant thereof of a coronavirus selected from the group consisting of SARS-CoV, MERS-CoV and SARS-CoV-2, preferably from SARS-CoV-2.

101. The complex of claim 96 wherein the coronavirus spike protein or fragment thereof has an amino acid sequence selected from the group consisting of the amino acids sequences according to SEQ ID NO: 1 to 15, preferably selected from SEQ ID NO. 1, 2, 3, 4, 9, 10 and 11.

102. The complex of claim 96 wherein the complex is immobilized, preferably on a surface of a test device.

103. The complex of claim 96 wherein the complex is bound to a receptor for the coronavirus spike protein or fragment or mutant thereof, preferably ACE2, wherein, optionally, the receptor is immobilized, preferably on a surface of a test device.

104. A method for producing a complex according to claim 96, the method being selected from the group consisting of: (1) a method comprising the step of expressing the coronavirus spike protein or a fragment or mutant thereof of said coronavirus spike protein, wherein the fragment or mutant at least contains the receptor binding domain of said coronavirus spike protein, in a recombinant host cell in the presence of linoleic acid or a derivative or a salt or a mimetic thereof, and, optionally purifying the complex from the host cell; (2) a method comprising the steps of: (i) introducing into host cells a heterologous nucleic acid encoding the coronavirus spike protein or a fragment or mutant thereof wherein said fragment or mutant at least contains the receptor binding domain of said coronavirus spike protein; (ii) culturing said host cells in the presence of linoleic acid or a derivative or a salt or a mimetic thereof; and, optionally (iii) purifying the complex from the host cells and/or the culture medium; (3) a method comprising the step of incubating an isolated coronavirus spike protein or fragment or mutant thereof wherein said fragment or mutant thereof at least contains a receptor binding domain of said coronavirus spike protein, with linoleic acid or a derivative or a salt or a mimetic thereof; and (4) a method comprising the steps of: (i) expressing the coronavirus spike protein or a fragment or mutant thereof wherein the fragment or mutant at least contains the receptor binding domain of said coronavirus spike protein in a recombinant host cell, preferably a linoleic acid-free host cell and medium; (ii) isolating the expressed coronavirus spike protein or a fragment or mutant thereof; and (iii) incubating the isolated coronavirus spike protein or fragment or mutant thereof with linoleic acid or a derivative or a salt or a mimetic thereof.

105. An in vitro assay for detecting whether a candidate molecule inhibits the binding of a complex according to claim 96 to a receptor protein for a coronavirus spike protein or fragment or mutant thereof, comprising the steps of: (a) contacting the complex with a receptor protein for the coronavirus spike protein; (b) contacting the complex and the receptor of (a) with the candidate molecule; and (c) detecting unbound receptor and/or unbound coronavirus spike protein or mutant or fragment thereof. wherein, optionally steps (a) and (b) are carried out simultaneously, or, optionally wherein step (b) is carried out before step (a)

106. An in vitro assay for detecting whether a candidate molecule inhibits the binding of a linoleic acid or derivative or salt or mimetic thereof to a coronavirus spike protein or fragment or mutant thereof wherein the fragment or mutant thereof at least contains the receptor binding domain of said coronavirus spike protein, comprising the steps of: (A) contacting a coronavirus spike protein or fragment or mutant thereof according to claim 96 with the candidate molecule and the linoleic acid or derivative or salt or mimetic thereof; and (B) measuring the amount of (B1) the candidate molecule bound to the coronavirus spike protein or fragment or mutant thereof; and/ or (B2) linoleic acid or derivative or salt or mimetic unbound to the coronavirus spike protein or fragment or mutant thereof.

107. The assay of claim 106, further comprising the step of determining a Kd value of the candidate molecule to the coronavirus spike protein or fragment or mutant thereof.

108. The assay of claim 107, further comprising the steps of: (I) performing the method with multiple different candidate molecules wherein the method is carried out for each candidate molecule in a single reaction; (II) determining a Kd value for each candidate molecule; (III) selecting a candidate molecule having a predetermined threshold Kd value to the coronavirus spike protein or mutant or fragment thereof.

109. The assay of claim 108 wherein the threshold Kd value is below 100 nM.

110. A method for the treatment and/or prevention of a coronavirus infection by administration of a composition comprising linoleic acid, or a derivative or salt or mimetic thereof, in form of an aerosol formulation or dry powder formulation to the respiratory tract of a subject, wherein the derivative, salt of mimetic binds to the coronavirus spike protein of said coronavirus and wherein the linoleic acid or derivative or salt of mimetic thereof is used in a non-vesicular form, the derivative, salt or mimetic being a compound characterised by the following general formula (I): ##STR00003## wherein Q is selected from O, S and NH; R1 is selected from OH, NH2, and SH; and R2 is a straight hydrocarbyl group having from 13 to 21 C atoms, preferably 17 C atoms.

111. The method of claim 110, wherein the composition is administered by nasal administration in a unit dose of 1 to 84 μg, preferably 20 μg, of said linoleic acid or derivative or salt or mimetic thereof, and/or wherein the composition is administered to the lower respiratory tract in a unit dose of 1 to 500 pg, preferably 20 pg, of said linoleic acid or derivative or salt or mimetic thereof.

112. The method of claim 110, wherein R2 has at least one unsaturated C—C bond, preferably two unsaturated C—C bonds, preferably the unsaturated C—C bonds are between C-8 and C-9 and between C-11 and C-12 of the hydrocarbyl group when counted from the carbon bound to the C=Q group in formula (I).

113. The method of claim 110, wherein the compound of formula (I) is selected from the group consisting of linoleic acid, oleic acid, arachidonic acid, elaidic acid, eicosapentaenoic acid, stearic acid, gamma-linoleic acid, calendic acid, arachidic acid, dihomo-gamma-linoleic acid, docosadienoic acid, adrenic acid, palm itic acid and behenic acid, preferably oleic acid.

114. The method of claim 110, wherein the linoleic acid or derivative or salt thereof is contained in a monophasic, preferably aqueous, solution or in a dry powder.

115. The method of claim 110 wherein the solution contains a fatty acid solubilizer, preferably selected from the group consisting of cyclodextrin, ethanol, propylene glycol and a polypropylene glycol, and mixtures of two or more thereof.

116. The method of claim 115 wherein the cyclodextrin is a β-cyclodextrin, preferably selected from the group consisting of O-methylated, acetylated, hydroxypropylated, hydroxyethylated, hydroxyisobutylated, glucosylated, maltosylated and sulfoalkylether-β-cyclodextrin and mixtures of two or more thereof.

117. The method of claim 116 wherein the molar ratio between the cyclodextrin and the linoleic acid or derivative or salt or mimetic thereof is at least 10 to 1, preferably 10:1 to 60:1.

118. The method of claim 110 wherein the coronavirus is a coronavirus causing respiratory disease in humans, inckusing but not limited to SARS-CoV, MERS-CoV and SARS-CoV-2.

119. The method of claim 110 wherein the aerosol formulation or dry powder formulation is administered by the use of a respiratory delivery device, preferably selected from the group consisting of a nebulizer, vaporizer, vapor inhaler, squeeze bottle, metered-dose spray pump, Bi-dir Multi-dose spray pump, a gas driven spray system/atomizer, electrically powered Nebulizers/Atomizers, mechanical powder sprayers, breath actuated inhaler, insufflator, meter dose inhaler, and a dry powder inhaler.

120. A method for selecting binder molecules, binding to the complex according to claim 96, from a library of multiple candidate binder molecules comprising the steps of: (α) contacting the complex with the library of multiple candidate binder molecules; and (β) detecting which of the multiple candidate binder molecules have bound to the complex.

121. Use of the complex according to claim 96 for the production of antibodies wherein a non-human animal is immunized with said complex.

122. A method for producing antibodies binding to a complex according to claim 96 comprising the steps of immunizing a non-human animal with said complex; and isolating antibodies binding to said complex form said animal.

123. A respiratory delivery device comprising a composition in the form of an aerosol formulation or a dry powder formulation containing linoleic acid, or a derivative or salt or mimetic thereof, in non-vesicular form and a fatty acid solubilizer wherein the linoleic acid, or a derivative or salt or mimetic binds to the spike protein of a coronavirus, the derivative, salt or mimetic being a compound characterised by the following general formula (I): ##STR00004## wherein Q is selected from O, S and NH; R1 is selected from OH, NH.sub.2, and SH; and R2 is a straight hydrocarbyl group having from 13 to 21 C atoms, preferably 17 C atoms.

124. The delivery device according to claim 123 wherein the composition further comprises one or more further ingredients selected from a mucoadhesive, at least one anti-oxidant and at least one propellant.

125. The delivery device of claim 124 wherein the mucoadhesive is selected from the group consisting of cellulose and derivatives thereof, more preferably methylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, carboxymethylcellulose, hydroxyethyl cellulose or a mixture of two or more thereof; and/or wherein the antioxidant is selected from the group consisting of ascorbic acid, tocopherols, EDTA, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, ascorbyl fatty acid esters and mixtures of two or more thereof; and/or wherein the propellant is a hydrofluoroalkane, preferably selected from the group consisting of hydrofluoroalkane, more preferably HFA 227, HFA 134a or a mixture thereof.

126. The delivery device according to claim 123, selected from the group consisting of a nebulizer, vaporizer, vapor inhaler, squeeze bottle, metered-dose spray pump, Bi-dir Multi-dose spray pump (OptiNose), a gas driven spray system/atomizer, electrically powered Nebulizers/Atomizers, mechanical powder sprayers, breath actuated inhaler, insufflator, meter dose inhaler, and a dry powder inhaler.

127. The delivery device of claim 123 wherein the device is adapted for intra-nasal delivery of said composition in a unit dose of 1 to 84 μg, preferably 20 μg, of said linoleic acid or derivative or salt or mimetic thereof; and/or wherein the device is adapted for pulmonary delivery of said composition in a unit dose of 1 to 500 μg, preferably 20 μg, of said linoleic acid or derivative or salt or mimetic thereof.

128. The delivery device of claim 123 wherein R2 has at least one unsaturated C—C bond, or two unsaturated C—C bonds, preferably the unsaturated C—C bonds are between C-8 and C-9 and between C-11 and C-12 of the hydrocarbyl group when counted from the carbon bound to the C=Q group in formula (I).

129. The delivery device of claim 123 wherein the compound is selected from the group consisting of oleic acid, arachidonic acid, elaidic acid, eicosapentaenoic acid, stearic acid, gamma-linoleic acid, calendic acid, arachidic acid, dihomo-gamma-linoleic acid, docosadienoic acid, adrenic acid, palm itic acid and behenic acid, preferably oleic acid.

130. The delivery device of claim 123 wherein the composition is a monophasic aqueous solution.

131. The delivery device of claim 123 wherein the composition includes a fatty acid stabilizer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0220] The Figures show:

[0221] FIG. 1A: LA-bound SARS-COV-2 S protein interaction with ACE2. In FIG. 1A the complex LA-bound SARSV-COV-2 S protein with ACE2 was analyzed by size exclusion chromatography (SEC) evidencing formation of a complex between the purified proteins. FIG. 1A Left: SEC profiles are shown with peak fractions labelled for ACE2 (III.), LA-bound Spike (II.) and a 1:1 mixture (I.) are shown. FIG. 1A Right: the peak fractions I., II. and III. were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie Brilliant Blue. Sections of the corresponding SDS-PAGE gels are shown. Molecular weights of a protein standard are given with numbers representing kilodaltons (kDa). Bands in the SDS-PAGE gel sections corresponding to Spike and ACE2 proteins are marked. FIG. 1B Chemical structure and calculated molecular weight of LA are indicated. FIG. 1C LC-MS analysis of highly purified S protein sample. C4 column elution profile (middle) and ESI-TOF spectra of the C4 peak elution fraction (below) are shown. The molecular weight corresponding to the peak in ESI-TOF is indicated.

[0222] FIG. 2: Cryo-EM image processing workflow of LA-bound SARS-COV-2 S protein. A motion-corrected cryo-EM micrograph is shown (scale bar 20 nm), reference-free 2D class averages (scale bar 10 nm), 3D classification and refinement resulting in cryo-EM maps corresponding to the open conformation and the closed conformation (not symmetrized and C3 symmetrized).

[0223] FIG. 3: Ribbon Diagram showing the receptor binding domain (RBD) of the SARS-CoV-2 S protein containing bound linoleic acid (LA), interacting with its target receptor for cell entry, angiotensin-converting enzyme 2 (ACE2). The part of the Spike RBD directly interacting with ACE2, the so-called Achilles Heel, is colored in dark grey and labelled (AH). The RBD and ACE2 (colored in grey) are shown in a cartoon representation; the LA is shown as spheres.

[0224] FIG. 4: Structure of SARS-CoV-2 S protein LA binding pocket seen in a close-up view. Portions of the two adjacent RBDs from LA-bound SARS-CoV-2 Spike are shown (colored in light grey), labelled RBD1 and RBD2. LA is shown in a stick representation. RBD1 is superimposed on the ‘apo’ form of the RBD (colored in dark grey) comprising no LA. The lateral movement of the gating helix (GH) to avoid a steric clash with LA is evident.

[0225] FIG. 5: The architecture of the three RBDs in the structure of ligand-free ‘apo’ SARS-CoV-2 S protein. A schematic drawing showing ‘apo’ SARS-CoV-2 S protein containing no LA (colored in light grey) is shown, superimposed on the structure of LA-bound SARS-CoV-2 S protein (colored in dark grey) looking down the Spike. LA in the RBD on the bottom is shown as spheres (omitted for better clarity in the other two RBDs from LA-bound Spike). Arrows indicate molecular movements accompanying the conformational switch resulting in a condensed RBD trimer in the LA-bound (‘holo’) form. The three RBDs forming the trimer in the apo form are denoted RBD1 apo, RBD2 apo and RBD3 apo, respectively. The three RBDs in the LA-bound form are denoted RBD1, RBD2 and RBD3, respectively. RBD and RBD1 apo, shown at the bottom, were used to align the apo and LA-bound forms of the SARS-CoV-2 S protein.

[0226] FIG. 6: In vitro enzyme-linked immunosorbent assay (ELISA) evidencing LA-bound SARS-CoV-2 S protein binding to immobilized ACE2 receptor is shown. The absorption at 450nm (normalized units) is plotted against the concentrations in a serial dilution of the S protein in phosphate buffered saline (PBS). Error bars indicate standard deviations from three independent replicates. The S protein competes with a purified RBD horse-radish peroxidase (HRP) fusion protein for ACE2 binding (see inset).

[0227] FIG. 7: Methods for production of Holo and Apo forms of SARS-CoV-2 S protein. Shown are schematic diagrams describing methods to produce SARS-CoV-2 S protein containing bound LA (Holo) and SARS-CoV-2 S protein without bound LA (Apo). Top: indicated schematically, SARS-CoV-2 S protein is produced in an expression system with supplemental LA provided, e.g. via cod liver oil, which results in the Holo form of the SARS-CoV-2 S protein. Indicated by the black rectangle is the LA binding pocket occupied by LA. Center: indicated schematically, SARS-CoV-2 S protein is produced in an expression system with supplemental LA provided which results in the Holo form of the SARS-CoV-2 S protein. Indicated by the black rectangle is the LA binding pocket. Stripping of bound LA can be carried out via e.g. detergent washing, to produce the Apo form of the SARS-CoV-2 S protein. Detergent stripping could be carried out on size exclusion column to permanently eliminate LA from the protein sample. Bottom: indicated schematically, SARS-CoV-2 S protein is produced in an expression system without supplemental LA provided, e.g. a mammalian transient transfection expression system with serum free media (24) to produce the Apo form of the SARS-CoV-2 S protein.

[0228] FIG. 8: Example 7. Screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. Left: indicated schematically is SARS-CoV-2 S protein, immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or a Fatty Acid Binding Protein (FABP). Indicated by the black rectangle is the LA binding pocket, which is occupied by LA. Center Black Arrow: a molar excess of a small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein with bound LA. Right: LA is displaced and replaced by drug candidate in the LA binding pocket. LA is then detected in solution following displacement via e.g. ELISA reaction, or via fluorescent FABP probes that change their flouresence when binding free fatty acids (25). A drug candidate capable of displacing LA from its binding pocket. The immobilized SARS-CoV-2 S protein in this example can comprise the trimeric S protein, or a fragment or mutant thereof, wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein.

[0229] FIG. 9: Example 8. Screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. Left: indicated schematically is SARS-CoV-2 S protein immobilized on to the surface of a microplate in aqueous solution, optionally containing a carrier protein for LA such as BSA or a FABP. Indicated by the black rectangle is the LA binding pocket, which in this embodiment is occupied by a labelled version of LA indicated as LA*. A labelled LA could be e.g. a conjugated fluorophore derivative of LA. Center: a small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein. Right: LA* is displaced and replaced by drug candidate in the LA binding pocket. LA* is then detected directly in solution following displacement via its label group. The immobilized SARS-CoV-2 S protein in this example can comprise the trimeric S protein, or a fragment or mutant thereof, wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein.

[0230] FIG. 10: Example 9. Screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. Left: indicated schematically SARS-CoV-2 S protein is immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or a FABP. Indicated by the black rectangle is the LA binding pocket which is occupied by LA. Center: LA is stripped from SARS-CoV-2 S protein, and a molar excess of small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein, together with labeled LA indicated as LA*. As an alternative to stripping LA from SARS-CoV-2 S protein, a sample of the protein produced in the absence of LA could be used to produce the Apo form of the protein. Right: LA* and drug candidate compete for the LA binding pocket. The ratio of bound to unbound LA* is LA is then detected in solution via the label group of LA*. The immobilized SARS-CoV-2 S protein in this example can comprise the trimeric S protein, or a fragment or mutant thereof, wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein.

[0231] FIG. 11: Example 10. Screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. Left: indicated schematically SARS-CoV-2 S protein is immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or a FABP. Indicated by the black rectangle is the LA binding pocket which is not occupied by LA. Right: LA and drug candidate compete for the LA binding pocket. LA is then detected in solution via e.g. ELISA reaction, or via fluorescent FABP probes that change their flouresence when binding free fatty acids (25). The immobilized SARS-CoV-2 S protein in this example can comprise the trimeric S protein, or a fragment or mutant thereof, wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein.

[0232] FIG. 12: Example 11. Screening method for discovery, i.e. identification, of inhibitors of the Holo SARS-CoV-2 S protein—ACE2 interaction. Shown is a schematic diagram describing a medium/high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of the Holo SARS-CoV-2 S protein—ACE2 interaction. Left: indicated schematically is the ELISA assay from FIG. 6. Holo SARS-CoV-2 S protein and Labeled RBD-HRP* are incubated together at concentrations that produce approximately EC30 signal in the immobilized ACE2 receptor binding ELISA assay. Center: Small molecule or biologic drug candidate is added to the EC30 component mixture from left. Right: The ELISA reaction is run as described in FIG. 6 at single point readout at the putative EC30 of an inactive drug candidate. An increase of A450 is indicative that the drug candidate is an inhibitor of binding of SARS-CoV-2 S protein to ACE2. This method is anticipated to be able to detect therapeutic drug candidate inhibitors which bind SARS-CoV-2 S protein outside it's LA binding pocket. The SARS-CoV-2 S protein in this example can comprise the trimeric S protein, or a fragment or mutant thereof, wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein.

[0233] FIG. 13: Example 12. Screening method for discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein—ACE2 interaction. Shown is a schematic diagram describing a medium/high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of the SARS-CoV-2 S protein—ACE2 interaction that are selective for either Apo or Halo form of the SARS-CoV-2 S protein. Left: indicated schematically is the ELISA assay from FIG. 6. Either Holo (top), or Apo (bottom) SARS-CoV-2 S protein and Labeled RBD-HRP* are incubated together at concentrations that produce approximately EC30 signal in the immobilized ACE2 receptor binding ELISA assay. Center: Small molecule or biologic drug candidate is added to the EC30 component mixture from left, to Holo mixture (top), or Apo mixture (bottom), where said reaction is carried out in separate wells in the microplate. Right: The ELISA reaction is run as described in FIG. 6 at single point readout at the putative EC30 of an inactive drug candidate, and the readout from Holo (top), or Apo (bottom) are compared for each putative drug candidate. An increase of A450 is indicative that the drug candidate is an inhibitor of binding of SARS-CoV-2 S protein to ACE2. This method is anticipated to be able to detect therapeutic drug candidate inhibitors which bind SARS-CoV-2 S protein outside it's LA binding pocket, and also to discern inhibitors that might have specificity for Holo or Apo form of SARS-CoV-2 S protein. The immobilized SARS-CoV-2 S protein in this example can comprise the trimeric S protein, or a fragment or mutant thereof, wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein.

[0234] FIG. 14: Example 13. Screening method for discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein using the SARS-CoV-2 Spike receptor binding domain (RBD) or variants of the RBD containing one or several mutations. Depicted are zoom-ins on the LA-binding ‘greasy’ tube entrance within the receptor binding domain of the SARS-CoV-2 S protein. The RBD is shown in a cartoon representation. Bound LA is represented by spheres. Amino acid residues in proximity of the carboxy headgroup of LA, one each in the individual zoom-ins, are represented by spheres and marked in the amino acid sequence segments (from primary sequence of SARS-CoV-2 S) above the zoom-ins in bold and by enlarged fonts. The screening methods of any of the Examples 1 to 6 could optionally utilize SARS-CoV-2 S RBD proteins comprising one or more mutations where either of the featured amino acids, or combinations thereof, of all of them, have been changed to a hydrophilic amino acid, for example Lys (K), Arg (R), Asn (N) or (Gln) (Q), or other hydrophilic amino acids including non-natural amino acids. The said RBD variants are then used in this Example in medium/high throughput-compatible process to discover potential small molecule or biologic drug candidates which interact with high affinity and high specificity with the RBD proteins described. These small molecules or biologic drug candidates are selective for either Apo or Halo form of the SARS-CoV-2 S protein.

[0235] FIG. 15: Example 14. Discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein using crystallization of the SARS-CoV-2 Spike RBD or variants of the RBD. The screening method in this example utilizes SARS-CoV-2 S protein RBD and/or the variants described in Example 13 (FIG. 14) in low/medium/high throughput-compatible crystallization and X-ray diffraction experiments including addition of one or several small molecule or biologic drug candidates in the crystallization experiments, to discover potential small molecule or biologic drug candidates which interact with high affinity and high specificity with the RBD proteins described. These small molecules or biologic drug candidates are selective for either Apo (top) or Holo form (bottom) of the SARS-CoV-2 S protein.

[0236] FIG. 16: Example 15. Discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein using crystallization of the SARS-CoV-2 Spike RBD or variants of the RBD. Depicted is a zoom-in on Arginine R408 and Glutamine Q409 in the SARS-CoV-2 S protein. R408 and Q409 are part of the LA binding pocket in the Spike RBD. Spike RBD is shown in a cartoon representation. Bound LA is represented by sticks. R408 and Q409 are marked. The screening method in this embodiment utilizes SARS-CoV-2 S protein in which R408, or Q409, or both have been mutated to a different amino acid residue, preferentially an alanine or a glycine, or any other amino acid residue that is not polar and/or positively charged. The said SARS-CoV-2 S protein variants are then used in this Example in medium/high throughput-compatible crystallization process as described in Example 14 to discover potential small molecule or biologic drug candidates which interact with high affinity and high specificity with the SARS-CoV-2 S proteins described. These small molecules or biologic drug candidates are selective for either Apo or Halo form of the SARS-CoV-2 S protein.

[0237] FIG. 17: Example 17. Phage display screening method. Shown is a schematic diagram describing phage display applied to discover antibodies that bind to holo (LA bound) SARS-CoV-2 spike protein. Left: indicated schematically SARS-CoV-2 spike protein is immobilized on to the surface of a microplate in aqueous solution. Indicated by the black rectangle is the LA binding pocket which is occupied by LA. Left Arrow: Indicated by the Y-shaped images, a phage display library is incubated with the immobilized SARS-CoV-2 spike protein with some antibodies binding to SARS-CoV-2 spike protein (Center). Non interacting phage display library species are washed away leading to enriched samples of SARS-CoV-2 spike protein with preferred antibody binding partners (Right). Not shown: standard and iterative phage display washing/panning cycles and nucleic acid identification methods are used to develop and identify high affinity binders to (LA bound) SARS-CoV-2 spike protein.

[0238] FIG. 18: High affinity binding of linoleic acid (LA) to immobilized SARS-CoV-2 Spike Protein receptor binding domain (RBD) quantified by surface plasmon resonance (SPR). LA was diluted to concentrations between 4 μM and 10 μM and flowed over 3,800 RU of biotinylated and lipidex-treated RBD immobilized on a streptavidin-coated sensor chip (light grey tracings). Dark grey lines correspond to a global fit of the data using a 1:1 binding model. Each experiment was repeated independently three times. All LA concentrations were used to calculate the KD, kon and koff values.

[0239] FIG. 19: High affinity binding of Oleic Acid (OA) to immobilized SARS-CoV-2 Spike Protein receptor binding domain (RBD) quantified by surface plasmon resonance (SPR). OA was diluted to concentrations between 1 uM and 3 uM and flowed over 3,200 RU of biotinylated and lipidex-treated RBD immobilized on a streptavidin-coated sensor chip (light grey tracings). Dark grey lines correspond to a global fit of the data using a 1:1 binding model. Each experiment was repeated independently three times. All OA concentrations were used to calculate the KD, kon and koff values, which are indicated in this figure.

[0240] FIGS. 20A and 20B: Cryo-EM structure of the SARS-CoV-2 Spike protein, oleic acid complex. FIG. 20A: Cryo-EM density of the Spike trimer is shown from a side view (left), and top view (right). Bound oleic acid is illustrated in the top view as spheres. FIG. 20B: Composite oleic acid binding pocket formed by adjacent Receptor Binding Domains (RBDs). Tube-shaped EM density is shown.

[0241] FIGS. 21A-21C: Minivirus displaying SARS-CoV-2 Spike protein on its surface is blocked from binding its receptor ACE2 by linoleic acid. FIG. 21A: Schematic illustration of a Minivirus with SARS-CoV-2 Spike protein on its surface, immobilized via their His-tag. FIG. 21B: Representative confocal microscopy images of MCF7 human epithelial cells incubated for 10 min with Miniviruses, showing attachment of the Miniviruses to the cell's surface. Miniviruses are individually visualized as small dots. The inset shows magnified area of attachment. FIG. 21C: Miniviruses allowed for a systematic assessment of changes in SARS-CoV-2 Spike protein-mediated cell interactions as a function of Free Fatty Acid (FFA) occupancy. Because recombinant native SARS-CoV-2 Spike protein binds the Free Fatty Acids Linoleic Acid and Oleic Acid during heterologous protein expression and purification, we first generated FFA-depleted Spike protein (ApoS) by treatment of the purified SARS-CoV-2 Spike protein with lipidex columns as described in Example 17. Compared to the native Spike protein, ApoS-decorated Miniviruses displayed increased binding to human epithelial cells (First two bars on the left of the chart). This is consistent with a model where FFAs lock a closed Spike protein conformation, inhibiting binding to its receptor, ACE2. Controlled loading of ApoS Miniviruses by incubating them with FFAs of differing length and saturation (i.e., palmitic acid (PA), oleic acid (OA), LA and arachidonic acid (AA)). Binding of OA, and LA, but not PA, in SARS-CoV-2 Spike protein was successfully verified by multiple reactions monitoring LC-MS/MS. Of note, addition of saturated PA did not significantly reduce Minivirus-ACE2 cell binding compared to ApoS. However, we found that the (poly)-unsaturated FFAs OA, LA and AA were able to reduce Minivirus binding compared to ApoS Miniviruses.

[0242] FIG. 22A and FIG. 22B: Electron tomography of cultured mammalian cells infected with live SARS-CoV-2 virus, in the presence or absence of linoleic acid. A human gut epithelial cell line Caco2 expressing ACE2 (Caco-2-ACE2) was infected with SARS-CoV-2 reporter virus (for details see Example 21). Tomographs were obtained in the absence (FIG. 22A), or presence (FIG. 22B) of 50uM linoleic acid. Clearly visible in untreated cells (FIG. 22A) are the presence of enveloped “replication factories” containing numerous SARS-CoV-2 virions. The dashed square in FIG. 22A (left image represents such a replication factory which is shown at higher magnification in FIG. 22A (right image. In FIG. 22B the same SARS-CoV-2 infected cells were co-incubated with 50uM linoleic acid. The dashed square in FIG. 22B (left image) is the site of a former SARS-CoV-2 replication factory. The dashed square in FIG. 22B(left image) reveals that linoleic acid penetrates the cells, forming lipid droplets inside the cell, which are well tolerated by the cell (significant cell death was not observed). At higher magnification in FIG. 22B (right image) it is revealed that the envelope of the former “replication factory” has been disrupted, and the viral particles themselves suffering visible deformation and destruction.

DETAILED DESCRIPTION OF THE INVENTION

[0243] The present invention is further illustrated by the following non-limiting examples:

EXAMPLES

Example 1

[0244] SARS-CoV-2 S protein production, with bound LA. The pFastBac Dual plasmid for SARS-CoV-2 S ectodomain for expression in insect cells 20 was kindly provided by Florian Krammer (Icahn School of Medicine, USA). In this construct, S comprises amino acids 1 to 1213 and is fused to a C-terminal thrombin cleavage site followed by a T4-foldon trimerization domain and a hexahistidine affinity purification tag. The polybasic cleavage site has been deleted (RRAR to A) in the construct 20. Protein was produced with the MultiBac baculovirus expression system (Geneva Biotech, Geneva, Switzerland) 40 in Hi5 cells using ESF921 media (Expression Systems Inc.). Supernatants from transfected cells were harvested 3 days post-transfection by centrifugation of the culture at 1,000g for 10 min followed by another centrifugation of supernatant at 5,000g for 30 min. The final supernatant was incubated with 7 ml HisPur Ni-NTA Superflow Agarose (Thermo Fisher Scientific) per 3 litres of culture for 1 h at 4° C. Subsequently, a gravity flow column was used to collect the resin bound with SARS-CoV-2 S protein, the resin was washed extensively with wash buffer (65 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.5), and the protein was eluted using a step gradient of elution buffer (65 mM NaH2PO4, 300 mM NaCl, 235 mM imidazole, pH 7.5). Elution fractions were analysed by reducing SDS-PAGE and fractions containing SARS-CoV-2 S protein were pooled, concentrated using 50 kDa MWCO Amicon centrifugal filter units (EMD Millipore) and buffer-exchanged in phosphate-buffered saline (PBS) pH 7.5. Concentrated SARS-CoV-2 S was subjected to size exclusion chromatography (SEC) using a Superdex 200 increase 10/300 column (GE Healthcare) in PBS pH 7.5. Peak fractions from SEC were analysed by reducing SDS-PAGE (See FIG. 1A).

[0245] Mass spectrometry analysis of SARS-CoV-2 S protein production, demonstrating bound LA. For mass spectrometry analysis, a Bruker maXis II ETD quadrupole-time-of-flight instrument with electrospray ionization (ESI) coupled to a Shimadzu Nexera HPCL was used. In order to extract the fatty acids from the protein sample, a chloroform extraction protocol was performed. For this, 100 μl of the protein sample was mixed with 400 pl chloroform for 2 hours on a horizontal shaker in a teflon-sealed glass vial at 25° C. The organic phase was then transferred to a new glass vial and the chloroform was evaporated for 30 min in a desiccator. Subsequently, 50 μl of a 20% (v/v) acetonitrile solution was added to dissolve the fatty acids. From this solution, a 1:100 dilution in 20% (v/v) acetonitrile was prepared and 20 μl were injected for LC-MS analysis. The samples were passed over a Phenomenex C4 Aeris column (100×2.1 mm, 3.6μ, 200 Å) heated to 50° C. using a gradient of 20% A to 98% B in 5 min (solvent A: H.sub.2O+0.1% formic acid; solvent B: ACN+0.1% formic acid). The system was operated with Bruker's O-TOF Control (V4.1) and Hystar (V4.1) software and data analysis as well as data post-processing was performed with Bruker's Data Analysis software (V4.4, SR1, See FIG. 1C).

Example 2

[0246] ACE2 protein production. The gene encoding for the ACE2 ectodomain (amino acids 1 to 597, 41) was codon optimized for insect cell expression, synthesized (Genscript Inc, New Jersey USA) and inserted into pACEBac1 plasmid (Geneva Biotech, Geneva, Switzerland). The construct contains an N-terminal melittin signal sequence for secretion and a C-terminal octahistidine affinity purification tag. ACE2 protein was produced and purified following the same protocol as for S protein (See FIG. 1A).

Example 3

[0247] SARS-CoV-2 Spike and ACE2 interaction analysis. Purified SARS-CoV-2 Spike and ACE2 proteins were combined in PBS pH 7.5 at a ratio of 1:1.5 Spike trimer to ACE2 monomer. The mixture was incubated on ice for 2 hours and subjected to SEC using a Superdex 200 increase 10/300column (GE Healthcare) equilibrated in PBS pH 7.5. Purified SARS-CoV-2 S protein and ACE2 proteins were individually run on the same column in the same buffer as controls. The contents of peak fractions were analyzed by reducing SDS-PAGE (See FIG. 1A).

Example 4

[0248] Cryo-EM Image Production.

[0249] Sample preparation and data collection. 4 μL of 1.25 mg/mL SARS-CoV-2 S protein was loaded onto a freshly glow discharged (2 min at 4 mA) Quantifoil R1.2/1.3 carbon grid (Agar Scientific), blotted using a Vitrobot MarklV (Thermo Fisher Scientific) at 100% humidity and 4° C. for 2 s, and plunge frozen. Data were acquired on a FEI Talos Arctica transmission electron microscope operated at 200 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV using the EPU software.

[0250] Data were collected in super-resolution at a nominal magnification of 130,000× with a virtual pixel size of 0.525 Å. The dose rate was adjusted to 6.1 counts/physical pixel/s. Each movie was fractionated in 55 frames of 200 ms. 3289 micrographs were collected in a single session with a defocus range comprised between −0.8 and −2 μm.

[0251] Data Processing. The dose-fractionated movies were gain-normalised, aligned, and dose-weighted using MotionCor2 42. Defocus values were estimated and corrected using CTFFIND4 43. 611,879 particles were automatically picked using Relion 3.0 software 44. Reference-free 2D classification was performed to select well-defined particles, after four rounds of 2D classification a total of 386,510 good particles were selected for further 3D classification. An initial model was generated using 50,000 particle images in Relion 3.0, and the selected particles from 2D classification were subjected to 3D classification using 8 classes. Classes 4 and 6 (see FIG. 2), showing prominent features representing a total of 202,082 particles were combined and used for 3D refinement. The 3D refined particles were then subjected to a second round of 3D classification using 5 classes. Class 4 and 5 were combined for the closed conformation map, class 3 represented the open conformation, comprising 136,405 and 57,990 particles respectively. The selected maps were subjected to 3D refinement without applying any symmetry with respective 3D models. The maps were subsequently subjected to local defocus correction and Bayesian particle polishing in Relion 3.0. Global resolution and B factor (−89Å2 and −116Å2 for closed and open maps respectively) of the map were estimated by applying a soft mask around the protein density, using the gold standard Fourier shell correlation (FSC) =0.143 criterion, resulting in an overall resolution of 3.03 Å and 3.7 Å respectively. C3 symmetry was applied to the closed conformation map using Relion 3.0, followed by 3D classification using 3 classes. Class 3 with 217,815 particles was selected for CTF refinement and Bayesian polishing, yielding a final resolution of 2.85 Å (B factor of −86.8). Local resolution maps were generated using Relion 3.0.

[0252] Cryo-EM model building and analysis. UCSF Chimera 45 and Coot 46 were used to fit atomic models (PDB ID 6VXX, 14) into the cryo-EM map. The model was subsequently manually rebuilt using Coot and the closed conformation map. This closed conformation model was used to build features specific of the open conformation map and to further improve the model by using the C3-symmetrised map. To guide the model building, sharpened 47 and unsharpened maps were used in different steps of the process. N-linked glycans were hand-built into the density where visible, and restraints for non-standard ligands were generated with eLBOW 48. The model for the closed conformation was real space refined with Phenix 49 and the quality was additionally analyzed using MolProbity 50 and EMRinger 51, to validate the stereochemistry of the components. The quasi-atomic model of the open conformation was generated by first fitting the atomic model of the closed conformation into the open cryo-EM map using UCSF Chimera. We then used COOT and UCSF Chimera to move one RBD into the open conformation, by aligning this RBD to the atomic model of a published open form of SARS-CoV-2 S (PDB ID 6VSB, 24). Finally, this open model was fitted into the cryo-EM open map with COOT and UCSF Chimera. Figures were prepared using UCSF chimera and PyMOL (Schrodinger, Inc). The resulting Cryo-EM model building enabled us to determine binding site of LA in the SARS-CoV-2 S protein, to identify conformational changes upon LA binding, and also to model SARS-CoV-2 S protein—ACE2 receptor interactions (See FIGS. 3, 4, and 5).

Example 5

[0253] ELISA activity assay, illustrated in FIG. 6. The SARS-CoV-2 sVNT kit was obtained from GenScript Inc. (New Jersey, USA) for ELISA activity assays. Serial dilution series were prepared (from 0-4096 nM) of purified SARS-CoV-2 S protein in PBS pH 7.5. This dilutions series was mixed with the same amount of HRP-RBD and incubated at 37° C. for 30 min. The mixture of each dilution of SARS-CoV-2 S proteins and HRP-RBD was then added to ELISA plate wells, which were coated with ACE2. Triplicates of each sample were added. The ELISA plate was then incubated at 37° C. for 15 min and subsequently washed 4 times with wash buffer. The signal was then developed by adding TMB solution to each well, incubating 15 minutes in the dark, followed by adding stop solution. The absorbance at 450 nm was immediately recorded. The data was plotted using Microsoft excel. The standard deviation of triplicates was added as error bars. This Example illustrates the functionality of our SARS-CoV-2 S protein in functional biochemical assays which are applicable to drug discovery, i.e. identification.

Example 6

[0254] FIG. 7 illustrates production methods for purified SARS-CoV-2 S protein. Shown is a schematic diagram illustrating 3 general strategies for producing either Holo (meaning LA-containing) SARS-CoV-2 S protein, or Apo (meaning not LA-containing) SARS-CoV-2 S protein, respectively. By means of these processes, Apo and Holo can be produced for side by side comparison in drug discovery, i.e. identification, processes targeting SARS-CoV-2 S protein in the below Examples.

Example 7

[0255] FIG. 8 illustrates a screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. In this method a SARS-CoV-2 S protein, or a fragment or mutant thereof wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein, is immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or Fatty Acid-Binding Protein (FABP). In this example, SARS-CoV-2 S protein, or a fragment or mutant thereof is bound by LA. A molar excess of a small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein (or a fragment or mutant thereof) with bound LA. In case of a bona fide inhibitor drug candidate, LA could be displaced and replaced by the drug candidate in the LA binding pocket. LA has extremely low water solubility, and BSA is often used as a carrier protein to provide aqueous bioavailability of LA and other free fatty acids. In the experimental setup of Example 7 which includes a carrier protein for LA such as BSA or a FABP, LA that is displaced from SARS-CoV-2 S protein by the drug candidate will have a new binding site available in solution provided by carrier protein BSA or FABP. LA can be detected in solution following displacement via e.g. ELISA reaction directly detecting LA, or, alternatively by the conformational change-driven change in FABP fluorescence induced by LA binding (25).

Example 8

[0256] FIG. 9 illustrates a screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. In this method a SARS-CoV-2 S protein, or a fragment or mutant thereof wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein, is immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or Fatty Acid-Binding Protein (FABP). In this example, SARS-CoV-2 S protein, or a fragment or mutant thereof is bound by a labelled analogue of LA, indicated as LA*. A molar excess of a small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein (or a fragment or mutant thereof) with bound LA*. In case of a bona fide inhibitor drug candidate, LA* could be displaced and replaced by the drug candidate in the LA binding pocket. In the experimental setup of Example 8 which includes a carrier protein for LA such as BSA or a FABP, LA* that is displaced from SARS-CoV-2 S protein by the drug candidate will have a new binding site available in solution provided by carrier protein BSA or FABP. LA* can be detected in solution following displacement via its label, e.g. a fluorescent label.

Example 9

[0257] FIG. 10 illustrates a screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. In this method a SARS-CoV-2 S protein, or a fragment or mutant thereof wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein, is immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or Fatty Acid-Binding Protein (FABP). In this example, SARS-CoV-2 S protein, is first stripped of bound LA, or produced by a method that does not result in bound LA (see Example 6). Next, a molar excess of a small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein (or a fragment or mutant thereof) together with a labelled LA, indicated as LA*, whereby LA* is optionally provided in complex with a carrier protein for LA* such as BSA or FABP. In case of a bona fide inhibitor drug candidate, the quantity of LA* bound to the LA binding pocket will be decreased. LA* can be detected in solution or bound to the SARS-CoV-2 S protein following the competition reaction via its label, e.g. a fluorescent label.

Example 10

[0258] FIG. 11 illustrates a screening method for discovery, i.e. identification, of inhibitors of LA binding to SARS-CoV-2 S protein. Shown is a schematic diagram describing a high throughput-compatible process to discover potential small molecule or biologic drug candidates which are inhibitors of LA binding to SARS-CoV-2 S protein. In this method a SARS-CoV-2 S protein, or a fragment or mutant thereof wherein said fragment or mutant at least contains a receptor binding domain of said coronavirus S protein, is immobilized on to the surface of a microplate in aqueous solution optionally containing a carrier protein for LA such as BSA or Fatty Acid-Binding Protein (FABP). In this example, SARS-CoV-2 S protein, is first stripped of bound LA, or produced by a method that does not result in bound LA (see Example 6). Next, a molar excess of a small molecule or biologic drug candidate is added to the solution of immobilized SARS-CoV-2 S protein (or a fragment or mutant thereof) together with a unlabelled LA, indicated as LA, whereby LA is optionally provided in complex with a carrier protein for LA such as BSA or FABP. In case of a bona fide inhibitor drug candidate, the quantity of LA bound to the LA binding pocket will be decreased. LA can be detected in solution or bound to the SARS-CoV-2 S protein following the competition reaction via e.g. ELISA reaction directly detecting LA, or, alternatively by the conformational change-driven change in FABP fluorescence induced by LA binding (25).

Example 11

[0259] FIG. 12 illustrates a screening method for discovery, i.e. identification, of inhibitors of the Holo SARS-CoV-2 S protein—ACE2 interaction based on the ELISA assay from FIG. 6. Here, Holo SARS-CoV-2 S protein and Labelled RBD-HRP* are incubated together at concentrations that produce approximately EC30 signal in the immobilized ACE2 receptor binding ELISA assay. Small molecule or biologic drug candidate is added to the EC30 component mixture, and the ELISA reaction is run as described in FIG. 6 at single point readout at the putative EC30 of an inactive drug candidate. An increase of A450 is indicative that the drug candidate is an inhibitor of binding of SARS-CoV-2 S protein to ACE2. This method is anticipated to be able to detect therapeutic drug candidate inhibitors which bind SARS-CoV-2 S protein either inside, or outside its LA binding pocket.

Example 12

[0260] FIG. 13 illustrates a screening method for discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein—ACE2 interaction based on the ELISA assay from FIG. 6. Here either Apo or Holo SARS-CoV-2 S protein and Labelled RBD-HRP* are incubated together at concentrations that produce approximately EC30 signal in the immobilized ACE2 receptor binding ELISA assay. Small molecule or biologic drug candidate is added to the EC30 component mixture, and the ELISA reaction is run as described in FIG. 6 at single point readout at the putative EC30 of an inactive drug candidate. An increase of A450 is indicative that the drug candidate is an inhibitor of binding of SARS-CoV-2 S protein to ACE2. This method is anticipated to be able to detect therapeutic drug candidate inhibitors which bind SARS-CoV-2 S protein either inside, or outside its LA binding pocket and also to discern inhibitors that might have specificity for Holo or Apo form of SARS-CoV-2 S protein.

Example 13

[0261] FIG. 14 illustrates a screening method for discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein using the SARS-CoV-2 Spike receptor binding domain (RBD) or variants of the RBD containing one or several mutations. The screening methods of any of the Examples 1-6 could optionally utilize SARS-CoV-2 S RBD proteins comprising one or more mutations where either of the featured amino acids, or combinations thereof, of all of them, have been changed to a hydrophilic amino acid, for example Lys (K), Arg (R), Asn (N) or (Gln (Q), or other hydrophilic amino acids including non-natural amino acids. The said RBD variants are then used in this Example in medium/high throughput-compatible process to discover potential small molecule or biologic drug candidates which interact with high affinity and high specificity with the RBD proteins described. These small molecules or biologic drug candidates are selective for either Apo or Halo form of the SARS-CoV-2 S protein.

Example 14

[0262] FIG. 15 illustrates a screening method for discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein using crystallization of the SARS-CoV-2 Spike RBD or variants of the RBD. The screening method in this example utilizes SARS-CoV-2 S protein RBD and/or the variants described in Example 13 (FIG. 14) in low/medium/high throughput-compatible crystallization and X-ray diffraction experiments including addition of one or several small molecule or biologic drug candidates in the crystallization experiments, to discover potential small molecule or biologic drug candidates which interact with high affinity and high specificity with the RBD proteins described. Fragment-based screening (FBS) is a widely applied method for the discovery, i.e. identification, of drug candidates. It has overtaken high throughput screening (HTS) as the most popular method for screening molecules, due to the significantly fewer compounds required for screening and synthesis, resulting in a higher hit rate for screening molecules than traditional screening methods. Here, single compound or multiple compound mixtures are incubated with the target protein: SARS-CoV-2 S protein RBD and/or the variants described in Example 13 (FIG. 14), and crystallization is employed to identify binders. In this Example, small molecules or biologic drug candidates targeting the LA binding pocket which are selective for either Apo or Holo form of the SARS-CoV-2 S protein can be discovered.

Example 15

[0263] FIG. 16 illustrates a screening method for discovery, i.e. identification, of Halo-specific or Apo-specific inhibitors of the Holo SARS-CoV-2 S protein using crystallization of the SARS-CoV-2 Spike RBD or variants of the RBD. The screening method in this embodiment utilizes SARS-CoV-2 S protein in which R408, or Q409, or both have been mutated to a different amino acid residue, preferentially an alanine or a glycine, or any other amino acid residue that is not polar and/or positively charged. The said SARS-CoV-2 S protein variants are then used in this Example in medium/high throughput-compatible crystallization process as described in Example 14. These small molecules or biologic drug candidates are selective for either Apo or Halo form of the SARS-CoV-2 S protein.

Example 16

[0264] FIG. 17 illustrates a screening method of the invention for identifying binder molecules to complexes of the invention, wherein antibodies as binder molecules and phage display are used as examples for binding an immobilized exemplary complex of the invention composed SARS-CoV-2 S protein and LA. A library of potential binder molecules, here a phage display antibody library is incubated with the immobilized complex and one or more candidate binding antibodies are bound to the complex. Unbound binder molecules of the library are removed by a washing step which results in enriched samples of binder molecules of the complex of the invention with bound binder molecules. The process is typically embodied in an iterative fashion using a first enriched pool of binder molecule obtain in the first cycle is used in a further cycle of incubation and washing, where the washing and/or incubation step is carried out under conditions allowing only binding of such first cycle-enriched candidate binder molecules having a higher affinity as compared to the first cycle and so on.

Example 17

[0265] Example 17 illustrates the high affinity binding of Linoleic Acid (LA) to the SARS-CoV-2 Spike Protein. Shown in FIG. 18 is a Surface Plasmon Resonance (SPR) analysis of the binding of Linoleic Acid (LA) to the SARS-CoV-2 Spike Protein receptor binding domain (RBD).

[0266] The methods utilized to conduct the SPR binding assay are as follows:

[0267] Binding assay. LA was diluted to concentrations between 4 μM and 10 μM and flowed over 3,800 RU of biotinylated and lipidex-treated RBD immobilized on a streptavidin-coated sensor chip. Black lines correspond to a global fit of the data using a 1:1 binding model. Each experiment was repeated independently three times. All LA concentrations were used to calculate the KD, kon and koff values. The results are shown in FIG. 18.

[0268] Receptor Binding domain (RBD) and biotinylated RBD expression cassette design. The gene encoding for the SARS-CoV-2 RBD (amino acids R319 to F541), fused at its N-terminus to the native spike signal sequence (amino acid sequence MFVFLVLLPLVSSQ), was codon optimized for insect cell expression, synthesized (Genscript Inc., New Jersey USA) and inserted into pACEBac1 plasmid (Geneva Biotech, Geneva, Switzerland). The resulting construct pACEBac1-5 RBD HIS comprises a C-terminal octa-histidine affinity purification tag.

[0269] RBD Protein expression and purification. Protein was produced with the MultiBac baculovirus expression system (Geneva Biotech, Geneva, Switzerland) in Hi5 cells using ESF921 media (Expression Systems Inc.). Supernatants from transfected cells were harvested 3 days post-transfection by centrifugation of the culture at 1,000 g for 10 min followed by another centrifugation of supernatant at 5,000 g for 30 min. The final supernatant was incubated with 7 ml HisPur Ni-NTA Superflow Agarose (Thermo Fisher Scientific) per 3 liters of culture for 1 h at 4° C. Subsequently, a gravity flow column was used to collect the resin bound with SARS-CoV-2 RBD protein, the resin was washed extensively with wash buffer (65 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.5), and the protein was eluted using a step gradient of elution buffer (65 mM NaH2PO4, 300 mM NaCl, 235 mM imidazole, pH 7.5). Elution fractions were analyzed by reducing SDS-PAGE and fractions containing SARS-CoV-2 RBD protein were pooled, concentrated using 50 kDa MWCO Amicon centrifugal filter units (EMD Millipore) and buffer-exchanged in phosphate-buffered saline (PBS) pH 7.5. Concentrated SARS-CoV-2 RBD protein was subjected to size exclusion chromatography (SEC) using a Superdex 200 increase 10/300 column (GE Healthcare) in PBS pH 7.5.

[0270] Biotinylation of RBD. Biotinylated RBD was generated by adding an avi tag for BirA mediated biotinylation as described in (M. Fairhead, M. Howarth, Site-specific biotinylation of purified proteins using BirA. Methods Mol. Biol. 1266, 171-184 (2015). doi:10.1007/978-1-4939-2272-7_12 Medline). Lipidex-treated biotinylated RBD was then prepared as described above for lipidex-treated S.

[0271] Lipidex-treatment of RBD protein to remove potential bound free fatty acid. Purified RBD protein was treated with lipidex-1000 resin (Perkin Elmer; cat no. 6008301), pre-equilibrated in PBS pH 7.5 overnight at 4 degrees C. on a roller shaker. Subsequently, lipidex-treated RBD was separated from the resin using a gravity flow column. Integrity of the protein was confirmed by size exclusion chromatography (SEC) using a S200 10/300 increase column (GE Healthcare) and SDS-PAGE.

Example 18

[0272] In order to assess the binding characteristics of fatty acids related to linoleic acid, the experiments of Example 17 were repeated, but using oleic acid (OA) instead of linoleic acid (LA). OA was diluted to concentrations between 1 μM and 3 μM and flowed over 3,200 RU of biotinylated and lipidex-treated RBD immobilized on a streptavidin-coated sensor chip. Black lines correspond to a global fit of the data using a 1:1 binding model. Each experiment was repeated independently three times. All OA concentrations were used to calculate the KD, kon and koff values. The results are shown in FIG. 19.

Example 19

[0273] Example 19 reveals the precise binding mode between Oleic Acid and the SARS-CoV-2 Spike Protein. The structure of Oleic Acid bound to the SARS-CoV-2 Spike trimer was determined at 2.6 A resolution by cryo-electron miscroscopy. The results are shown in FIGS. 20A and 20B.

[0274] The methods utilized to determine the structure are as follows:

[0275] Protein expression and purification. Spike protein was expressed and purified as described (Toelzer et al Science 2020) except that minimal media devoid of supplemented free fatty acids was used (Expression system Inc). No FFA (OA or else) was supplemented at any step.

[0276] Cryo-EM sample preparation and data collection. 4 μL of ˜1 mg/mL Spike was loaded onto a freshly glow discharged (2 min at 4 mA) C-flat R1.2/1.3 carbon grid (Agar Scientific), blotted using a Vitrobot MarklV (Thermo Fisher Scientific) at 100% humidity and 4° C. for 2 s, and plunge frozen. Data were acquired on a FEI Talos Arctica transmission electron microscope operated at 200 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV using the EPU software. Data were collected in super-resolution at a nominal magnification of 130000× with a virtual pixel size of 0.525 A. The dose rate was adjusted to 6.1 counts/physical pixel/s. Each movie was fractionated in 55 frames of 200 ms. 8,639 micrographs were collected in a single session with a defocus range comprised between −0.8 and −2 μm.

[0277] Cryo-EM data processing. The dose-fractionated movies were gain-normalized, aligned, and dose-weighted using MotionCor2. Defocus values were estimated and corrected using the Gctf program. >1 Mio particles were automatically picked using Relion 3.0 software. The auto-picked particles were extracted with a box size of 110 px (2× binning). Reference-free 2D classification was performed to select well-defined particles. After four rounds of 2D classification >400.000 particles were selected for subsequent 3D classification. The initial 3D model was filtered to 60 A during 3D classification in Relion using 8 classes. A box size of 220 px (1.05 A/px, unbinned) was used. 3D-refined particles were then subjected to a second round of 3D classification yielding 200.000 particles. These particles were subjected to 3D refinement without applying any symmetry. The maps were subsequently subjected to local defocus correction and Bayesian particle polishing in Relion 3.1. Global resolution and B factor (−97.6 Å2) of the map were estimated by applying a soft mask around the protein density, using the gold-standard Fourier shell correlation (FSC) =0.143 criterion, resulting in an overall resolution of 3.0 A. C3 symmetry was applied to the Bayesian polished C1 map using Relion 3.1 yielding a final resolution of 2.6 A (B factor of −106.7 Å2).

[0278] Cryo-EM model building and analysis. For model building, UCSF Chimera was used to fit an atomic model of the SARS-CoV2 Spike locked conformation (Toelzer et al Science 2020) into the C3 symmetrized cryo-EM map. The model was rebuilt using sharpened and unsharpened maps in Coot [and then fitted into the C1 cryo-EM map. Namdinator and Coot were used to improve the fit and N-linked glycans were built into the density for both models where visible. Restraints for non-standard ligands were generated with eLBOW. The model for C1 and C3-symmetrized closed conformation was real space refined with Phenix, and the quality was additionally analyzed using MolProbity and EMRinger, to validate the stereochemistry of the components. Figures were prepared using UCSF chimera and PyMOL (Schrodinger, Inc).

Example 20

[0279] Minivirus displaying SARS-CoV-2 Spike protein on its surface is blocked from binding its receptor ACE2 by linoleic acid. (A) Schematic illustration of a Minivirus with SARS-CoV-2 Spike protein on its surface, immobilized via their His-tag. (B) Representative confocal microscopy images of MCF7 human epithelial cells incubated for 10 min with Miniviruses, showing attachment of the Miniviruses to the cell's surface. Miniviruses are individually visualized as small dots. The inset shows magnified area of attachment. (C) Miniviruses allowed for a systematic assessment of changes in SARS-CoV-2 Spike protein-mediated cell interactions as a function of Free Fatty Acid (FFA) occupancy. Because recombinant native SARS-CoV-2 Spike protein binds the Free Fatty Acids Linoleic Acid and Oleic Acid during heterologous protein expression and purification, we first generated FFA-depleted Spike protein (ApoS) by treatment of the purified SARS-CoV-2 Spike protein with lipidex columns as described in Example 17. Compared to the native Spike protein, ApoS-decorated Miniviruses displayed increased binding to human epithelial cells (First two bars on the left of the chart). This is consistent with a model where FFAs lock a closed Spike protein conformation, inhibiting binding to its receptor, ACE2. Controlled loading of ApoS Miniviruses by incubating them with FFAs of differing length and saturation (i.e., palmitic acid (PA), oleic acid (OA), LA and arachidonic acid (AA)). Binding of OA, and LA, but not PA, in SARS-CoV-2 Spike protein was successfully verified by multiple reactions monitoring LC-MS/MS. Of note, addition of saturated PA did not significantly reduce Minivirus-ACE2 cell binding compared to ApoS. However, we found that the (poly)-unsaturated FFAs OA, LA and AA were able to reduce Minivirus binding compared to ApoS Miniviruses.

Example 21

Example 21 reveals via electron tomography the effect of linoleic acid on cultured mammalian cells infected with live SARS-CoV-2 virus.

[0280] FIGS. 22A and 22B shows that the stereotypical enveloped mini-organelles known as “replication factories” inside SARS-CoV-2 are wrecked by the presence of linoleic acid, and the viral particles themselves also suffer visible deformation and destruction by the presence of linoleic acid.

[0281] The methods utilized to carry out the electron tomography are as follows:

[0282] Cells and virus propagation. A human gut epithelial cell line Caco2 expressing ACE2 (Caco-2-ACE2) (a kind gift of Dr Yohei Yamauchi, University of Bristol) were cultured at 37° C. in 5% CO2 in Dulbecco's modified Eagle's medium plus GlutaMAX (DMEM, Gibco, ThermoFisher) supplemented with 10% fetal bovine serum (FBS, Gibco, ThermoFisher) and 0.1 mM non-essential amino acids (NEAA, Sigma Aldrich). A SARS-CoV-2 reporter virus expressing a gene encoding the fluorescent protein turboGFP in place of the ORF7 gene (termed rSARS-CoV-2/Wuhan/ORF7-tGFP) was generated using a SARS-CoV-2 (Wuhan isolate) reverse genetics system by yeast-based transformation associated recombination cloning (manuscript in preparation). The virus was propagated in cells grown in infection medium (Eagle's minimum essential medium plus GlutaMAX (MEM, Gibco) supplemented with 2% FBS and NEAA). Cells were incubated at 37° C. in 5% CO2 until cytopathic effects were observed at which time the supernatant was harvested and filtered through a 0.2 um filter.

[0283] Viral detection by fluorescence. Caco-2-ACE2 cells were seeded onto glass coverslips coated with poly-D-lysine in 24 well plates or in μClear 96-well Microplates (Greiner Bio-one) in DMEM supplemented with 10% FBS until cell coverage on the coverslips reached 25%. The cells were inoculated with rSARS-CoV-2/Wuhan/ORF7-tGFP at MOI 5 in MEM supplemented with 2% FBS for 60 minutes at room temperature before the media was removed and replaced with infection medium containing 50 μM linoleic acid and 0.25% DMSO, or 0.25% DMSO only. Control wells were treated the same but received no infectious inoculum. Cells were incubated at 37° C. in 5% CO2 for 36 hours until turboGFP expression was detectable in cells in the 96 well plate by fluorescence imaging with an ImageXpress Pico Automated Cell Imaging System (Molecular Devices). Samples were inactivated and fixed by submersion in 4% paraformaldehyde (PFA) for 60 minutes at room temperature. All work with infectious recombinant SARS-CoV-2 was done inside a class III microbiological safety cabinet in a containment level 3 facility at the University of Bristol, UK.

[0284] Electron tomography. 300 nm sections collected on Pioloform-coated slot grids (Agar Scientific) were incubated in a solution of 15 nm gold fiducial markers (Aurion) for 5 min on each side. Tilt series (−65° to +65° at 1.5° increments) were acquired at 19,000× magnification (0.5261 nm/px) using a FEI Tecnai 20 transmission electron microscope operated at 200 kV and equipped with a 4k by 4k FEI Eagle camera. Electron tomograms were reconstructed using fiducial markers for alignment in IMOD.

[0285] Listing of SEQ ID NOs:

TABLE-US-00003 SARS-CoV-2 Spike protein used for structural studies SEQ ID NO: 1 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPD KVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGY FKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNN LDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS PASVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQL NRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFS QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTIT SGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV KQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQS LQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFC GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPSGRLVPRGSPGSGYIPEAPRDGQAYVRKDG EWVLLSTFLGHHHHHH SARS-CoV-2 Spike protein full-length SEQ ID NO: 2 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPD KVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGY FKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNN LDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS PRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFC TQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQ KLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGR LQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRV DFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNT FVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHT SPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSC CSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT SARS-CoV Spike protein full-length SEQ ID NO: 3 MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGV YYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPV IPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNS TNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCT FEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGY QPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSP AQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQ NPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNIT NLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTF FSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPG QTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKY RYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLND YGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLI KNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTD SVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQD VNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAE HVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMS LGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDC NMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNT REVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFI EDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGL TVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPF AMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESL TTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLN DILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEI RASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPH GVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFN GTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVY DPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVN IQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWL GFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDE DDSEPVLKGVKLHYT MERS-CoV Spike protein full-length SEQ ID NO: 4 MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFF DKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGD HGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRI GAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGK MGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNS YTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFM YTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQ FATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQP LTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGV YSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFK RLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSS LILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLI LATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNA NQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGST VAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQL GNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVG YYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACE HISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNS SLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLA SIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQ KVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANL RQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSIS TGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASA RDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGW TAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANK FNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASEL SNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNA FVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQG THIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAAN PTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSL NTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFK NVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESY IDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCC TGCGTNCMGKLKCNRCCDRYEEYDLEPHKVHVH HCoV-OC43 Spike protein full-length SEQ ID NO: 5 MFLILLISLPTAFAVIGDLKCPLDTSYKGTFNNKDTGPPF ISTDTVDVTNGLGTYYVLDRVYLNTTLFLNGYYPTSGSTY RNMALKGTDKLSTLWFKPPFLSDFINGIFAKVKNTKVFKD GVMYSEFPAITIGSTFVNTSYSVVVQPRTINSTQDGDNKL QGLLEVSVCQYNMCEYPHTSCHPKLGNHFKELWHLDTGVV SCLYKRNFTYDVNANYLYFHFYQEGGTFYAYFTDTGVVTK FLFNVYLGMALSHYYVMPLTCISRRDIGFTLEYWVTPLTS RQYLLAFNQDGIIFNAVDCMSDFMSEIKCKTQSIAPPTGV YELNGYTVQPIADVYRRKPDLPNCNIEAWLNDKSVPSPLN WERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCF SSITIDKFAIPNGRKVDLQLGNLGYLQSSNYRIDTTATSC QLYYNLPAANVSVSRFNPSTWNKRFGFIEDSVFVPQPTGV FTNHSVVYAQHCFKAPKNFCPCKLNGSCPGKNNGIGTCPA GTNYLTCDNLCTLDPITFKAPGTYKCPQTKSLVGIGEHCS GLAVKSDYCRGNSCTCQPQAFLGWSADSCLQGDKCNIFAN LILHDVNSGLTCSTDLQKANTDIILGVCVNYDLYGISGQG IFVEVNATYYNSWQNLLYDSNGNLYGFRDYITNRTFMIRS CYSGRVSAAFHANSSEPALLFRNIKCNYVFNNSLTRQLQP INYSFDSYLGCWNAYNSTAISVQTCDLTVGSGYCVDYSKN RRSRRAITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIP SEFTIGNMEEFIQTSSPKVTIDCAAFVCGDYAACKLQLVE YGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKL KDGVNFNVDDINFSPVLGCLGSECSKASSRSAIEDLLFDK VKLSDVGFVEAYNNCTGGAEIRDLICVQSYKGIKVLPPLL SENQISGYTLAATSASLFPPWTAAAGVPFYLNVQYRINGL GVTMDVLSQNQKLIANAFNNALYAIQQGFDATNSALVKIQ AVVNANAEALNNLLQQLSNRFGAISASLQEILSRLDALEA EAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKV NECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFNYVP TKYVTAKVSPGLCIAGNRGIAPKSGYFVNVNNTWMYTGSG YYYPEPITENNVVVMSTCAVNYTKAPYVMLNTSIPNLPDF KEELDQWFKNQTSVAPDLSLDYINVTFLDLQVEMNRLQEA IKVLNHSYINLKDIGTYEYYVKWPWYVWLLICLAGVAMLV LLFFICCCTGCGTSCFKKCGGCCDDYTGYQELVIKTSHDD HCoV-HKU1 Spike protein full-length SEQ ID NO: 6 MLLIIFILPTTLAVIGDFNCTNFAINDLNTTVPRISEYVV DVSYGLGTYYILDRVYLNTTILFTGYFPKSGANFRDLSLK GTTYLSTLWYQKPFLSDFNNGIFSRVKNTKLYVNKTLYSE FSTIVIGSVFINNSYTIVVQPHNGVLEITACQYTMCEYPH TICKSKGSSRNESWHFDKSEPLCLFKKNFTYNVSTDWLYF HFYQERGTFYAYYADSGMPTTFLFSLYLGTLLSHYYVLPL TCNAISSNTDNETLQYWVTPLSKRQYLLKFDNRGVITNAV DCSSSFFSEIQCKTKSLLPNTGVYDLSGFTVKPVATVHRR IPDLPDCDIDKWLNNFNVPSPLNWERKIFSNCNFNLSTLL RLVHTDSFSCNNFDESKIYGSCFKSIVLDKFAIPNSRRSD LQLGSSGFLQSSNYKIDTTSSSCQLYYSLPAINVTINNYN PSSWNRRYGFNNFNLSSHSVVYSRYCFSVNNTFCPCAKPS FASSCKSHKPPSASCPIGTNYRSCESTTVLDHTDWCRCSC LPDPITAYDPRSCSQKKSLVGVGEHCAGFGVDEEKCGVLD GSYNVSCLCSTDAFLGWSYDTCVSNNRCNIFSNFILNGIN SGTTCSNDLLQPNTEVFTDVCVDYDLYGITGQGIFKEVSA VYYNSWQNLLYDSNGNIIGFKDFVTNKTYNIFPCYAGRVS AAFHQNASSLALLYRNLKCSYVLNNISLTTQPYFDSYLGC VFNADNLTDYSVSSCALRMGSGFCVDYNSPSSSSSRRKRR SISASYRFVTFEPFNVSFVNDSIESVGGLYEIKIPTNFTI VGQEEFIQTNSPKVTIDCSLFVCSNYAACHDLLSEYGTFC DNINSILDEVNGLLDTTQLHVADTLMQGVTLSSNLNTNLH FDVDNINFKSLVGCLGPHCGSSSRSFFEDLLFDKVKLSDV GFVEAYNNCTGGSEIRDLLCVQSFNGIKVLPPILSESQIS GYTTAATVAAMFPPWSAAAGIPFSLNVQYRINGLGVTMDV LNKNQKLIATAFNNALLSIQNGFSATNSALAKIQSVVNSN AQALNSLLQQLFNKFGAISSSLQEILSRLDALEAQVQIDR LINGRLTALNAYVSQQLSDISLVKFGAALAMEKVNECVKS QSPRINFCGNGNHILSLVQNAPYGLLFMHFSYKPISFKTV LVSPGLCISGDVGIAPKQGYFIKHNDHWMFTGSSYYYPEP ISDKNVVFMNTCSVNFTKAPLVYLNHSVPKLSDFESELSH WFKNQTSIAPNLTLNLHTINATFLDLYYEMNLIQESIKSL NNSYINLKDIGTYEMYVKWPWYVWLLISFSFIIFLVLLFF ICCCTGCGSACFSKCHNCCDEYGGHHDFVIKTSHDD HCoV-229E Spike protein full-length SEQ ID NO: 7 MFVLLVAYALLHIAGCQTTNGLNTSYSVCNGCVGYSENVF AVESGGYIPSDFAFNNWFLLTNTSSVVDGVVRSFQPLLLN CLWSVSGLRFTTGFVYFNGTGRGDCKGFSSDVLSDVIRYN LNFEENLRRGTILFKTSYGVVVFYCTNNTLVSGDAHIPFG TVLGNFYCFVNTTIGNETTSAFVGALPKTVREFVISRTGH FYINGYRYFTLGNVEAVNFNVTTAETTDFCTVALASYADV LVNVSQTSIANIIYCNSVINRLRCDQLSFDVPDGFYSTSP IQSVELPVSIVSLPVYHKHTFIVLYVDFKPQSGGGKCFNC YPAGVNITLANFNETKGPLCVDTSHFTTKYVAVYANVGRW SASINTGNCPFSFGKVNNFVKFGSVCFSLKDIPGGCAMPI VANWAYSKYYTIGSLYVSWSDGDGITGVPQPVEGVSSFMN VTLDKCTKYNIYDVSGVGVIRVSNDTFLNGITYTSTSGNL LGFKDVTKGTIYSITPCNPPDQLVVYQQAVVGAMLSENFT SYGFSNVVELPKFFYASNGTYNCTDAVLTYSSFGVCADGS IIAVQPRNVSYDSVSAIVTANLSIPSNWTTSVQVEYLQIT STPIVVDCSTYVCNGNVRCVELLKQYTSACKTIEDALRNS ARLESADVSEMLTFDKKAFTLANVSSFGDYNLSSVIPSLP TSGSRVAGRSAIEDILFSKLVTSGLGTVDADYKKCTKGLS IADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGIALGG LTSAVSIPFSLAIQARLNYVALQTDVLQENQKILAASFNK AMTNIVDAFTGVNDAITQTSQALQTVATALNKIQDVVNQQ GNSLNHLTSQLRQNFQAISSSIQAIYDRLDTIQADQQVDR LITGRLAALNVFVSHTLTKYTEVRASRQLAQQKVNECVKS QSKRYGFCGNGTHIFSIVNAAPEGLVFLHTVLLPTQYKDV EAWSGLCVDGTNGYVLRQPNLALYKEGNYYRITSRIMFEP RIPTMADFVQIENCNVTFVNISRSELQTIVPEYIDVNKTL QELSYKLPNYTVPDLVVEQYNQTILNLTSEISTLENKSAE LNYTVQKLQTLIDNINSTLVDLKWLNRVETYIKWPWWVWL CISVVLIFVVSMLLLCCCSTGCCGFFSCFASSIRGCCEST KLPYYDVEKIHIQ HCoV-NL63 Spike protein full-length SEQ ID NO: 8 MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIV TGLLPTHWFCANQSTSVYSANGFFYIDVGNHRSAFALHTG YYDANQYYIYVTNEIGLNASVTLKICKFSRNTTFDFLSNA SSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTF YVPAAYKLTKLSVKCYFNYSCVFSVVNATVTVNVTTHNGR VVNYTVCDDCNGYTDNIFSVQQDGRIPNGFPFNNWFLLTN GSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGS DVNCNGYQHNSVVDVMRYNLNFSANSLDNLKSGVIVFKTL QYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTINTT HVSTFVGILPPTVREIVVARTGQFYINGFKYFDLGFIEAV NFNVTTASATDFWTVAFATFVDVLVNVSATNIQNLLYCDS PFEKLQCEHLQFGLQDGFYSANFLDDNVLPETYVALPIYY QHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCVRTS HFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNF QKFKTICFSTVEVPGSCNFPLEATWHYTSYTIVGALYVTW SEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYDYVGTGI IRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQ PDQVAVYQQSIIGAMTAVNESRYGLQNLLQLPNFYYVSNG GNNCTTAVMTYSNFGICADGSLIPVRPRNSSDNGISAIIT ANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRC KNLLKQYTSACKTIEDALRLSAHLETNDVSSMLTFDSNAF SLANVTSFGDYNLSSVLPQRNIRSSRIAGRSALEDLLFSK VVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGV ADAERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNY VALQTDVLQENQKILAASFNKAINNIVASFSSVNDAITQT AEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQAIS NSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNK YTEVRGSRRLAQQKINECVKSQSNRYGFCGNGTHIFSIVN SAPDGLLFLHTVLLPTDYKNVKAWSGICVDGIYGYVLRQP NLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFV NISRVELHTVIPDYVDVNKTLQEFAQNLPKYVKPNFDLTP FNLTYLNLSSELKQLEAKTASLFQTTVELQGLIDQINSTY VDLKLLNRFENYIKWPWWVWLIISVVFVVLLSLLVFCCLS TGCCGCCNCLTSSMRGCCDCGSTKLPYYEFEKVHVQ SARS-CoV-2 Spike protein RBD domain SEQ ID NO: 9 SETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLC PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI SARS-CoV Spike protein RBD domain SEQ ID NO: 10 AELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLC PFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFST FKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQI  MERS-CoV Spike protein RBD domain SEQ ID NO: 11 SQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECD FSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFT CSQISPAAIASNCYSSLILDYFSYPLSMKSDL HCoV-OC43 Spike protein RBD domain SEQ ID NO: 12 SEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPDLPNC NIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADS FTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDL HCoV-HKU1 Spike protein RBD domain SEQ ID NO: 13 SEIQCKTKSLLPNTGVYDLSGFTVKPVATVHRRIPDLPDC DIDKWLNNFNVPSPLNWERKIFSNCNFNLSTLLRLVHTDS FSCNNFDESKIYGSCFKSIVLDKFAIPNSRRSDL HCoV-229E Spike protein RBD domain SEQ ID NO: 14 NRLRCDQLSFDVPDGFYSTSPIQSVELPVSIVSLPVYHKH TFIVLYVDFKPQSGGGKCFNCYPAGVNITLANFNETKGPL HCoV-NL63 Spike protein RBD domain SEQ ID NO: 15 EKLQCEHLQFGLQDGFYSANFLDDNVLPETYVALPIYYQH TDINFTATASFGGSCYVCKPHQVNISLNGNTSV

REFERENCES CITED

[0286] 1 Varga, Z. et al. Endothelial cell infection and endotheliitis in COVID-19. Lancet 395, 1417-1418, doi :10.1016/SO140-6736(20)30937-5 (2020). [0287] 2 Tay, M. Z., Poh, C. M., Renia, L., MacAry, P. A. & Ng, L. F. P. The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol 20, 363-374, doi:10.1038/541577-020-0311-8 (2020). [0288] 3 Zhou, P. et aL A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273, doi:10.1038/s41586-020-2012-7 (2020). [0289] 4 Yan, B. et al. Characterization of the Lipidomic Profile of Human Coronavirus-Infected Cells: Implications for Lipid Metabolism Remodeling upon Coronavirus Replication. Viruses 11, doi:10.3390/v11010073 (2019). [0290] 5 Zumla, A., Chan, J. F., Azhar, E. I., Hui, D. S. & Yuen, K. Y. Coronaviruses—drug discovery and therapeutic options. Nat Rev Drug Discov 15, 327-347, doi:10.1038/nrd.2015.37 (2016). [0291] 6 Wu, J. T. et al. Estimating clinical severity of COVID-19 from the transmission dynamics in Wuhan, China. Nat Med 26, 506-510, doi:10.1038/541591-020-0822-7 (2020). [0292] 7 Grasselli, G. et al. Baseline Characteristics and Outcomes of 1591 Patients Infected With SARS-CoV-2 Admitted to ICUs of the Lombardy Region, Italy. JAMA, doi:10.1001/jama.2020.5394 (2020). [0293] 8 Cummings, M. J. et al. Epidemiology, clinical course, and outcomes of critically ill adults with COVID-19 in New York City: a prospective cohort study. Lancet, doi :10.1016/SO140-6736(20)31189-2 (2020). [0294] 9 Hoffmann, M. et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271-280 e278, doi:10.1016/j.ce11.2020.02.052 (2020). [0295] 10 Matsuyama, S. et al. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Acad Sci USA 117, 7001-7003, doi:10.1073/pnas.2002589117 (2020). [0296] 11 Hoffmann, M., Kleine-Weber, H. & Pohlmann, S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell 78, 779-784 e775, doi:10.1016/j.molce1.2020.04.022 (2020). [0297] 12 Du, L. et al. The spike protein of SARS-CoV--a target for vaccine and therapeutic development. Nat Rev Microbiol 7, 226-236, doi:10.1038/nrmicro2090 (2009). [0298] 13 Letko, M., Marzi, A. & Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5, 562-569, doi:10.1038/s41564-020-0688-y (2020). [0299] 14 Walls, A. C. et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281-292 e286, doi:10.1016/j.ce11.2020.02.058 (2020). [0300] 15 Kindler, E. & Thiel, V. To sense or not to sense viral RNA--essentials of coronavirus innate immune evasion. Curr Opin Microbiol 20, 69-75, doi:10.1016/j.mib.2014.05.005 (2014). [0301] 16 Kikkert, M. Innate Immune Evasion by Human Respiratory RNA Viruses. J Innate Immun 12, 4-20, doi:10.1159/000503030 (2020). [0302] 17 Zang, R. et al. TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol 5, doi:10.1126/sciimmunol.abc3582 (2020). [0303] 18 Qin, C. et al. Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin Infect Dis, doi:10.1093/cid/ciaa248 (2020). [0304] 19 Ziegler, C. G. K. et al. SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues. Cell 181, 1016-1035 e1019, doi:10.1016/j.ce11.2020.04.035 (2020). [0305] 20 Shen, B. et al. Proteomic and Metabolomic Characterization of COVID-19 Patient Sera. Cell, doi:10.1016/j.ce11.2020.05.032 (2020). [0306] 21 Goodwin, C. M., Xu, S. & Munger, J. Stealing the Keys to the Kitchen: Viral Manipulation of the Host Cell Metabolic Network. Trends Microbiol 23, 789-798, doi:10.1016/j.tim.2015.08.007 (2015). [0307] 22 Schoggins, J. W. & Randall, G. Lipids in innate antiviral defense. Cell Host Microbe 14, 379-385, doi:10.1016/j.chom.2013.09.010 (2013). [0308] 23 Nagamine, T., Inaba, T. & Sako, Y. A nuclear envelop-associated baculovirus protein promotes intranuclear lipid accumulation during infection. Virology 532, 108-117, doi:10.1016/j.viro1.2019.04.006 (2019). [0309] 24 Zaman, M. M. et al. Linoleic acid supplementation results in increased arachidonic acid and eicosanoid production in CF airway cells and in cftr−/− transgenic mice. Am J Physiol Lung Cell Mol Physiol 299, L599-606, doi:10.115.sup.2/.sub.ajplung.00346.2009 (2010). [0310] 25 Richieri GV, Ogata RT, Kleinfeld AM. The measurement of free fatty acid concentration with the fluorescent probe ADIFAB: a practical guide for the use of the ADIFAB probe. Mol Cell Biochem. 1999;192(1-2):87-94. [0311] 26 Toelzer et al. Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein. Science. 370, 725-730 (2020).