Patent Application
20240035012
The present invention relates to engineered ACE2 oligomers and composition comprising the oligomers. The present invention also relates to compositions and methods for preventing or treating coronavirus infection and detecting coronavirus.
Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has resulted in a severe global pandemic. Following SARS-CoV, SARS-CoV-2 is yet another beta-coronavirus emerged to threaten human health. SARS-CoV-2 and SARS-CoV are very similar, sharing 79.5% sequence identity (1), having similar spike protein structures (2-4), and having the same cell surface receptor angiotensin converting enzyme II (ACE2) (1, 5). Unfortunately, seventeen years after severe acute respiratory syndrome (SARS) pandemic, no targeted vaccines or therapeutics were approved for SARS which would have a high probability to treat COVID-19. Many neutralizing antibodies against SARS-CoV-2 are currently being urgently developed (6-10), some of these might become available later this year or next year. However, RNA viruses are known to have higher mutation rates (11, 12), mutation strains making current SARS-CoV-2 neutralizing antibodies ineffective could develop in the future, and many SARS-CoV-2 mutations have already been identified such as D614G(13-16). The appearance of COVID-19 after SARS indicates other related coronavirus pandemic will likely happen in the future too. Thus, therapeutics that are broadly effective against SARS-CoV-2 and mutants, even other SARS-CoV-2 related coronaviruses are highly desirable. Both SARS-CoV-2 and SARS-CoV bind ACE2 for cell entry, SARS-CoV-2 mutants and future related coronavirus will likely bind ACE2 for infection too. Therefore, decoys proteins engineered based on ACE2 could serve as the most broadly neutralizing proteins against these viruses and will be least likely to face mutational escape.
Furthermore, ACE2 biological function supports using ACE2 decoy proteins for SARS-CoVs infection treatment. Coronavirus infection or even spike protein binding can cause shedding of ACE2 from cell surface resulting decreased ACE2 expression level and accumulation of plasma angiotensin II (17-19) and this is closely related with acute lung injury(17, 20-22). Replenishing soluble ACE2 could alleviate acute respiratory distress syndrome (ARDS) (17, 21-23). In fact, it has been shown ACE2 peptidase domain could inhibit SARS-CoVs infection in cell assays and organoids (24-26), one clinical trial(NCT04335136) was also registered to use recombinant ACE2 to treat COVID-19. However, recombinant soluble ACE2 only has moderate binding affinity to SARS-CoV-2 spike protein (˜30 nM) (27) and can only inhibit virus at high concentration(24, 26, 28, 29), thus it may not be an optimal molecule to inhibit virus infection. Engineered ACE2 bearing multiple mutations and dimeric ACE2-ig have been shown to have better inhibition activities (25, 28-30). Spike proteins of SARS-CoVs function as trimers (2-4), we envisioned an engineered trimeric ACE2 protein could potentially bind up to three receptor binding domains (RBD) on spike protein to drastically increase binding affinity through avidity effect and to potently inhibit SARS-CoVs.
SARS-CoV-2 enters cells via ACE-2, which binds the trimeric spike protein with moderate affinity (KD˜30 nM). Despite a constant background mutational rate, the virus must retain binding with ACE2 for infectivity, providing a functional constraint for SARS-CoV-2 inhibitors. We engineered a trimeric ACE2 (T-ACE2) that binds spike protein with extremely high affinity (KD<1 pM), while retaining ACE2 native sequence. T-ACE2 can potently neutralize SARS-CoV-2, SARS-CoV, eight SARS-CoV-2 mutants and a SARSr-CoV tested. The cryo-EM structure of the complex revealed T-ACE2 can induce spike protein to transit to three RBDs up conformation for binding. We believe T-ACE2 represents a valuable approach for developing broadly neutralizing proteins against SARS-CoVs and mutants.
In one aspect, the present inventions provide ACE2 oligomers, wherein the ACE2 oligomer is formed by monomers, and each monomer comprises a soluble ACE2, a linker and an oligomerization motif. In some embodiments, the monomer comprises from N-terminal to C-terminal a soluble ACE2, a linker and an oligomerization motif. In some embodiments, the ACE 2 oligomer comprises an ACE2 trimer. In some embodiments, the ACE2 oligomer is an ACE2 trimer, tetramer, pentamer, hexamer and heptamer. In some embodiments, the oligomerization motif is a coil coiled motif, a foldon motif or a three helix bundle motif.
In some embodiments, the linker is a flexible linker or a rigid linker. In some embodiments, the linker is selected from the group consisting of GS(EAAAK)5GS (SEQ ID NO: 40), AH5 (SEQ ID NO: 41), GGGH5 (SEQ ID NO: 42), H3 (SEQ ID NO: 43), H4 (SEQ ID NO: 44), H6 (SEQ ID NO: 45), H7 (SEQ ID NO: 46), AP12 (SEQ ID NO: 47), AP 15 (SEQ ID NO: 48), (GGGGS)5 (SEQ ID NO: 49), and (EAAAK)5 (SEQ ID NO: 50). In some embodiments, the linker has 1, 2, 3, 4 or 5 amino acid substitution as compared to SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, the linker has the same length as SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, the linker comprises SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and has 1, 2, 3, 4 or 5 additional amino acids at one or both ends of SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, the linker comprises (EAAAK)n, wherein n can be any integer from 3 to 15. In some embodiments, the linker comprises (AP)n, wherein n can be any integer from 8 to 22.
In some embodiments, the soluble ACE2 comprises a sequence as set forth in SEQ ID NO: 3, 51, 52, 53, 54 or 55. In some embodiments, the soluble ACE2 comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 3, 51, 52, 53, 54 or 55. In some embodiments, the soluble ACE2 has the same length as SEQ ID NO: 3, 51, 52, 53, 54 or 55. In some embodiments, the soluble ACE2 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids at one or both ends of SEQ ID NO: 3, 51, 52, 53, 54 or 55.
In another aspect, the present inventions provide a composition comprising the ACE2 oligomers. In some embodiments, the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier.
In another aspect, the present inventions provide a use of the oligomer or the composition comprising the oligomer in manufacturing a medicament for treating coronavirus infection. In another aspect, the present inventions provide a use of the oligomer or the composition comprising the oligomer in manufacturing a composition for preventing coronavirus infection. In another aspect, the present inventions provide a use of the oligomer or the composition comprising the oligomer in manufacturing a composition for detecting coronavirus in a sample. In some embodiments, the coronavirus is SARS-CoV, SARS-CoV-2 and/or SARSr-CoV. In some embodiments, the coronavirus is a mutant of SARS-CoV, a mutant of SARS-CoV-2, and/or a mutant of SARSr-CoV.
In another aspect, the present inventions provide an ACE2 oligomer as described above or a composition comprising the ACE2 oligomer for treating or preventing coronavirus infection. In another aspect, the present inventions provide an ACE2 oligomer as described above or a composition comprising the ACE2 oligomer for detecting coronavirus in a sample. In some embodiments, the coronavirus is SARS-CoV, SARS-CoV-2 and/or SARSr-CoV. In some embodiments, the coronavirus is a mutant of SARS-CoV, a mutant of SARS-CoV-2, and/or a mutant of SARSr-CoV.
In another aspect, the present inventions provide a method of treating coronavirus infection, comprising administering to a subject a therapeutically effective amount of the ACE2 oligomer or the composition as defined above. In another aspect, the present inventions provide a method of preventing coronavirus infection, comprising administering to a subject a prophylactically effective amount of the ACE2 oligomer or the composition as defined above. In another aspect, the present inventions provide a method of detecting coronavirus in a sample, comprising obtaining a sample, and contacting the sample with the ACE2 oligomer or the composition as described above. In some embodiments, the coronavirus is SARS-CoV, SARS-CoV-2 and/or SARSr-CoV. In some embodiments, the coronavirus is a mutant of SARS-Cov, a mutant of SARS-CoV-2, and/or a mutant of SARSr-CoV.
L-M. SARS-CoV-2 inhibition by ACE2-rigid-foldon (T-ACE2), ACE2-H3-foldon (H3), ACE2-GGGH5-foldon (G3H5), ACE2-AP12-foldon (AP12), ACE2-AP15-foldon (APIS), ACE2-H6-foldon (H6), ACE2-AH5-foldon (AH5), ACE2-H4-foldon (H4), ACE2-H7-foldon (H7). The cells used in L were Caco-2 cells, and the cells used in M were huh-7 cells.
N. SARS-CoV-2 inhibition by ACE2-rigid-foldon (T-ACE2), ACE2-AP15-foldon (APIS), ACE2 M1-AP15-foldon (M1), ACE2 M2-AP15-foldon (M2), ACE2 M3 -AP15-foldon (M3), ACE2 M4-AP15-foldon (M4) and ACE2 M5-AP15-foldon (M5). The cells used in N were Caco-2 cells.
O. SARS-CoV-2 inhibition by ACE2-rigid-foldon with GFP cleaved (T-ACE2-Cut), ACE2-AP15-foldon with GFP cleaved (AP15-Cut), ACE2 M1-AP15-foldon with GFP cleaved (M1-Cut), ACE2 M2-AP15-foldon with GFP cleaved (M2-Cut), ACE2 M3-AP15-foldon with GFP cleaved (M3-Cut), ACE2 M4-AP15-foldon with GFP cleaved (M4-Cut) and ACE2 M5-AP15-foldon with GFP cleaved (M5-Cut). The cells used in O were Caco-2 cells.
“ACE2” or “angiotensin converting enzyme II”, is a type I cell-surface glycoprotein and is found in human and mammals (such as primate, bat, cat, dog, horse, mouse, rat, hamster, pig, cattle). Unless otherwise specified, ACE2 as used herein encompasses wild-type ACE2 and all the naturally-existing variants from any human and mammal species, as well as engineered ACE2. Human ACE2 is typically composed of 805 amino acids, with amino acids 1-17 being a N-terminal signal peptide, amino acids 18-740 being extracellular, amino acids 741-761 being transmembrane, and amino acids 762-805 being cytoplasmic. ACE2 comprises a peptidase domain (PD) (residues 18-615) with its HEXXH zinc binding metalloprotease motif, a Collectrin (a regulator of renal amino acid transport and insulin)-like domain (CLD) (residues 616-768) that includes a ferredoxin-like fold “Neck” domain, that end with an hydrophobic transmembrane hydrophobic helix region of 22 amino acid residues followed by an intracellular segment of 43 amino acid residues. Many human ACE2 variants have been identified, for example, those that include any one or any combination of the following mutations: S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P, H378R, K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L or D509Y (Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility Stawiski et al., 2020 (https://doi.org/10.1101/2020.04.07.024752)). ACE2 is found to be expressed in lungs, arteries, heart, kidney, intestines etc. and has diverse biological functions, including regulation of blood pressure through the renin-angiotensin-aldosterone system (RAAS). ACE2 also serves as the entry point into cells for some coronaviruses, including HCoV-NL63, SARS-CoV, and SARS-CoV-2. More specifically, the binding of the spike protein of SARS-CoV and SARS-CoV-2 to the enzymatic domain of ACE2 on the surface of cells results in endocytosis and translocation of both the virus and the enzyme into endosomes located within cells.
In some embodiments, ACE2 may be a human ACE2. In some embodiments, the human ACE2 may be the ACE2 of the sequence set forth under SEQ ID NO: 1 (wild-type). In some embodiments, the human ACE2 may be any naturally-existing or engineered ACE2 mutants or variants that retain a certain level of binding affinity to a spike protein of a coronavirus as compared to SEQ ID NO: 1, for example, mutants or variants having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and retaining at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the binding affinity, or has increased binding affinity, to a spike protein of a coronavirus as compared to the ACE2 of SEQ ID NO: 1. In some embodiments, the ACE2 mutant may have the length of SEQ ID NO: 1 and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions as compared to SEQ ID NO: 1. In some embodiments, the ACE2 mutant may have the same length as SEQ ID NO: 1 and have K31F, H34I, and E35Q substitutions as compared to SEQ ID NO: 1 (Proc Natl Acad Sci U S A. 2020 Nov 10;117(45):28046-28055. Wells JA.). In some embodiments, the ACE2 mutant may have the same length as SEQ ID NO: 1 and have T27Y, L79Y, and N330Y substitutions as compared to SEQ ID NO: 1 (Science. 2020 Sep4;369(6508):1261-1265. Procko E.). In some embodiments, the ACE2 mutant may have the same length as SEQ ID NO: 1 and have T27Y, and H34A substitutions as compared to SEQ ID NO: 1 (Sci Rep 11, 12740 (2021) Tanaka, S.). In some embodiments, the ACE2 mutant may have the same length as SEQ ID NO: 1 and have T27Y, K31F, H34I, E35Q, L79Y, and N330Y substitutions as compared to SEQ ID NO: 1. In some embodiments, the ACE2 mutant may have the same length as SEQ ID NO: 1 and have T27Y, H34A, L79Y, and N330Y substitutions as compared to SEQ ID NO: 1.
“Soluble ACE2” as used herein refers to an ACE2 as described above but lacks the transmembrane and cytoplasmic residues. In some embodiments, soluble ACE2 may comprise the entire extracellular domain. In some embodiments, soluble ACE2 may comprise a part of the extracellular domain. In some embodiments, soluble ACE2 may be the ACE2 that is composed of residues 1-740 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), or may be any functional fragments thereof. Functional fragments mean any fragments that retain at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the binding affinity, or has increased binding affinity, to a spike protein of a coronavirus as compared to SEQ ID NO: 3 (SEQ ID NO: 3 denotes amino acids 18-615 of SEQ ID NO: 1). Soluble ACE2 also encompasses variants or mutants of the above functional fragments that have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the above functional fragments, and retaining at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the binding affinity, or has increased binding affinity, to a spike protein of a coronavirus as compared to the ACE2 of SEQ ID NO: 3. In some embodiments, the soluble ACE2 may be SEQ ID NO: 3. In some embodiments, the soluble ACE2 may be of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the soluble ACE2 may have the length of SEQ ID NO: 3 and have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitution as compared to SEQ ID NO: 3. In some embodiments, the soluble ACE2 may be M1 (SEQ ID NO: 51), which has residues 18-615 of SEQ ID NO: 1 and has K31F, H34I, and E35Q substitutions as compared to SEQ ID NO: 1. In some embodiments, the soluble ACE2 may be M2 (SEQ ID NO: 52), which has residues 18-615 of SEQ ID NO: 1 and hasT27Y, L79Y, and N330Y substitutions as compared to SEQ ID NO: 1. In some embodiments, the soluble ACE2 may be M3 (SEQ ID NO: 53), which has residues 18-615 of SEQ ID NO: 1 and has T27Y, and H34A substitutions as compared to SEQ ID NO: 1. In some embodiments, the soluble ACE2 may be M4 (SEQ ID NO: 54), which has residues 18-615 of SEQ ID NO: 1 and has T27Y, K31F, H34I, E35Q, L79Y, and N330Y substitutions as compared to SEQ ID NO: 1. In some embodiments, the soluble ACE2 may be M5 (SEQ ID NO: 55), which has residues 18-615 of SEQ ID NO: 1 and has T27Y, H34A, L79Y, and N330Y substitutions as compared to SEQ ID NO: 1.
“Coronaviruses” are a group of related RNA viruses that are roughly spherical particles with bulbous surface projections and cause diseases in mammals and birds. Coronaviruses identified thus fir include SARS-Cod' i 2003. HCoV NL63 in 2004, HICoV HKU1 in 2005, MERS-CoV in 2012, and SARS-CoV-2 in 2019. In some embodiments, the coronaviruses comprise SARS-CoV and mutants (or variants) thereof, SARS-CoV-2 and mutants (or variants) thereof, and SARS-related coronaviruses (SARSr-CoV) and mutants (or variants) thereof SARSr-CoV refers to any coronavirus strain that enters a host cell through ACE2. In some embodiments, mutants or variants of SARS-CoV, SARS-CoV-2 or SARSr-CoV refer to coronavirus strains having a. genome that is of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99..2%, 99.3%, 99.4%, 99.5%, 99.6%, 99,7%, 99.8% or 99.9% sequence identity to the genome of SARS-CoV, SARS-CoV-2 or SARSr-CoV. In some embodiments, mutants or variants of SARS-CoV, SARS-CoV-2 or SARSr-CoV exhibit different or substantially the same activities and properties as SARS-COV, SARS-CoV-2 or SARSr-CoV, In some embodiments, SARS-CoV-2 may refer to the strain of the first reported genome (SARS-CoV-2 Wuhan-Hu-1). In some embodiments, mutants or variants of SARS-CoV-2 have a mutation in the spike protein. in some embodiments, mutations in the spike protein include a substitution selected from the group consisting of V341I, A344S, F342L, V367F, R408I, A435S, N439K, G476S, V483A, and D614G, as compared to the sequence of the spike protein of SAR S-CoV-2 Wuhan-Hu-1.
“ACE2 monomer” refers to a monomeric peptide or protein that comprises a soluble ACE2, a linker, and an oligomerization domain. In some embodiments, the ACE2 monomer comprises from N-terminal to C-terminal a soluble ACE2, a linker and an oligomerization domain. In some embodiments, the soluble ACE2 is directly connected to the linker through a peptide bond. In some embodiments, the linker is directly , connected to the oligomerization domain through a peptide bond. Different ACE2 monomers may have different soluble ACE2, different linker and/or different oligotnerization domain. In some embodiments, the ACE2 monomer may further comprise a label for being used in coronavirus detection. In some embodiments, the label may be a fluorescence label. In some embodiments, the label may be a fluorescence protein. In some embodiments, the label may be a quantum dot.
“ACE2 oligomer” refers to oligomers formed from association of ACE2 monomers through the oligomerization domain, the association may be covalent bond and/or non-covalent interactions (e.g., electrostatic interactions (e.g., ionic, hydrogen bonding, halogen bonding), van der Waals forces (e.g., dipole-dipole, dipole-induced dipole, London dispersion forces), n-effects, hydrophobic effect) In some embodiments, the ACE2 oligomer may comprise an ACE2 timer. In some embodiments, the ACE2 oligomer may be an ACE2 heptamer, hexamer, pentamer, tetramer or trimer. In some embodiments, the ACE2 oligomer may be formed from association of identical ACE2 monomers. In some embodiments, the ACE oligomer may be formed from association of different ACE2 monomers. In some embodiments, the ACE2 oligomer may be formed through spontaneous association of the oligomerization domain of the ACE2 monomers.
“Oligomerization motif” or “oligomerization domain” refers to a motif or domain that interacts with one another and brings the monomer into association. Different oligomerization motifs or domains are known in the art. For example, naturally-existing or de novo designed coiled coil motifs that allow 2-7 alpha-helices being coiled together, to form, for example, helical bundles or helical barrels (Robust De Novo-Designed Homotetrameric Coiled Coils. Biochemistry, Edgell et al., 2020 (https://doi.org/10.1021/acs.biochem.0c00082); A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology, Fletcher et al., 2012 (https://doi.org/10.1021/sb300028q); Navigating the Structural Landscape of De Novo α-Helical Bundles, Rhys et al., 2019 (https://doi.org/10.1021/jacs.8b13354); Computational design of water-soluble alpha-helical barrel, Thomson et al., 2014 (DOI: 10.1126/science.1257452); Maintaining and breaking symmetry in homomeric coiled-coil assemblies, Rhys et al., 2018 (https://doi.org/10.1038/s41467-018-06391-y). In some embodiments, the oligomerization motif may be a heptamerization motif, a hexamerization motif, a pentamerization motif, a tetramerization motif or a trimerization motif. In some embodiments, the oligomerization motif may be a coiled coil motif, a foldon motif or a three helix bundle motif.
“Linker” as used herein refers to a region that links two protein domain (e.g., a soluble ACE2 and an oligomerization motif) together. Linkers used in fusion protein technology are typically categorized into “flexible linker”, “rigid linker” and “in vivo cleavable linker”, and the standards for such categorization are well-known in the art (Fusion Protein Linkers: Property, Design and Functionality, Chen et al., 2012 (10.1016/j.addr.2012.09.039)). Flexible linkers confer flexibility in the structure, and examples of flexible linkers known in the art include (GGGGS)3, (Gly)8, (Gly)6, GGGGS, (GGGGS)n (n=1, 2, 4). Flexible linker as provided in the present disclosure includes (GGGGS)5 (SEQ ID NO: 50). Rigid linkers confer rigidity in the structure, and examples of rigid linkers known in the art include (EAAAK)n (n=1-3), A(EAAAK)4ALEA(EAAAK)4A, AEAAAKEAAAKA, PAPAP, (Ala-Pro)n (10-34 aa). Rigid linkers as provided in the present disclosure include GS(EAAAK)5GS (SEQ ID NO: 40), AH5 (SEQ ID NO: 41), GGGH5 (SEQ ID NO: 42), H3 (SEQ ID NO: 43), H4 (SEQ ID NO: 44), H6 (SEQ ID NO: 45), H7 (SEQ ID NO: 46), AP12 (SEQ ID NO: 47), AP15 (SEQ ID NO: 48), and (EAAAK)5 (SEQ ID NO: 51)).
“Treating a coronavirus infection” means reducing the amount of coronavirus or completely eliminating the presence of coronavirus in a subject, and/or alleviating one or more symptoms associated with coronavirus infection or completely eliminating the symptoms in a subject, as compared to the results in the absence of the treatment.
“Preventing a coronavirus infection” means preventing the infection of coronavirus in a subject, as compared to the results in the absence of the treatment.
“Detecting coronavirus” means detecting the presence, level or amount of coronavirus, and/or the activity of coronavirus in a sample. In some embodiments, the sample is a “biological sample” that may include body fluids (such as sputum, semen, lymph, sera, plasma, urine, synovial fluid and cerebro-spinal fluid), cell samples or tissue samples obtained from human and animals (mammals, poultry, livestock, birds etc.). In some embodiments, the samples may be an “environmental sample” or “non- biological sample” including feces, surgical fluids, water (drinking water, sea water, river water etc.), soil, food (meat, seafood, vegetables, fruits, diary etc.) and any other samples obtained from the environment. Methods of pretreating the sample such that it is suitable for detection are known in the art.
Any methods that involve using specific and/or high-affinity interactions between two protein molecules for detecting a target (such as a protein or a virus expressing a target protein) can be readily applied to the detection method of the present inventions, wherein the sensor is ACE2 oligomer and the target is coronavirus, and the specific and high-affinity interaction is between the ACE2 oligomer and the spike protein of the virus. Such detection methods are already known in the art. For examples, the detection method may involve competitive chromatography and involve attaching a florescent label such as fluorescence proteins or quantum dots (CN111273016A) to the ACE2 oligomer.
The term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, buffers and excipients, including buffered saline solution, water, and emulsions (such as an oil/water or water/oil emulsion), and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, 19th ed. 1995). Preferred pharmaceutical carriers depend upon the intended mode of administration of the active ingredient agent.
A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary according to factors such as the state of infection, disease or disorder; age; sex; and weight of the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmaceutical composition is outweighed by the therapeutically beneficial effects.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. A prophylactically effective amount may vary according to factors such as the state of infection, disease or disorder; age; sex; and weight of the individual. A prophylactically effective amount is also one in which any toxic or detrimental effects of the pharmaceutical composition is outweighed by the prophylactically beneficial effects.
As used herein, “sequence identity”, “% sequence identity”, or “% identical” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Sequences are generally aligned for maximum correspondence over a designated region, e.g., a region at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucleotides in length, and can be up to the full-length of the reference amino acid or nucleotide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Examples of algorithms that are suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Further exemplary algorithms include ClustalW (Higgins D., et al. (1994) Nucleic Acids Res 22: 4673-4680), available at www.ebi.ac.uk/Tools/clustalw/index.html. The percent sequence identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent sequence identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The terms “a”, “an”, and “the” as used herein are intended to mean “one and more than one” or “at least one”, unless the context clearly suggests a singular meaning.
The term “and/or” as used herein are intended to include any and all possible combinations of one or more of the listed items.
The terms “comprises” and “comprising” as used herein are intended to indicate the presence of an element, component, feature, step etc., but not to exclude the presence of any other elements, components, features, steps etc. In the present invention, when it is mentioned that a product comprises certain components, a substance comprises a structure, a method comprises a step etc., it should be understood that it also recites the product which is composed only of these components, a substance that is composed only of the structure, a method that is composed only of the step etc.
To develop trimeric ACE2 decoy proteins, we chose a C-terminal domain of T4 fibritin (foldon) (31, 32) or a three helix bundle (3HB) (33, 34) as trimerization motifs since these have been successfully demonstrated to form stable protein trimers (27, 31, 32). We then looked at the reported SARS-CoVs spike protein structures to determine the linker between trimerization motifs and ACE2 (27, 35-39). SARS-CoV spike protein mostly adopt one or two RBDs up conformations and can engage one or two ACE2 monomers (35, 36). A very small population of SARS-CoV spike protein can have three RBDs up conformation to bind three ACE2 monomers. SARS-CoV-2 structures mostly have closed conformation or one RBD up conformation (27, 37, 38). From these structural analyses, we estimated distances between RBDs on the same spike protein could range from 60 Å to 100 Å when they are in the up conformations. Moreover, structures from SARS-CoV viral particle revealed there are about 100 spike protein trimers displayed on the 100 nm diameter viral particle surface giving inter spike protein distance around 200 Å (3, 40, 41).
To retain the possibility for intra-spike or inter-spike avidity, we chose a flexible (GGGGS)5 linker or a more rigid linker selected from the group consisting of GS(EAAAK)5GS linker (SEQ ID NO: 40), AH5 linker (SEQ ID NO: 41), GGGHS linker (SEQ ID NO: 42), H3 linker (SEQ ID NO: 43), H4 linker (SEQ ID NO: 44), H6 linker (SEQ ID NO: 45), H7 linker (SEQ ID NO: 46), AP12 linker (SEQ ID NO: 47), and APIS linker (SEQ ID NO: 48) to construct trimeric ACE2 (42). We used ACE2 peptidase domain (18-615) (SEQ ID NO: 3, 51, 52, 53, 54 or 55) to construct all trimeric ACE2 decoy proteins, linkers were inserted after ACE2, followed by the trimerization motifs. We therefore constructed seventeen ACE2 monomers, for forming trimeric ACE2 proteins. The structure of the monomers are: ACE2-flexible-3HB (SEQ ID NO: 11) (flexible denotes the (GGGGS) s linker), ACE2-rigid-3HB (SEQ ID NO: 7) (rigid denotes the GS(EAAAK)5GS linker), ACE2-flexible-foldon (SEQ ID NO: 9), ACE2-rigid-foldon (SEQ ID NO: 5), ACE2-AH5-foldon (SEQ ID NO: 15), ACE2-GGGHS-foldon (SEQ ID NO: 17), ACE2-H3-foldon (SEQ ID NO: 19), ACE2-H4-foldon (SEQ ID NO: 21), ACE2-H6-foldon (SEQ ID NO: 23), ACE2-H7-foldon (SEQ ID NO: 25), ACE2-AP12-foldon (SEQ ID NO: 27), ACE2-AP15-foldon (SEQ ID NO: 29) , ACE2 M1-AP15-foldon (SEQ ID NO: 31), ACE2 M2-AP15-foldon (SEQ ID NO: 33), ACE2 M3-AP15-foldon (SEQ ID NO: 35), ACE2 M4-AP15-foldon (SEQ ID NO: 37), and ACE2 M5-AP15-foldon (SEQ ID NO: 39) (the structure is shown from N terminus to C terminus). In addition, we constructed two trimeric ACE2 proteins with a short linker GGGS (ACE2-short-3HB, ACE2-short-foldon) and a monomeric ACE2 as control proteins.
Note that all the ACE2 monomers were first constructed and obtained with an HRV3C cleavage sequence, an eGFP tag and a His8 tag (together termed the C-terminal tag) following the trimerization domain. Unless specified otherwise, the ACE2 monomer or trimer used below comprise the C-terminal tag.
We first used ELISA assay to determine binding affinities between ACE2 proteins and the prefusion stabilized trimeric SARS-CoV-2 spike protein ectodomain (S-ECD) (27). ACE2 monomer binds S-ECD with IC50˜27 nM. For trimeric ACE2 proteins, we saw massive binding affinity enhancement. Rigid linker constructs have highest binding affinities, ACE2-rigid-3HB and ACE2-rigid-foldon both bind S-ECD with IC50˜30 pM (
We further analyzed ACE2 proteins binding using biolayer interferometry (ForteBio Octet RED96) (
Next, we assessed the inhibitory activities of these trimeric ACE2 decoy proteins using SARS-CoV-2 and SARS-CoV pseudotyped viruses. ACE2 monomer can only inhibit SARS-CoV-2 pseudotyped virus at high concentration with IC50>50 nM. Trimeric ACE2 with flexible linkers shown much better inhibition activity, ACE2-flexible-3HB can inhibit SARS-CoV-2 with IC50 of 3.46 nM, ACE2-flexible-foldon has better inhibition activity with IC50 of 1.58 nM (
Because of the significantly advantageous effect attained by T-ACE2, we designed more trimeric ACE2 using different rigid linkers and compared these trimeric ACE2 with T-ACE2. Specifically, we made ACE2-H3-foldon (H3), ACE2-GGGHS-foldon (G3H5), ACE2-AP12-foldon (AP 12), ACE2-AP 1 5 -fol don (APIS), ACE2-H6-fol don (H6), ACE2-AH5-foldon (AH5), ACE2-H4-foldon (H4), and ACE2-H7-foldon (H7).
We also designed more trimeric ACE2 using the APIS linker and different soluble ACE2 mutants. Specifically, we made ACE2 M1-AP15-foldon (M1), ACE2 M2-AP15-foldon (M2), ACE2 M3-AP15-foldon (M3), ACE2 M4-AP15-foldon (M4), and ACE2 M5-AP15-foldon (M5). We also made T-ACE2 with the C-terminal tag cleaved (T- ACE2-Cut), AP 15 with the C-terminal tag cleaved (AP15-Cut), M1 with the C-terminal tag cleaved (M1-Cut), M2 with the C-terminal tag cleaved (M2-Cut), M3 with the C-terminal cleaved (M3-Cut), M4 with the C-terminal tag cleaved (M4-Cut), and M5 with the C-terminal tag cleaved (MS-Cut).
We then asked whether T-ACE2 can also inhibit SARS-CoV-2 mutants and related coronaviruses. We tested T-ACE2 inhibition activities on eight naturally occurring SARS-CoV-2 mutants including seven RBD domain mutations (14, 16), and D614G mutation (43); and the SARSr-CoV (WIV1) (
We further tested T-ACE2 inhibition of authentic SARS-CoV-2 virus (
We hypothesized properly designed trimeric ACE2 might engage more than one RBD from trimeric spike protein and thus dramatically increase binding affinity through avidity effect. To confirm this unique engagement, we determined the complex structure of T-ACE2/S-ECD using cryo-EM. In the complex structure, spike protein adopts only one conformation: the three RBDs up conformation. The complex is near perfect three-fold symmetric. Significantly, all these three RBDs bind to three ACE2s simultaneously, binding interactions between ACE2 and RBD are essentially the same as previous reports and the three individual monomer from the complex aligns quite well (
The distance between the C-terminal end of the three ACE2s is around 110 Å. If the trimerization motif sits right in the middle, then ideal linker length between trimerization motif and ACE2 would be around 60 Å, corresponding to (GGGGS)3 linker, thus the (GGGGS)5 flexible linker in our proteins is long enough for three ACE2 binding but is not optimal. The more rigid (EAAAK) 5 linker is shorter than (GGGGS)5 and can effectively separate different functional domains of fusion proteins (48). We think the (EAAAK)5 linker length is probably around 60 Å making it an optimal linker for T-ACE2, the rigidity nature of this (EAAAK) 5 linker probably helps to keep ACE2 right around RBD for immediate rebinding even if one of the ACE2 falls off spike protein. This probably explains the strong trimer-trimer avidity binding.
Since the beginning of COVID-19, tremendous efforts have been made to develop therapeutics especially neutralizing antibodies to treat COVID-19. However, the widespread and ongoing crisis of COVID-19 indicates SARS-CoV-2 will not be eliminated soon, thus unexpected mutations making current neutralizing antibodies ineffective could develop in the future. Furthermore, the emergence of COVID-19 after SARS suggests similar coronavirus pandemic might happen in the future. These calls for therapeutic approaches widely useful for current and future similar coronaviruses and mutants.
Several engineered ACE2 proteins bearing different number of mutations have been shown to increase spike protein binding affinities and virus neutralization activities (29, 49). Here, we engineered trimeric ACE2 proteins and showed T-ACE2, AH5, GGGH5, H3, H4, H6, H7, AP12, AP15, M1, M2, M3, M4, M5, AP15-Cut, M1-Cut, M2-Cut, M3-Cut, M4-Cut and M5-Cut can bind spike protein with extremely high affinity to potently inhibit all tested viruses including SARS-CoV-2, SARS-CoV, eight naturally occurred SARS-CoV-2 mutants and a SARSr-CoV. We demonstrated T-ACE2 can induce spike protein to transit to the unique three RBDs up conformation and bind all three RBDs simultaneously. The rigid linker employed in T-ACE2 has been injected into mice and didn't seem to show strong immunogenicity (50), 3HB and foldon trimerization motifs have been observed to cause immunogenicity, but introducing glycans can silence the immunogenicity without disrupting the trimer formation (51). We believe proteins engineered based wild type ACE2 such as T-ACE2 would be the most broadly SARS-CoVs neutralizing proteins and will be most resistant to mutational escape. We speculate properly designed higher oligomeric ACE2s may also have additional inter-molecular avidity binding with spike proteins on virus surface thus may have even higher virus inhibition activities. The extremely high binding affinity between T-ACE2 and spike protein (KD<1pM) suggests T-ACE2 could be useful for virus detection methods development. The nature that T-ACE2 was engineered based on native ACE2 sequence also makes such detection methods widely useful for all SARS-CoVs and related viruses.
Whether this T-ACE2 induced spike protein conformation change represents a transition state during virus infection cannot be definitively answered here. Full length ACE2 protein functions as dimer (44). The two monomers from this ACE2 dimer are in two-fold symmetry, they are also in close distance (distance between D615 is about 53 Å), so it's hard to imagine this dimeric ACE2 can engage more than one RBD from the same spike protein with current structural understandings. It is though possible cell surface ACE2 dimers might cluster together to induce more RBDs to adopt up conformation and eventually help virus to transit from prefusion state to postfusion state.
To construct trimeric ACE2s, we inserted the linker (GGGGS) 5 (SEQ ID NO: 49), GS(EAAAK)5GS (SEQ ID NO: 40), AH5 (SEQ ID NO: 41), GGGH5 (SEQ ID NO: 42), H3 (SEQ ID NO: 43), H4 (SEQ ID NO: 44), H6 (SEQ ID NO: 45), H7 (SEQ ID NO: 46), AP12 (SEQ ID NO: 47), AP15 (SEQ ID NO: 48) or GGGS after ACE2 (18-615), followed by trimerization motifs, an HRV3C cleavage sequence, an eGFP tag and a His8 tag. Monomeric ACE2 (SEQ ID NO: 13) was constructed as ACE2 (18-615)-(GGGGS)5-HRV3C-eGFP-His8 for direct comparison.
The ACE2 peptidase domain (18-615) (derived from full-length ACE2 (accession number: NM_001371415)) (SEQ ID NO: 3) was cloned from the plasmids donated by Peihui Wang's lab, mutants of ACE2 peptidase domain (SEQ ID Nos: 51-55) were constructed in our own lab based on previous publications. The genes of 3HB and foldon were synthesis by Genewiz, Suzhou, China. All the gene fragments were assembled by the Gibson assembly kit (Cat.C112-01, Vazyme). The assembled fragments were subcloned into pEGFP between XhoI and EcoRI respectively. The cloned plasmids were transformed into E.coli DH5α for amplification. Amplified plasmids were extracted using GoldHi EndoFree Plasmid Maxi Kit (Cat. CW2104M, CWBio).
HEK 293F cells (Invitrogen) were cultured in Freestyle medium (Gibco, Lot.2164683) at 37 ° C. under 6% CO2 in a CRYSTAL shaker (140 rpm). The cells were transiently transfected with the ACE2 plasmids and polyethylenimine (PEI) (Polysciences, Cat.24765-1) when the cell density reached approximately 1.0×106/mL. 1 mg plasmids were premixed with 2.6 mg PEI in 50 ml of fresh medium for 15 minutes before adding to one liter cell culture. The transfected cells were cultured for 96 hours before harvesting.
For purification of ACE2 proteins, the cell supernatants were harvested by centrifugation at 1000 g for 5 minutes. Then the supernatants were loaded on Ni-NTA beads (Smart-Lifesciences, Cat. SA004100), washed with washing buffer (5 mM imidazole, 1× PBS). Proteins were then eluted with elution buffer (50 mM imidazole, 1× PBS).
The eluted proteins were concentrated and subject to size-exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare) in the PBS buffer. The peak fractions were collected and concentrated for further analysis. The protein molecular weight was analyzed by a size exclusion chromatography (AdvanceBio SEC 300Å) in PBS buffer pH 7.4. The standard proteins were purchased from GE. The results are shown in
To remove C-terminal tags of ACE2 proteins, 16 ug HRV3C protease (expressed and purified in house) was add to lmg ACE2 protein and incubated at 4 ° C. overnight, followed by size-exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare) purification and analysis .
4-12% SDS-PAGE gels or 12% SDS-PAGE gels were purchased from Genscript (Suzhou). Protein gels were run at 80 V for 5 minutes then turn to 130 V for 45 minutes in 1× MOPS buffer. When the electrophoresis was finished, the protein gels were stained in staining buffer (1.25 grams coomassie Blue R-25 dissolved in 1 L buffer containing 300 ml ethanol, 100 mL acetic acid, and 600 mL water) for 30 minutes. Then the stained gels were destained in destaining buffer (1 L containing 300mL ethanol, 100 mL acetic acid, and 600 mL water) for 2 hours.
96-well ELISA plates (JET BIOFIL, #FEP-100-096) were coated with 50 μL per well of different S-ECD protein concentrations (
To determine the binding affinities of different ACE2 proteins, 96-well ELISA plates (JET BIOFIL, #1-EP-100-096) were coated with 50 μL per well of S-ECD (3 μg/mL) in coating buffer (NCM Biotech, #F30500) overnight at 4° C. Plates were washed with phosphate-buffered saline with 0.1% Tween-20 (PBST) four times then blocked with 2% bovine serum albumin (BSA, SIGMA, #B2064-50G) in PBST for 2 hours at room temperature. After blocking, the plates were washed with PBST four times then incubated with 70 μL per well of series diluted ACE2 samples in PBST for 2 hours at 37° C. Plates were washed with PBST four times then incubated with 70 μL per well of 1:2,000 dilution of Anti-GFP antibody (Rabbit PAb, Sino Biological, #13105-RP01) for 1 hour at 37° C. Plates were again washed four times then incubated with 70 μL per well of 1:10,000 dilution of HRP-conjugated Goat Anti-Rabbit IgG (Beyotime, #A0208) for 1 hour at 37° C. After final four times washing, plates were added with 100 μL per well of TMB single-component substrate solution (Solarbio, #PR1200) and the reaction was stopped by the addition of 50 μL per well of 1M hydrochloric acid. The absorbance at 450 nm was measured on a Microplate reader (Thermo, Varioskan LUX).
Purified S-ECD protein was biotinylated at a theoretical 1:3 molar ratio with EZ-Link NHS-PEG12-Biotin (Thermo Fisher Scientific, CAT#: 21313) according to the manufacturer's instructions. The unreacted biotin was removed by ultrafiltration with an Amicon column (30 KDa MWCO, Millipore, CAT: UFC5010BK).
For kinetic analyses, S-ECD was captured on streptavidin biosensors. Biotinylated S-ECD was diluted to 20 μL g/mL in dilution buffer (PBS with 0.02% Tween 20 and 0.1% BSA). Then sensors baseline were equilibrated in the dilution buffer for 90 seconds. Then the S-ECD was loaded until the thickness signal is 0.6 nm or 0.3 nm (low loading). After loading, the sensor was washed for 60 seconds in the dilution buffer. The sensors were then immersed into wells containing ACE2 proteins for 100 seconds (association phase), followed by immersion in dilution buffers for an additional 300 seconds (dissociation phase). The background signal was measured using an reference sensor with S-ECD loading but no ACE2 protein binding and was subtracted from corresponding ACE2 binding sensor. Curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software. Mean kon, koff values were determined by averaging all binding curves that matched the theoretical fit with an R2 value of 0.95.
Human hepatoma Huh-7 cells were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Human colorectal adenocarcinoma Caco-2 cells were obtained from the American Type Culture Collection (ATCC). Human primary embryonic kidney cells (293T) (CRL-3216™) were obtained from the American Type Culture Collection (ATCC). These cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal bovine serum (FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin at 37° C. under 5% CO2.
The envelop-encoding plasmids of SARS-CoV-2-S, SARS-CoV-S, and SARSr-CoV-S (Rs3367 and WIV1) and luciferase-expressing vector (pNL4-3.Luc.R-E-) were maintained in house. The plasmids encoding mutant SARS-CoV-2-S (V341I, F342L, V367F, R4081, A435S, G476S, V483A, or D614G) were constructed using a site mutation kit (Yeasen, China) and confirmed by sequencing.
These pseudoviruses were generated according to the previous study (52, 53). Briefly, the envelop-encoding plasmid (20 μg) and pNL4-3.Luc.R-E- (10 μg) were co-transfected into 293T cells cultured at 10 cm cell culture dish using Vigofect transfection reagent (Vigorous Biotechnology, China). After 10 hours, the cell culture medium was changed with fresh DMEM containing 10% FBS. Supernatants containing pseudovirus were harvested 48 hours later, filtered with 0.45 μm filter (Millipore), and using for single-cycle infection.
The pseudovirus inhibition assay was conducted as previously described (52, 53). Briefly, 1×104 Huh-7 cells (or Caco-2 cells) were seeded into the 96-well cell culture plate and cultured for 12 hours. The recombinant proteins (ACE2 trimers) were diluted with DMEM and mixed with pseudovirus, incubated at 37° C. for 30 minutes, and added to Huh-7 cells (or Caco-2 cells). After 12 hours of infection, the culture medium was replaced with fresh DMDM containing 10% FBS, and cells were cultured for an additional 48 hours. Then cells were lysed with Cell Lysis Buffer (Promega, Madison, WI, USA), and the luciferase activity was detected using the Luciferase Assay System (Promega, Madison, WI, USA).
Cell lines and Virus
African green monkey kidney Vero-E6 cell line was cultured with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal bovine serum (FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin at 37° C. under 5% CO2. SARS-CoV-2 (SARS-CoV-2/SH01/human/2020/CHN, GenBank No. MT121215) was isolated from a COVID-19 patient in Shanghai, China. The virus was purified and propagated in Vero-E6 cells, then stocked at −80° C. Viral titer was measured by the 50% Tissue culture infective dose (TCID50) method. All experiments involving live SARS-CoV-2 virus were performed in Biosafety Level 3 Laboratory (BSL-3), Fudan University.
The live SARS-CoV-2 inhibition assay was performed as previously described(54). Briefly, 3×104 Vero-E6 cells were seeded into the 96-well cell culture plate and cultured for 12 hours. Recombinant proteins (ACE2 trimers) were diluted with FBS-free DMEM and mixed with 100 TCID50 SARS-CoV-2, incubated at 37° C. for 30 minutes. Then, the protein-virus mixtures were added to Vero-E6 cells and incubated at 37° C. for 1 hour. After removing the mixtures, cells were cultured with fresh DMEM containing 2% FBS for a further 48 hours. Then, the supernatants were collected to detect viral RNA titer.
RNA extraction and Quantitative Real-Time PCR (qPCR) Assay
Total viral RNA in supernatants were extracted using Trizol LS reagent (Invitrogen, USA) according to manufacturer's manual. Then qPCR was conducted with a One-Step PrimeScrip RT-PCR Kit (Takara, Japan) following the manufacturer's instructions. qPCR reaction was performed with the program of 95° C. for 10 seconds, 42° C. for 5 minutes; 40 cycles of 95° C. for 5 seconds, 50° C. for 30 seconds, 72° C. for 30 seconds on Bio-Rad CFX96. Viral loads were determined by a standard curve prepared with a plasmid containing SARS-CoV-2 nucleocapsid protein (N) gene (purchased form BGI, China). Primers and probe targeting SARS-CoV-2 N gene were ordered from Genewiz (Suzhou, China) and the sequences as follows:
The purification of the extracellular domain (ECD) (Genebank ID: QHD43416.1) (1-1208 aa) of S protein was as previously (55). The purified S-ECD was mixed with the T-ACE2 at a molar ratio of about 1:2 for one hour at 4° C. To remove excessive T-ACE2, the mixture was subjected to size-exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare) in buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl. Peak fractions of S-ECD in complex with T-ACE2 were collected for EM analysis.
The peak fractions of the complex were concentrated to about 1.5 mg/mL and mixed with 0.05% Octyl Maltoside, Fluorinated (Anatrace) before applied to the grids. Aliquots (3.3 μL) of the protein complex were placed on glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3). The grids were blotted for 2.5 s or 3.0 s and flash-frozen in liquid ethane cooled by liquid nitrogen with Vitrobot (Mark IV, Thermo Scientific). The cryo-EM samples were transferred to a Titan Krios operating at 300 kV equipped with Cs corrector, Gatan K3 Summit detector and GIF Quantum energy filter. Movie stacks were automatically collected using AutoEMation (56), with a slit width of 20 eV on the energy filter and a defocus range from −1.2 μm to −2.2 μm in super-resolution mode at a nominal magnification of 81,000x. Each stack was exposed for 2.56 s with an exposure time of 0.08 s per frame, resulting in a total of 32 frames per stack. The total dose rate was approximately 50 e−/Å2 for each stack. The stacks were motion corrected with MotionCor2 (57) and binned 2-fold, resulting in a pixel size of 1.087 Å/pixel. Meanwhile, dose weighting was performed (58). The defocus values were estimated with Gctf (59).
Particles were automatically picked using Relion 3.0.6 (60-63) from manually selected micrographs. After 2D classification with Relion, good particles were selected and subject to two cycle of heterogeneous refinement without symmetry using cryoSPARC (64).The good particles were selected and subjected to Non-uniform Refinement (beta) with C1 symmetry, resulting in the 3D reconstruction for the whole structures, which was further subject to 3D classification, 3D auto-refinement and post-processing with Relion. For interface between RBD and ACE2, the datasets were subject to focused refinement with adapted mask on each RBD and ACE2 sub-complex to improve the map quality. Then the dataset of three RBD and ACE2 sub-complexes were combined and subject to focused refinement with Relion, resulting in the 3D reconstruction of better quality on the interface between S-ECD and ACE2.
The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion (65) with high-resolution noise substitution (66). Refer to
For model building of the complex of S-ECD with ACE2, the atomic model of the published structure S-ECD (PDB ID: 7C2L) and ACE2 molecular (PDB ID: 6M18) were used as templates, which were molecular dynamics flexible fitted (MDFF) (67) into the whole cryo-EM map of the complex and the focused-refined cryo-EM map of the RBD-ACE2 sub-complex, respectively. And the fitted atomic models were further manually adjusted with Coot (68). Each residue was manually checked with the chemical properties taken into consideration during model building. Several segments, whose corresponding densities were invisible, were not modeled. Structural refinement was performed in Phenix (69) with secondary structure and geometry restraints to prevent overfitting. To monitor the potential overfitting, the model was refined against one of the two independent half maps from the gold-standard 3D refinement approach. Then, the refined model was tested against the other map. Statistics associated with data collection, 3D reconstruction and model building were summarized in Table 1.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/110613 | Aug 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/113962 | 8/23/2021 | WO |
