STATEMENT OF PRIORITY

[0001] This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 63/146,796, filed on February 8, 2020, the entire contents of which are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING [0002] A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5470.890.WO_ST25.txt, 47,026 bytes in size, generated on July 12, 2021 and filed via EFS- Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.

FIELD OF THE INVENTION

[0003] The present invention relates to fusion proteins that include reporter enzyme fragments. The present invention also relates to fusion proteins that may be useful for the detection of SARS-CoV-2 neutralizing antibodies and other neutralizing antibodies. The invention further relates to polynucleotides and vectors encoding the fusion proteins, cells comprising the fusion proteins, and methods of using the fusion proteins to study binding of test compounds, as well as diagnostic methods using the same.

BACKGROUND OF THE INVENTION

[0004] The novel coronavirus termed SARS-CoV-2, the virus responsible for Coronavirus disease 2019 (COVID-19), mediates entry into host cells by its transmembrane spike (S) glycoprotein. The S glycoprotein includes two functional subunits. The first subunit, SI, is responsible for binding to the host cell receptor and includes the receptor-binding domain (RBD). The second subunit, S2, is responsible for fusion of the viral and cellular membranes. The angiotensin-converting enzyme 2 (ACE2) receptor on host cells has been identified as the cellular receptor onto which the RBD of the SARS-CoV-2 binds to initiate entry into the host cell. Once suitable binding of S glycoprotein(s) to the ACE2 receptor occurs, the membrane fusion process may be activated, and endocytosis of the viral particle may be initiated. This mechanism of entry into the host cell is the same or similar for other coronaviruses, including the coronavirus responsible for the SARS pandemic of 2002, also referred to as SARS-CoV or SARS-CoV-1.

[0005] Detection and quantification of SARS-CoV-2 neutralizing antibodies in COVID-19 patients and vaccinated persons would be desirable, both to guide treatment of patients and to help control the COVID-19 pandemic. Additionally, detection and quantification methods for SARS-CoV-2 neutralizing antibodies that are sensitive and relatively inexpensive may also be desirable.

SUMMARY OF THE INVENTION

[0006] The present invention addresses previous shortcomings in the art by providing a relatively inexpensive and sensitive assay method for detection and quantification of SARS- CoV-2 neutralizing antibodies. The assay is based on protein-fragment complementation (PFC) assays.

[0007] Provided according to embodiments of the invention are complementary fusion protein pairs comprising a first fusion protein that includes a first reporter enzyme fragment linked to a receptor binding domain (RBD) polypeptide, or a functional fragment thereof, and a second fusion protein that includes a second reporter enzyme fragment linked to a receptor polypeptide, or a functional fragment thereof. Thus, when the RBD peptide of the first fusion protein and the receptor polypeptide of the second fusion protein bind, the first reporter enzyme fragment and the second reporter enzyme fragment may also bind, thereby, under certain conditions, reporting the RBD and receptor interaction via a signal from the reporter enzyme.

[0008] While any suitable RBD (e.g., viral RBD) and receptor pair may be used in fusion proteins of the invention, in some embodiments, the first fusion protein includes a receptor binding domain (RBD) protein of a coronavirus, or a functional fragment thereof. In particular embodiments, the coronavirus is SARS-CoV-2 or SARS-CoV-1. Also provided is a second fusion protein including an ACE2 polypeptide, or a functional fragment thereof. In some embodiments, the ACE2 polypeptide is a human ACE2 polypeptide. While any suitable reporter enzyme may be used in fusion proteins of the invention, in some embodiments, the first fusion protein includes a first luciferase fragment. Also provided is a second fusion protein including a second luciferase fragment.

[0009] Also provided according to embodiments of the invention are polynucleotides encoding the first fusion protein and/or the second fusion protein of the invention. Vectors and host cells that include such polynucleotides are also provided in some embodiments. Further provided are cells that include at least one fusion protein of the invention. Also provided are methods of producing fusion protein(s) that include expressing the first fusion protein, the second fusion protein, or both, as encoded by at least one polynucleotide and/or at least one vector of the invention.

[0010] Provided according to some embodiments of the invention are methods for determining the binding ability of a test compound to a receptor binding domain and/or a receptor domain. Such methods include contacting a first fusion protein of an embodiment of the invention, a second fusion protein of an embodiment of the invention, and the test compound in a composition for a time sufficient for binding of the first fusion protein to the second fusion protein; adding a reporter enzyme substrate (e.g., a luciferase substrate) to the composition; measuring a level of reporter (e.g., bioluminescence) of the composition; and comparing the measured reporter level with a reporter level produced by the first fusion portion and the second fusion protein in the absence of the test compound to determine an amount of binding of the test compound to the first fusion protein and/or second fusion protein.

[0011] Also provided are methods for determining the presence of a binding inhibitor, such as a neutralizing antibody, in a test sample, the methods including contacting a first fusion protein of an embodiment of the invention, a second fusion protein of an embodiment of the invention, and the test sample in a composition for a time sufficient for binding of the first fusion protein to the second fusion protein; adding a reporter enzyme substrate (e.g., a luciferase substrate) to the composition; measuring a level of reporter (e.g., bioluminescence) in the composition; and comparing the measured reporter level with a reporter level produced in a composition comprising the first fusion portion and the second protein in the absence of the test sample to determine whether a binding inhibitor such as a neutralizing antibody is present in the test sample.

[0012] Further provided according to embodiments of the invention are kits that include a first fusion protein of an embodiment of an invention, a second fusion protein of an embodiment of the invention, a polynucleotide of an embodiment of the invention, a vector of an embodiment of the invention, and/or a cell of an embodiment of the invention.

[0013] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure l is a schematic illustrating a configuration wherein a RBD-luciferase fragment fusion protein and an ACE2-luciferase fusion protein are not predicted by computer software modeling to bind and emit a bioluminescence signal.

[0015] Figure 2 is a schematic illustrating the potential binding of a first fusion protein and a second fusion protein as predicted by computer modeling software.

[0016] Figure 3 illustrates the effect of co-incubation of the supernatants of certain fusion proteins. Two pairs of fusion proteins were tested, fusion proteins A and B, and fusion proteins C and D. Co-incubation of fusion proteins C and D (also referred to as 4+5) showed marked increase in gLuc expression over time, while fusion proteins A and B (also referred to as 2+3) did not show an increase in gLuc expression over time.

[0017] Figure 4 illustrates the effect of temperature on gLuc expression of a complementary fusion protein pair according to an embodiment of the invention.

[0018] Figure 5 shows a comparison of the gLuc expression (relative light units or RLU) for a complementary fusion protein pair in the absence of test compounds, and in the presence of test compounds of hlgG-Fc (5 pg/ml), CoV2-RBD (5 pg/ml), CoVl-RBD (5 pg/ml), NL63- RBD (5 pg/mL), ACE2-Fc (5 pg/ml), CR3022 (1 pg/ml) and CR3022 (10 pg/ml).

[0019] Figures 6A and 6B show the gLuc expression for a complementary pair of fusion proteins according to an embodiment of the invention, both in the absence of a test compound, and in the presence of hlgG (1 pg/ml), hlgG (10 pg/ml), CR3022 (1 pg/ml), CR3022 (10 pg/ml), and mAb240C (10 pg/ml), after 15 minutes (A) and 30 minutes (B).

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

[0022] Nucleotide sequences are presented herein by single strand only, in the 5’ to 3’ direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0023] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying, and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0024] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0026] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. [0027] The term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5' and/or 3' or N-terminal and/or C-terminal ends of the recited sequence such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids on both ends added together. The term “materially altered,” as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in an activity (e.g., catalytic activity or ligand binding activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0028] The term “percent sequence identity” as used herein refers to the degree of sequence identity between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of Karlin & Attschul (1990) Proc. Natl. Acad. Set. 87: 2264-2268, modified as in Karlin & Attschul (1993) Proc. Natl. Acad. Set. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al. (1997) Nuc. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used.

[0029] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g, chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide or nucleotide sequence refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where singlestranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

[0030] An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5’ end and one on the 3’ end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated polynucleotide includes some or all of the 5’ non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.

[0031] The term “isolated” also can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.

[0032] An “isolated” cell refers to a cell that is separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention. In some embodiments, an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo.

[0033] The term “fragment,” as applied to a nucleic acid, nucleotide sequence, or polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g. , 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of less than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0034] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g. , 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention. In other embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of less than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, or less consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0035] As used herein, the term “complementary” includes separate DNA sequences and amino acid residues that may align end to end, overlap or contain gaps when compared to the native sequences of a reporter enzyme, but when together reconstitute reporter enzyme function.

[0036] The term “reporter enzyme fragment” or “luciferase fragment” refers to a fragment, as defined herein, of a full-length reporter enzyme or luciferase enzyme, respectively, wherein the sequence of this protein is derived from a section of full-length reporter protein or luciferase enzyme, respectively. The reporter enzyme fragment or luciferase fragment can be contacted with an additional fragment of the same protein to form an active reporter enzyme or luciferase. [0037] As used herein, “luciferase” or “Luc” refers to an oxidative enzyme that produces bioluminescence. Any suitable luciferase may be used, including, but not limited to, firefly luciferase, sea pansy luciferase (Renilla reniformis). copepod (Metrida longa and Gaussia), bacterial (e.g., Vibrio fischeri. Vibrio haweyi, and Vibrio harveyi . and dinoflagellate. In particular embodiments, the luciferase is Gaussia luciferase, also referred to herein as “gLuc.” [0038] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass peptides, unless indicated otherwise.

[0039] As used herein, the term “N-terminus” refers to the end of an amino acid chain (e.g., a polypeptide) that is terminated by a free amine group (-NH2) and the “C-terminus” refers to the end of the amino acid chain that is terminated by a free carboxyl (-COOH) group. Peptide sequences are written from N-terminus to C-terminus, from left to right. If a reporter enzyme is divided into two fragments, the fragment including the N-terminus of the enzyme, and/or amino acids closest to the N-terminus, is referred to the “N-terminal reporter enzyme fragment” and the fragment including the C-terminus of the enzyme, and/or the amino acids closest to the C-terminus, is considered the “C-terminal reporter enzyme fragment.” For Gaussia luciferase, described in embodiments herein, the N-terminal luciferase fragment may be referred to as “N- terminal luciferase fragment” or “GLN” and the C-terminal luciferase fragment may be referred to as “C-terminal luciferase fragment” or “GLC.”

[0040] A “fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides and/or peptides not found fused together in nature are fused together in the correct translational reading frame. In some embodiments, fusion proteins may further include amino acids that are useful for identifying and/or purifying the fusion protein. [0041] As used herein, a “functional” polypeptide or "functional fragment" is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g., enzyme activity, receptor binding). In particular embodiments, the “functional” polypeptide or “functional fragment” substantially retains all of the activities possessed by the unmodified peptide. By “substantially retains” biological activity, it is meant that the polypeptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A “non-functional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as protein binding and enzyme activity can be measured using assays that are well known in the art and as described herein.

[0042] By the term “express” or “expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.

[0043] A “vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A “replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral (e.g, plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker. A “recombinant” vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.

[0044] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acidprotein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, expression control sequences, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (e.g., delivery to specific tissues, duration of expression, etc.).

[0045] Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu etal., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263: 14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0046] In some embodiments, a polynucleotide of this invention can be delivered to a cell in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et al., Proc. Natl. Acad. Sci. USA 54:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 55:8027 (1988); and Ulmer et al., Science 259 : 1745 (1993)). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al., Science 337:3%! (1989)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Patent No. 5,459,127. The use of lipofection to introduce exogenous nucleotide sequences into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. In representative embodiments, transfection is directed to particular cell types in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra). Targeted peptides, e.g, hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

[0047] In various embodiments, other molecules can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from nucleic acid binding proteins (e.g., WO96/25508), and/or a cationic polymer (e.g., WO95/21931).

[0048] It is also possible to introduce a vector in vivo as naked nucleic acid (see U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated nucleic acid delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3: 147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).

[0049] The term “transfection” or “transduction” means the uptake of exogenous or heterologous nucleic acid (RNA and/or DNA) by a cell. A cell has been “transfected” or “transduced” with an exogenous or heterologous nucleic acid when such nucleic acid has been introduced or delivered inside the cell. A cell has been “transformed” by exogenous or heterologous nucleic acid when the transfected or transduced nucleic acid imparts a phenotypic change in the cell and/or a change in an activity or function of the cell. The transforming nucleic acid can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell or it can be present as a stable plasmid.

[0050] As used herein, the term “biological sample” refers to a sample that contains or consists of material obtained or derived from an organism, e.g., a mammal, e.g., a human, nonhuman primate, rodent (e.g., mouse, rabbit, rat), dog, cat, or other domesticated animal or bird, etc. A biological sample can comprise, for example, any of the following: whole blood, fractionated blood, plasma, serum, other blood fraction or blood product, aqueous humor, vitreous humor, tears, synovial fluid, cerebrospinal fluid, saliva, mucous, sweat, milk, urine, fecal material, pericardial fluid, gastric fluid, peritoneal fluid, pleural fluid, cyst fluid, broncheolar lavage fluid, nasal lavage, lymphatic fluid, mammary fluid, duct fluid, tears, tissue extract, glandular secretion, tissue extract, whole cell lysate or fraction thereof, cell or tissue extract, tissue homogenate, or cell culture supernatant. In some embodiments a biological sample is essentially cell-free. In some embodiments a biological sample contains no more than 1% cells by weight or volume. The term “biological sample” can also refer to a sample derived by processing a biological sample comprising any of the afore-mentioned materials, e.g., by fractionating or diluting the sample, isolating or purifying one or more substances (e.g., proteins) from the sample, etc. If a biological sample is being tested, for example, to determine the absence or presence of a test compound such a SARS-CoV-2 neutralizing antibody, it may be referred to as a test sample.

[0051] As used herein, the term “test compound” refers to any compound that may be tested to determine binding with one or more fusion proteins of the invention. In some embodiments, the test compound is a polymer such as a protein. In some embodiments, the test compound is an antibody or other protein active in biological processes.

[0052] As used herein, two or more moieties are “linked” if they are physically associated or connected with one another to form a molecular structure that is sufficiently stable so that they remain associated under the conditions in which the linkage is formed and, preferably, under the conditions in which the molecular structure thus formed is used. In certain embodiments of the invention, the linkage is a covalent linkage. In other embodiments, the linkage is noncovalent. Moieties may be linked either directly or indirectly. When two moieties are directly linked, they are either covalently bonded to one another or are in sufficiently close proximity such that intermolecular forces between the two moieties maintain their association. When two moieties are indirectly linked, they are each linked either covalently or noncovalently to a third moiety, which maintains the association between the two moieties. In general, when two moieties are referred to as being linked by a “linker” or “linking moiety” or “linking portion”, the linkage between the two linked moieties is indirect, and typically each of the linked moieties is covalently bonded to the linker. The linker can be any suitable moiety that reacts with the two moieties to be linked within a reasonable period of time, under conditions consistent with stability of the moieties (which may be protected as appropriate, depending upon the conditions), and in sufficient amount, to produce a reasonable yield.

[0053] “Subject”, as used herein, refers to an individual from whom a biological sample is obtained and/or on whom a test is performed, e.g., for experimental, diagnostic, and/or therapeutic purposes. Unless otherwise indicated, subjects are mammals, e.g., humans, nonhuman primates, domesticated mammals such as dogs, or cats, rodents, such as rabbits, mice, etc. In particular embodiments, the subject is human.

[0054] “Protein-fragment complementation (PFC)” assays use a reporter enzyme that is divided into two inactive components wherein each inactive component is linked to a polypeptide of interest. When the two inactive components are reconstituted, the enzymatic activity may thus be resumed. As such, when a first fragment of the reporter enzyme is fused to a first polypeptide, and the second fragment of the reporter enzyme is fused to a second polypeptide, if correctly designed, when the first and second polypeptides bind, the reporter enzyme fragments also functionally reconstitute and enzymatic activity of the reporter enzyme resumes, signaling the binding of the first and second polypeptides. Examples of reporter enzymes include, but are not limited to, P-galactosidase, dihydrofolate reductase (DHFR), P- lactamase, and luciferase. In the case of luciferase, when the luciferase fragments functionally reconstitute, and the appropriate luciferase substrate is present, a bioluminescence signal is emitted.

Fusion Proteins

[0055] Provided according to embodiments of the invention are complementary fusion protein pairs comprising a first fusion protein that includes a first reporter enzyme fragment linked to a receptor binding domain (RBD) polypeptide, or a functional fragment thereof, and a second fusion protein that includes a second reporter enzyme fragment linked to a receptor polypeptide, or a functional fragment thereof. Thus, when the RBD peptide of the first fusion protein and the receptor polypeptide of the second fusion protein bind, the first reporter enzyme fragment and the second reporter enzyme fragment may also bind, thereby, under certain conditions, reporting the RBD and receptor interaction via a signal from the reporter enzyme.

[0056] While any suitable RBD (e.g., viral RBD) and receptor pair may be used in fusion proteins of the invention, in some embodiments, the first fusion protein includes a receptor binding domain (RBD) protein of a coronavirus, or a functional fragment thereof. In particular embodiments, the coronavirus is SARS-CoV-2 or SARS-CoV-1. Also provided is a second fusion protein including a receptor for a coronavirus such as an ACE2 polypeptide, or a functional fragment thereof. In some embodiments, the ACE2 polypeptide is a human ACE2 polypeptide. While any suitable reporter enzyme may be used in fusion proteins of the invention, in some embodiments, the first fusion protein includes a first luciferase fragment. Also provided is a second fusion protein including a second luciferase fragment.

[0057] The first luciferase fragment and the second luciferase fragment are complementary so that when apart, the fragments do not process a substrate (e.g., luciferin) to produce bioluminescence, but when bound together, they reconstitute the active luciferase and may cause a suitable luciferase substrate to emit a bioluminescence signal. Other reporter enzymes will work in a similar fashion and may or may not require a substrate to produce a signal indicating that the two fragments have reconstituted. As used herein, the fusion proteins may be referred to as a first fusion protein and a second fusion protein, but this designation is only to differentiate between the different types of fusion proteins and no order or preference is indicated by this designation.

First Fusion Protein

[0058] Provided according to embodiments of the invention is a first fusion protein that includes a first reporter enzyme fragment linked to a receptor binding domain (RBD) polypeptide (e.g., a viral RBD polypeptide), or a functional fragment thereof. In some embodiments of the invention, the first reporter enzyme fragment is a first luciferase fragment. In some embodiments, the RBD polypeptide, or a functional fragment thereof, is from a coronavirus, such as SARS-CoV-2 or SARS-CoV-1, as described above. In some embodiments, the first luciferase fragment is a Gaussia luciferase (gLuc) fragment. In some embodiments, an N-Terminal luciferase fragment (as defined herein) is linked to the RBD or a functional fragment thereof in the first fusion protein (GLN-RBD or RBD-GLN). In some embodiments, the N-Terminal luciferase fragment is linked to the RBD or a functional fragment thereof via the C-terminus of the N-Terminal luciferase fragment and the N-terminus of the RBD or the functional fragment thereof (GLN-RBD).

[0059] In some embodiments, the RBD may be any portion of a SARS-CoV-2 SI subunit protein that provides binding to a human ACE2 receptor and the functional fragment may be any portion of the RBD that is functional, as defined herein. In some embodiments, the portion of the fusion protein including the RBD or functional fragment thereof comprises, consists of, or consists essentially of, an amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 1 (RBD)

SFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG

[0060] However, any suitable variant now known or later discovered of an RBD of a coronavirus including any SARS-CoV-2 variant may be used. In some embodiments, the RBD (or functional fragment) portion of the first fusion protein has a percent sequence identity of at least 90%, 92%, 95%, 98%, or 99% to SEQ ID NO. 1.

[0061] In some embodiments, the first luciferase fragment comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 2. However, any luciferase fragment that is complementary with the second luciferase fragment of the second fusion protein may be used. In some embodiments, the first luciferase fragment portion of the first fusion protein has a percent sequence identity of at least of 90%, 92%, 95%, 98%, or 99% to SEQ ID NO. 2.

SEQ ID NO: 2 (GLN)

K PTENNEDFNI VAVASNFATT DLDADRGKLP GKKLPLEVLK EMEANARKAG CTRGCLICLS HIKCTPKMKK FIPGRCHTYE GDKESAQGGI G

[0062] Additionally, in some embodiments, the first fusion protein includes a linker intervening between the RBD and reporter enzyme (e.g., luciferase) fragment. In some embodiments, an N-Terminal luciferase fragment and the RBD (or functional fragment thereof) are linked at the C-terminus of the N N-Terminal luciferase fragment and the N-terminus of the RBD or functional fragment thereof (GLN-linker-RBD). Various linkers are known and could be applied equivalently. For example, the linker can comprise, consist essentially of, or consist of a peptide of about 2 to about 12 amino acids or more, e.g., about 8 to about 12 amino acids, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more amino acids. In another embodiment, the linker comprises repeats of the amino acid sequence Gly-Ser, e.g., 2, 3, 4, 5, or 6 repeats or more. In a further embodiment, the linker comprises repeats of a thermostable helix from ribosomal protein L9, e.g., 2, 3, 4, 5, or 6 repeats or more. A linker sequence may, for example, be the sequence of SEQ ID NO: 3, the SEQ ID NO: 4, or the like.

SEQ ID NO: 3

GGGGSGGGGS

SEQ ID NO: 4

GGSGGSGGSGG

[0063] In addition, in some embodiments, a signal peptide may be included at the N-terminus of the first fusion protein. Any suitable signal peptide may be included. In some embodiments, the signal peptide comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 5.

SEQ ID NO: 5

MVNGVK VLF A LICIAVAEA

[0064] Further, in some embodiments, the first fusion protein may include one or more amino acids that may facilitate purification, e.g., protein tags, such as polyhistidine (e.g., six or more sequential histidine amino acids), FLAG, glutathione, and Myc. Fusion proteins of the invention can further be modified for use in cells in vitro, ex vivo, or in vivo by the addition, e.g., at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in the cell or in vivo. This can be useful in those situations in which the protein termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the protein to be administered. This can be done either chemically during the synthesis of the fusion protein or by recombinant DNA technology by methods familiar to artisans of average skill. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the fusion proteins can be covalently or noncovalently coupled to pharmaceutically acceptable “carrier” proteins or other molecules (e.g., PEG) prior to administration.

[0065] In some embodiments, the first fusion protein comprises, consists essentialy of, or consists of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9. SP refers to signal protein, GLN refers to the N-Terminal luciferase fragment, RBD is the RBD of the SARS-CoV-2 SI protein, and H7 is a histidine tag.

SEQ ID NO: 6 (SP-GLN-linker-RBD-H7)

MVNGVKVLFA LICIAVAEAK PTENNEDFNI VAVASNFATT DLDADRGKLP

GKKLPLEVLK EMEANARKAG CTRGCLICLS HIKCTPKMKK FIPGRCHTYE GDKESAQGGI GGGSGGSGGS GGSFTVEK GIYQTSNFRV QPTESIVRFP

NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY

GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT

GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG HHHH HHH

SEQ ID NO: 7 (SP-GLN-linker-RBD)

MVNGVKVLFA LICIAVAEAK PTENNEDFNI VAVASNFATT DLDADRGKLP GKKLPLEVLK EMEANARKAG CTRGCLICLS HIKCTPKMKK FIPGRCHTYE GDKESAQGGI GGGSGGSGGS GGSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG

SEQ ID NO: 8 (GLN-linker-RBD)

K PTENNEDFNI VAVASNFATT DLDADRGKLP GKKLPLEVLK EMEANARKAG CTRGCLICLS HIKCTPKMKK FIPGRCHTYE GDKESAQGGI GGGSGGSGGS GGSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG

SEQ ID NO.: 9 (GLN-linker-RBD-H7) K PTENNEDFNI VAVASNFATT DLDADRGKLP GKKLPLEVLK EMEANARKAG CTRGCLICLS HIKCTPKMKK FIPGRCHTYE GDKESAQGGI GGGSGGSGGS

GGSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV

YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN

YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT

NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG HHHH HHH

[0066] In some embodiments, the first fusion protein has a percent sequence identity of at least 90%, 92%, 95%, 98%, or 99% to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and/or SEQ ID NO.: 9.

Second Fusion Protein

[0067] Provided according to some embodiments of the invention is a second fusion protein that includes a second reporter enzyme fragment linked to a receptor polypeptide (e.g., viral receptor polypeptide), or a functional fragment thereof. In some embodiments, the second reporter enzyme is a second luciferase fragment. In some embodiments, the receptor polypeptide is a coronavirus receptor polypeptide such as an ACE2 polypeptide, or a functional fragment thereof. In some embodiments, the ACE2 polypeptide is a human ACE2. In some embodiments, the second luciferase fragment is a Gaussia luciferase (gLuc) fragment, e.g., that complements the gLuc fragment in the first fusion protein. In some embodiments, a C-Terminal luciferase fragment is linked to an ACE2 polypeptide or a functional fragment thereof. In some embodiments, the C-Terminal luciferase fragment is linked to the ACE2, or a functional fragment thereof, via the C-terminus of the C-Terminal luciferase fragment and the N-terminus of the ACE2 or the functional fragment thereof (GLC-ACE2). In some embodiments, the ACE2 polypeptide or a functional fragment thereof may be any human ACE2 to which the SARS-CoV-2 RBD binds, or a fragment thereof that is functional as defined herein. In some embodiments, the ACE2 polypeptide or functional fragment thereof portion of the second fusion protein comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 10.

SEQ ID NO: 10 (ACE2) TIEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL

NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL

PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL

TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS

IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGVVEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK NSFVGWSTDW SPYAD

[0068] However, any suitable variant now known or later discovered of an ACE2 polypeptide, enzyme, and/or receptor may be used. In some embodiments, the ACE2 polypeptide, or fragment thereof, portion of the second fusion protein has a percent sequence identity of at least 90%, 92%, 95%, 98%, or 99% to SEQ ID NO. 10.

[0069] In some embodiments, the second luciferase fragment comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 11. However, any luciferase fragment that is complementary with the first luciferase fragment in the first fusion protein may be used. In some embodiments, the second luciferase fragment has a percent sequence identity of at least 90%, 92%, 95%, 98%, or 99% to SEQ ID NO. 11.

SEQ ID NO: 11 (GLC)

AIVDIPE IPGFKDLEPM EQFIAQVDLC VDCTTG CLKGLANVQC SDLLKKWLPQ

RCATFASKIQ GQVDKIKGAG GD

[0070] Additionally, in some embodiments, the second fusion protein includes a linker intervening between the receptor polypeptide and the reporter enzyme (e.g., luciferase) fragment. In some embodiments, the second fusion protein includes a linker intervening between the C-Terminal luciferase fragment and the ACE2 receptor polypeptide, or a fragment thereof. In some cases, the linker is between the C-terminus of the C-Terminal luciferase fragment and the N-terminus of the ACE2 receptor polypeptide, or a fragment thereof. Various linkers are known and could be applied equivalently. For example, the linker can comprise, consist essentially of, or consist of a peptide of about 2 to about 12 amino acids or more, e.g., about 8 to about 12 amino acids, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more amino acids. In another embodiment, the linker comprises repeats of the amino acid sequence Gly- Ser, e.g., 2, 3, 4, 5, or 6 repeats or more. In a further embodiment, the linker comprises repeats of a thermostable helix from ribosomal protein L9, e.g., 2, 3, 4, 5, or 6 repeats or more. A linker sequence may have, for example, a sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or the like. [0071] addition, in some embodiments, a signal peptide may be included at the N-terminus of the second fusion protein. Any suitable signal peptide may be included. In some embodiments, the signal peptide of the second fusion protein comprises, consists essentially of, or consists of a sequence of SEQ ID NO: 5, as described above.

[0072] Further, in some embodiments, the second fusion protein may include one or more peptides that may facilitate purification, e.g., protein tags, such as polyhistidine (e.g., six or more sequential histidine amino acids), FLAG, glutathione, and Myc,. Fusion proteins of the invention can further be modified for use in cells in vitro, ex vivo, or in vivo by the addition, e.g., at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in the cell or in vivo. This can be useful in those situations in which the protein termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the protein to be administered. This can be done either chemically during the synthesis of the fusion protein or by recombinant DNA technology by methods familiar to artisans of average skill. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the fusion proteins can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins or other molecules (e.g., PEG) prior to administration.

[0073] In some embodiments, the second fusion protein comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and/or SEQ ID NO: 15. SP refers to the signal peptide, GLC is the C-Terminal luciferase fragment, ACE2 is the ACE2 receptor polypeptide, and H7 is a histidine tag. SEQ ID NO: 12 (SP-GLC-linker-ACE2-H7)

MVNGVKVLFA LICIAVAEAK PTEAIVDIPE IPGFKDLEPM EQFIAQVDLC VDCTTGCLKGLANVQC SDLLKKWLPQ RCATFASKIQ GQVDKIKGAG GDDTGGSGGS GGSGGT IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE

IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF

HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL

PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGVVEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA

GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK

NSFVGWSTDW SPYADHHHHHH H

SEQ ID NO: 13 (SP-GLC-linker-ACE2)

MVNGVKVLFA LICIAVAEAK PTEAIVDIPE IPGFKDLEPM EQFIAQVDLC VDCTTGCLKGLANVQC SDLLKKWLPQ RCATFASKIQ GQVDKIKGAG GDDTGGSGGS GGSGGT IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE

IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF

HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL

PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGVVEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA

GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK

NSFVGWSTDW SPYAD

SEQ ID NO: 14 (GLC-linker-ACE2)

AIVDIPE IPGFKDLEPM EQFIAQVDLC VDCTTGCLKGLANVQC SDLLKKWLPQ RCATFASKIQ GQVDKIKGAG GDDTGGSGGS GGSGGT IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGVVEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK NSFVGWSTDW SPYAD

SEQ ID NO: 15 (GLC-linker-ACE2-H7)

AIVDIPE IPGFKDLEPM EQFIAQVDLC VDCTTGCLKGLANVQC SDLLKKWLPQ RCATFASKIQ GQVDKIKGAG GDDTGGSGGS GGSGGT IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGVVEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK NSFVGWSTDW SPYADHHHHHH H

[0074] In some embodiments, the second fusion protein has a percent sequence identity of at least 90%, 92%, 95 %, 98%, or 99% to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and/or SEQ ID NO: 15.

[0075] Also provided according to embodiments of the invention are compositions that include at least one first fusion protein of the invention and/or at least one second fusion protein of the invention. In some embodiments, the compositions include a carrier, e.g., a pharmaceutically acceptable carrier, which may include, but is not limited to, salts, solvents, buffers, detergents, neutral proteins, e.g. albumin, which may be used to facilitate specific binding interactions, maintain protein stability, reduce non-specific or background interactions, and the like. In some embodiments, such compositions further include a biological sample and/or a test compound as defined herein.

Polynucleotide, Vectors, and Cells

[0076] One aspect of the invention relates to polynucleotides encoding the fusion proteins of the invention. In one embodiment, the polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence that encodes at least one of the fusion proteins of the invention. Polynucleotides of this invention include RNA, DNA (including cDNAs) and chimeras thereof. The polynucleotides can further comprise modified nucleotides or nucleotide analogs. It will be appreciated by those skilled in the art that there can be variability in the polynucleotides that encode the fusion proteins of the present invention due to the degeneracy of the genetic code. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same polypeptide, is well known in the literature.

[0077] The isolated polynucleotides encoding the fusion proteins of the invention will typically be associated with appropriate expression control sequences, e.g., promoters, enhancers, transcription/translation control signals and polyadenylation signals.

[0078] A variety of promoter/enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter can be constitutive or inducible, depending on the pattern of expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. The promoter is chosen so that it will function in the target cell(s) of interest.

[0079] To illustrate, the polynucleotide encoding the fusion protein can be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, an albumin promoter, an Elongation Factor 1-a (EFl -a) promoter, a PyK promoter, a MFG promoter, or a Rous sarcoma virus promoter.

[0080] Inducible promoter/enhancer elements include hormone-inducible and metalinducible elements, and other promoters regulated by exogenously supplied compounds, including without limitation, the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (see WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA 93:3346 (1996)); the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA 59:5547 (1992)); the tetracycline-inducible system (Gossen et al., Science 268A166 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15: 239 (1997); Wang et al., Gene Ther., 4:432 (1997)); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865 (1997)).

[0081] Moreover, specific initiation signals are generally required for efficient translation of inserted protein coding sequences. These translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0082] The present invention further provides cells comprising the polynucleotides and fusion proteins of the invention. The cell may be a cultured cell or a cell ex vivo or in vivo, e.g., for use in therapeutic methods, diagnostic methods, screening methods, methods for studying the biological action of viruses, methods of producing fusion proteins, or methods of maintaining or amplifying the polynucleotides of the invention, etc. The cell can be e.g., a bacterial, fungal (e.g., yeast), plant, insect, avian, mammalian, or human cell.

[0083] The polynucleotide can be incorporated into an expression vector. Expression vectors compatible with various host cells are well known in the art and contain suitable elements for transcription and translation of nucleic acids. Typically, an expression vector contains an “expression cassette," which includes, in the 5' to 3' direction, a promoter, a coding sequence encoding a fusion protein operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.

[0084] Non-limiting examples of promoters of this invention include CYC1, HIS3, GALI, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and alkaline phosphatase promoters (useful for expression in Saccharomycesy, A0X1 promoter (useful for expression in Pichia , P-lactamase, lac, ara, tet, trp, IPL, IPR, T7, tac, and trc promoters (useful for expression in Escherichia colfy, light regulated-, seed specific-, pollen specific-, ovary specific-, pathogenesis or disease related-promoters, cauliflower mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5 -bisphosphate carboxylase, shoot-specific promoters, root specific promoters, chitinase, stress inducible promoters, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells).

[0085] Further examples of animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the El A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EFl) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein- L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, a- actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis and/or disease-related promoters, and promoters that exhibit tissue specificity, such as the elastase I gene control region, which is active in pancreatic acinar cells; the insulin gene control region active in pancreatic beta cells, the immunoglobulin gene control region active in lymphoid cells, the mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; the albumin gene promoter, the Apo Al and Apo All control regions active in liver, the alpha-fetoprotein gene control region active in liver, the alpha 1 -antitrypsin gene control region active in the liver, the beta-globin gene control region active in myeloid cells, the myelin basic protein gene control region active in oligodendrocyte cells in the brain, the myosin light chain-2 gene control region active in skeletal muscle, and the gonadotropic releasing hormone gene control region active in the hypothalamus, the pyruvate kinase promoter, the villin promoter, the promoter of the fatty acid binding intestinal protein, the promoter of smooth muscle cell a-actin, and the like. In addition, any of these expression sequences of this invention can be modified by addition of enhancer and/or regulatory sequences and the like.

[0086] Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor I (EFl) enhancer, yeast enhancers, viral gene enhancers, and the like. [0087] Termination control regions, i.e., terminator or polyadenylation sequences, may be derived from various genes native to the preferred hosts. In some embodiments of the invention, the termination control region may comprise or be derived from a synthetic sequence, a synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.

[0088] Expression vectors can be designed for expression of polypeptides in host cells, e.g., prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in bacterial cells such as E. coli, insect cells (e.g., the baculovirus expression system), yeast cells, plant cells or mammalian cells. Some suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Examples of bacterial vectors include pQE70, pQE60, pQE-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., EMBO J. 6:229 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933 (1982)), pJRY88 (Schultz et al., Gene 54: 113 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of nucleic acids to produce proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156 (1983)) and the pVL series (Lucklow and Summers Virology 170:31 (1989)).

[0089] Examples of mammalian expression vectors include pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6: 187 (1987)). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.

[0090] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid- protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

[0091] In addition to the regulatory control sequences discussed above, the recombinant expression vector can contain additional nucleotide sequences. For example, the recombinant expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector.

[0092] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques, including, without limitation, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and viral -mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al.. Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012), and other laboratory manuals.

[0093] If stable integration is desired, often only a small fraction of cells (in particular, mammalian cells) integrate the foreign DNA into their genome. In order to identify and select integrants, a nucleic acid that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the nucleic acid of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that comprising the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0094] The polynucleotide can also be introduced into a plant, plant cell or protoplast and, optionally, the isolated nucleic acid encoding the polypeptide is integrated into the nuclear or plastidic genome. Plant transformation is known in the art. 5ee, in general, Meth. Enzymol. Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554. [0095] In one aspect, the invention relates to methods of producing the fusion proteins of the invention, comprising expressing the fusion protein encoded by the polynucleotides and/or vectors described above. In one embodiment, the fusion proteins can be expressed in vitro, e.g., by in vitro transcription and/or translation. In another embodiment, the fusion protein can be expressed in a cell, e.g., an isolated cell, such as a cell line or a primary cell or a cell in an isolated tissue. In some embodiments, the cell can be a bacterial, fungal (e.g., yeast), insect, plant, or animal (e.g., mammalian) cell. In a different embodiment, the cell can be present in an animal or plant, e.g., for in vivo production of the fusion protein.

[0096] In one embodiment, the fusion protein is produced recombinantly by preparing a polynucleotide encoding the fusion protein. Coding sequences for the fusion proteins of the invention can be prepared using techniques well known in the art, including cutting and splicing polynucleotides encoding domains of the fusion protein or chemically synthesizing all or part of the coding sequence. In another embodiment, the fusion protein can be prepared at the protein level, e.g., by linking peptides or chemically synthesizing all or part of the amino acid sequence.

Assays and Methods of Using Fusion Proteins

[0097] Also provided according to embodiments of the invention are methods of using fusion proteins of the invention. As described above, the first fusion protein of the invention and the second fusion protein of the invention may be used to “report” the binding of a RBD polypeptide (or functional fragment thereof) linked to a first reporter enzyme fragment with a receptor (or functional fragment thereof) linked to a second reporter enzyme fragment. Thus, in some embodiments, methods include contacting a first fusion protein of the invention with a second fusion protein of the invention in a composition. The composition may include only the first and second fusion proteins or it may include additional components such as water, buffer, salts, and the like, including cellular or other biological material present in supernatants, as described above.

[0098] When a reporter enzyme such as luciferase is used, methods further include contacting the composition with a luciferase substrate, and measuring the bioluminescence emitted by the composition. In some embodiments, such methods are performed with a test compound and/or a biological sample included in the composition. If the test compound or a compound in the biological sample binds to the first fusion protein, the second fusion protein, or both, in a manner that inhibits interaction of the RBD polypeptide and the receptor, then the complementary fusion protein pairs will not bind together, the functional luciferase will not reconstitute, and no bioluminescence will result. In some embodiments, bioluminescence is measured using relative light units (RLU), for example, by using a method described in Li F., Cheng L., Murphy C.M., Reszka-Blanco N.J., Wu Y., Chi L., Hu J., Su L., Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs, Sci Rep. 2016 Nov 7;6:36483, and/or Murphy C.M., Xu Y., Li F., Nio K., Reszka-Blanco N., Li X., Wu Y., Yu Y., Xiong Y., Su L., Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication, Cell Rep. 2016 Sep 13; 16(11):2846-54. Thus, the difference between the bioluminescence (RLU) in the absence and the presence of a test compound may provide information about the capability of the test compound or a compound in the biological/test sample to interfere with the binding of the RBD to the ACE2 receptor.

[0099] In some embodiments of the invention, provided are methods of the invention that include the steps of (a) contacting the first fusion protein, the second fusion protein, and a test compound in a composition for a time sufficient for the first fusion protein and the second fusion protein to bind together; (b) optionally adding a reporter enzyme substrate (e.g., luciferase substrate) to the composition, (c) measuring the level of reporter (e.g., bioluminescence) in the composition, and (d) comparing the measured reporter level in the presence of the test compound with the reporter level in the absence of the test compound (but with the first and second fusion proteins). As described above, the reporter level will decrease if the test compound binds to the first fusion protein and/or the second fusion protein and inhibits interaction, and thus, a decrease in reporter level in the presence of the test compound may be used as an indication of the test compound’s ability to inhibit interaction of the RBD/receptor pair of interest.

[0100] In some embodiments of the invention, provided are methods of the invention that include the steps of (a) contacting the first fusion protein, the second fusion protein, and a biological sample in a composition for a time sufficient for the first fusion protein and the second fusion protein to bind together; (b) optionally adding a reporter enzyme substrate (e.g., luciferase substrate) to the composition, (c) measuring the level of reporter (e.g., bioluminescence) in the composition, and (d) comparing the measured reporter level in the presence of the biological sample with the reporter level in the absence of the biological sample (but with the first and second fusion proteins). As described above, the reporter level will decrease if there is a compound in the biological sample that binds to the first fusion protein and/or the second fusion protein and inhibits interaction, and thus, a decrease in reporter level in the presence of the biological sample may be used as an indication that a compound in the biological sample inhibits interaction of the RBD/receptor pair of interest. Thus, in some embodiments, methods of the invention may be used to detect binding inhibitors of the RBD/receptor pair, including neutralizing antibodies, in a biological sample.

[0101] In particular embodiments of the invention, a complementary pair of fusion proteins includes a first fusion protein that includes a first luciferase fragment and a RBD polypeptide for SARS-CoV-2, or a functional fragment thereof. The complementary pair of fusion proteins also includes a second fusion protein that includes a second luciferase fragment and a human ACE2 polypeptide, or a functional fragment thereof. In the methods above, a biological sample may be included in the composition including the first and second fusion proteins, and a decrease in bioluminescence in the presence of the biological sample may indicate that a neutralizing antibody for SARS-CoV-2 is present in the biological sample. This may provide a rapid and inexpensive method for determining whether a subject has neutralizing antibodies for SARS-CoV-2 in their serum or other biological fluids.

[0102] In some method embodiments described herein, contacting step (a) may include incubating the first fusion protein and the second fusion protein (and optionally a test compound and/or biological sample) for a predetermined time, such as a time in a range of about 15 minutes to about 12 hours, and in some embodiments, for a time in a range of about 15 minutes to 6 hours, and in some embodiments, for a time in a range of about 15 to about 30 minutes. In some embodiments, the composition is incubated at a temperature in a range of about 20 °C to about 40 °C, and in some embodiments, at a temperature in a range of about 25 °C to about 37 °C.

[0103] In some embodiments, methods further include the steps of expressing the first fusion protein from at least one cell of the invention, and expressing the second fusion protein from at last one cell of the invention prior to the contacting of the first and second fusion proteins. In some embodiments, supernatant from the expression of the first fusion protein may be combined with supernatant from the expression of the second fusion protein to form at least part of the test composition.

[0104] The term “luciferase substrate” refers to so called luciferins, which are compounds that are oxidized by an active luciferase to form a light emitting molecule. The luciferase substrate includes but is not limited to coelenterazine compounds, which are also referred to as CTZ or CLZN, including but not limited to benzylcoelenterazine (also known as coelenterazine h, 2,8-dibenzyl-6-(4- hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H)-one, CAS: 50909-86-9). Alternatively, native coolenterazine may be used (6-(4-hydroxyphenyl)- 2-[(4- hydroxyphenyl)methyl]-8-(phenylmethyl)-7H-imidazo[3,2-a] pyrazin-3-one, CAS: 55779-48- 1), as well as other luciferins from the Col el enterzine class including the non-native derivates, e.g., Coelenterazine 400a (Bisdeoxycoelenterazine, 2,8-dibenzyl-6-phenyl- imidazo[l,2A]pyrazin-3-(7H)-l, CAS 70217-82-2), e-Coelenterazine (Coelenterazine-E, Benz[f]imidazol[l,2-a]quinoxalin-3(6H)-one,5,l l-dihydro-8-hydroxy-2-[(4-hydroxyphenyl- methyl]-12-(phenylmethyl), CAS: 114496-02-5), Coelenterazine-Fluoride (Coelenterazine F, 8-benzyl-2-(4-fluorobenzyl)-6-(4-hydroxyphenyl)imidazo[l,2- a]pyrazin-3(7H)-one, CAS: 123437-16-1), e-Coelenterazine-F (Benz[f]imidazol[l,2-a]quinoxalin-3(6H)-one,5,l 1- dihydro- 8-hydroxy-2-[(4-fluorophenyl-methyl]-12-(phenylmethyl)), v-Coelenterazine (Coelenterazine- v, 16-benzyl-5-hydroxy-13-[(4-hydroxyphenyl)methyl]-l 1,14,17- triazatetracyclo[8.7.0.0 ^A {2,7}.0 ^A { 1 l,15}]heptadeca-l(10),2(7),3,5,8,13,15-heptaen-12-one), Coelenterazine hep (2-benzyl-8-(cyclopentylmethyl)-6-(4-hydroxyphenyl)imidazo[ 1 ,2- a]pyrazin-3(7H)-one CAS: 123437-32-1), Coelenterazine cp (8-(cyclopentylmethyl)-2-(4- hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H )-one, CAS: 123437-25-2), Coelenterazine fcp (8-(cyclopentylmethyl)-2-(4-fluorobenzyl)-6-(4-hydroxyphenyl )imidazo- [l,2-a]pyrazin-3(7H)-one CAS: 123437-33-2), Coelenterazine ip (8-(isopropylmethyl)-2-(4- hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H )-one).

[0105] The present invention finds use in research applications, as well as diagnostic and medical applications. Suitable subjects include all organisms, e.g., bacteria, fungi, plants, insects, avians, fish, and mammals. The term “avian” as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. The term “mammal” as used herein includes, but is not limited to, humans, bovines, ovines, caprines, equines, felines, canines, murines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. In other embodiments, the subject is an animal model of disease.

[0106] A further aspect of the invention relates to kits for carrying out the methods of the invention. The kits can comprise the fusion proteins, polynucleotides, vectors, and/or cells of the invention. The kits can comprise further components useful for carry out the methods of the invention, including without limitation, containers, buffers, ligands, reagents, fluorescent dyes, antibodies, cells, probes, primers, vectors, etc.

[0107] The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1

Materials and Methods

Modeling and 3D simulation of Fusion Protein Pairs

[0108] Protein modeling software was used to determine the three-dimensional structure of various fusion proteins that include the RBD of SARS-CoV-2 or human ACE2 with complementary luciferase fragments. Such modeling determined that certain fusion proteins may be suitable while others may not be suitable. For example, referring to FIG. 1, conformation modeling determined that certain fusion protein combinations would not allow for binding of the RBD with the ACE2 polypeptide while also allowing luciferase reconstitution. In FIG. 1, it can be seen that a fusion protein pair including a GLN-RBD polypeptide and an ACE2-GLC polypeptide would not allow for GLN-GLC reconstitution because the GLC at the C-terminus of ACE2 is kept away from the RBD-binding site.

[0109] However, 3D modeling predicted fusion protein pairs that should theoretically bind and provide a luciferase bioluminescence signal. FIG. 2 shows the theoretical possibility of the potential pairing of a fusion protein including GLC-RBD binding to a GLN-ACE2 protein. Such theoretical three-dimensional modeling determined possible complementary fusion protein pairs that might allow binding of the RBD and ACE2 domains while also allowing the functional luciferase to reconstitute.

[0110] Two theoretical complementary fusion protein pairs were determined to be: SP-GLC-RBD-H7 (“A”) and SP-GLN-ACE2-H7 (“B”); and SP-GLN-RBD-H7 (“C”) and SP-GLC-ACE2-H7 (“D”); wherein SP refers to the signal peptides for secreted expression of the gLucN or gLucC fusion proteins and H7 is the histidine tag for purification. Such fusion pair proteins were then prepared and tested for gLuc activity.

Fusion Protein Expression and Testing

[OHl] Fusion proteins A, B, C, and D were each separately synthesized by expression from HEK293 cells. The supernatant including fusion protein A (10 pl) was co-incubated with the supernatant including fusion protein B (10 pl) for a predetermined time (15 min to 6 h) at 37 °C before adding the gLuc substrate to produce bioluminescence. The supernatant including fusion protein C (10 pl) was also co-incubated with the supernatant including fusion protein D (10 pl) for a predetermined time (15 min to 6 h) at 37 °C before adding the gLuc substrate to produce bioluminescence. The bioluminescence for each sample was detected (using a GloMax® 20/20 Luminometer, Promega Corporation) and the results are shown in FIG. 3. As can be seen in FIG. 3, the A and B fusion proteins did not appear to reconstitute the gLuc activity, even after co-incubation for 6 hours. However, fusion proteins C and D showed gLuc activity, which indicates that this complementary fusion protein pair was able to bind and reconstitute to form the active gLuc enzyme.

[0112] The temperature dependence of the binding of fusion proteins C and D was also determined. The supernatant including fusion protein C (10 pl) was also co-incubated with the supernatant including fusion protein D (10 pl) for 12 hours at different temperatures. FIG. 4 shows that reconstitution and gLuc activity was significant at 25 °C and 37 °C, but there was no significant activity at either -4 °C or 50 °C.

[0113] Fusion proteins were then tested to determine whether the complementary fusion protein pairs may be used in an assay to detect neutralizing antibodies for SARS-CoV-2. As shown in FIG. 5, the gLuc activity of fusion proteins C and D can be blocked when a recombinant CoVl or CoV2 RBD or recombinant ACE was co-incubated with the fusion proteins. In this assay, the supernatant including fusion protein C (5 pl) and the recombinant protein (or mAb; 10 pl) was co-incubated with the supernatant including fusion protein D (5 pl) for 15 min at 37 °C before adding the gLuc substrate for detection. FIG. 5 also shows that the CR3022 antibody also blocked gLuc activity in this assay. Of note, a human immunoglobulin antibody (hlgG-Fc) and another coronavirus RBD (NL63-RBD) did not block gLuc activity.

[0114] Two monoclonal antibodies (CR3022 and mAb240C) with known CoV2 neutralizing activity were also found to block the gLuc activity. As can be seen in FIG. 6A, after 15 minutes, the gLuc activity was blocked using CR3022 at both 1 pg/ml and 10 pg/ml concentrations and mAb240C at 10 pg/ml. Referring to FIG. 6B, at 30 minutes, the gLuc activity was blocked by 10 pg/ml of CR3022 or 10 pg/ml mAb240C.

[0115] Thus, the data suggests that the present fusion proteins and methods may be used to detect the presence of SARS-CoV-2 neutralizing antibodies in biological samples.

[0116] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.