FIELD

The invention relates to compositions, such as nucleic acids, polypeptides, and fragments thereof, and methods of use thereof relating to Severe Acute Respiratory Syndrome (SARS) coronavirus, preferably SARS-CoV-2. The compositions may be useful as diagnostic reagents, in kits comprising such reagents, and for methods to identify a history of a SARS or SARS-like coronavirus infection in a person or animal.

BACKGROUND

Coronaviruses are a large family of viruses, some of which cause respiratory illnesses in humans ranging from common colds to more severe conditions such as Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS). The novel coronavirus involved in the outbreak starting in 2019-2020 has been named SARS-CoV-2 by the World Health Organization (WHO). The disease it causes has been named “coronavirus disease 2019” (or “COVID-19”). There are currently no specific treatments, drugs, or vaccines available to treat or prevent COVID-19.

There is an unmet urgent need for prophylactic or therapeutic compositions against the SARS-CoV-2 virus as well as methods and compositions to identify a history of infection with the virus in people and animals. For example, there is an unmet need for an assay to detect a history of SARS-CoV-2 infection serologically in a subject who has been immunized with an immunogenic composition that includes or expresses an antigen that is derived from the SARS- CoV-2 spike (S) glycoprotein. The assay should distinguish a history of infection with SARS- CoV-2 from a history of infection with common seasonal coronaviruses.

SUMMARY OF THE INVENTION

To meet these and other needs, a SARS-CoV-2 protein other than spike (S) protein that is highly immunogenic and well enough behaved biochemically to be an easily produced target antigen for immunoassays is sought.

In one aspect the invention relates to a peptide having the sequence SEQ ID NO: 1. In another aspect, the invention relates to a peptide having the sequence SEQ ID NO: 5 (also shown in FIG. 3B). In another aspect, the invention relates to a peptide having the sequence SEQ ID NO: 2. In another aspect, the invention relates to a peptide having the sequence SEQ ID NO: 6, also shown in FIG. 6. In another aspect, the invention relates to a nucleic acid molecule that encodes any one of the peptides described herein.

In some embodiments, the peptide further includes a linker sequence. In some embodiments, the linker sequence is GRS.

In some embodiments, the peptide further includes a 3C protease cleavage sequence.

In some embodiments, the peptide further includes a peptide sequence tag.

In another aspect, the invention relates to a method of detecting a previous SARS-CoV- 2 infection in a biological sample. The method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the person or animal from which the sample was obtained.

In another aspect, the invention relates to a method of detecting a SARS-CoV-2 specific antibody in a biological sample. The method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 specific antibody in the sample.

In yet another aspect, the invention relates to a method of detecting a previous SARS- CoV-2 infection in a human or animal from which a biological sample was obtained. The method includes a) contacting the sample with a C-terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the person or animal from which the sample was obtained.

In yet another aspect, the invention relates to a method of detecting a SARS-CoV-2 antibody in a biological sample. The method includes a) contacting the sample with a C- terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 specific antibody in the sample.

In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 1 . In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 5 (also shown in FIG. 3B).

In some embodiments, the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 2. In some embodiments, the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 6, also shown in FIG. 6.

In some embodiments, the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV spike polypeptide has been administered.

In some embodiments, the sample is from a human or animal prior to administration of an immunogenic composition eliciting an immune response against a SARS-CoV spike polypeptide.

In some embodiments, the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV spike polypeptide had not been administered.

In some embodiments, the polypeptide or variant thereof is attached to a solid support prior to contact with the sample.

In some embodiments, the sample is selected from any one of blood, serum, plasma, saliva, urine, mucus, fecal matter, and tissue extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts sequence features of SARS-CoV-2 nucleocapsid protein. FIG. 1 is taken from Figure 1A-C of Kang et al., "Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites," version posted March 7, 2020, on bioRxiv preprint server, Figure 1A-C of which is incorporated by reference.

FIG. 2 depicts Conserved sequence motif of N protein. Only the N-terminal region is shown. SEQ ID Nos of the motifs from the respective viruses listed in FIG. 2 are as follows:

SARS-CoV2 Wuhan-Hu-1 (SEQ ID NO: 10); Bat-SL-CoV-ZC45 (SEQ ID NO: 10); Bat-SL-CoV-ZX21(SEQ ID NO: 10); SARS-CoV Tor2 (SEQ ID NO: 11); SARS-CoV BJ01 (SEQ ID NO: 11); SARS-CoV INP_828858.1 (SEQ ID NO: 11); HCoV-OC43 Human Coronavirus OC43 (SEQ ID NO: 12); Bat Coronavirus HKU9-1 (SEQ ID NO: 13); HCoV-NL63 Human Coronavirus NL63 (SEQ ID NO: 14); HCoV229E Human Coronavirus 229E (SEQ ID NO: 15); HCoV-HKU-1 Human Coronavirus (SEQ ID NO: 16); SARS-CoV GZ02 (SEQ ID NO: 17); SARS-CoV Rs_672/2006 (SEQ ID NO: 18); Bat-SL-CoV RsSHC014 (SEQ ID NO: 19); Bat Coronavirus HKUS-1(SEQ ID NO: 20); Bat Coronavirus HKU4-1 (SEQ ID NO: 21); 2019- nCoV_HKU-SZ (SEQ ID NO: 22); Coronavirus BtRs-BetaCoV/Yn2018D (SEQ ID NO: 23); Bat- SL-Rs4255 (SEQ ID NO: 23); Bat-SL-Rs4231 (SEQ ID NO: 24); Bat Coronavirus RaTG13 (SEQ ID NO: 25)

FIG. 3A depicts an exemplary full length N protein with a linker, 3C protease cleavage sequence, twin-strep tag, and His-8 tag (SEQ ID NO: 8).

FIG. 3B depicts an exemplary NTD-modified Full length N protein with a linker, 3C protease cleavage sequence, twin-strep tag, and His-8 tag (SEQ ID NO: 5).

FIG. 4 depicts structural coordinates for the SARS-CoV-2 N-terminal domain.

FIG. 5 depicts a structure of the C-terminal domain of SARS-CoV N.

FIG. 6 depicts an exemplary CTD-dimer N protein construct, including a dimerization interface, 3C protease cleavage sequence, twin-strep tag, and His-8 tag (SEQ ID NO: 6).

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for an N-terminal domain (NTD)-modified full length Nucleocapsid (N) protein.

SEQ ID NO: 2 sets forth the amino acid sequence for a C-terminal domain (CTD)-dimer N protein.

SEQ ID NO: 3 sets forth the amino acid sequence for a full length N protein, wild-type.

SEQ ID NO: 4 sets forth the amino acid sequence corresponding to residues 110-117 of the sequence shown in SEQ ID NO: 3.

SEQ ID NO: 5 sets forth the amino acid sequence for an exemplary N-terminal domain (NTD)- modified full length Nucleocapsid (N) protein, shown in FIG. 3B.

SEQ ID NO: 6 sets forth the amino acid sequence for an exemplary C-terminal domain (CTD)- dimer N protein, shown in FIG. 6. SEQ ID NO: 7 sets forth the amino acid sequence for an exemplary CTD-dimer N protein.

SEQ ID NO: 8 sets forth the amino acid sequence for an exemplary full length N protein, shown in FIG. 3A.

SEQ ID NO: 9 and SEQ ID NO: 26 set forth the respective 9 amino acid sequence in the N protein.

SEQ ID NOs: 10-25 set forth the respective amino acid sequences shown in FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION SARS-CoV-2 Related Peptides and Nucleic Acids

The major structural proteins common to coronaviruses include the spike (S), membrane (M), and nucleocapsid (N). The spike glycoprotein (S) has been characterized as a surface antigen of Coronaviridae viruses and it binds to the host cell receptor, angiotensin converting enzyme-2 (ACE-2). The S protein is a known target for neutralizing immune responses. The membrane (M) glycoproteins of coronaviruses are also surface exposed and may be useful for immunoassays.

The nucleocapsid (N) protein is the most abundant protein of coronaviruses. It packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly, among other functions. Its N-terminal region includes mostly positively charged amino acids, and is responsible for RNA binding. The C-terminus is capable of self-association. Between these two structural domains, there lies a highly disordered region, which serves as a linker. Accordingly, the N-protein can be classified into three distinct regions, referred to herein as the NTD (N terminal domain), a SR rich linker, and the CTD (C-terminal domain).

Described herein are variants of an N protein. In some embodiments, a variant of the N protein includes a modification by amino acid additions to the N-terminus, C-terminus, and/or middle of the peptide. In some preferred embodiments, additions are to the N-terminus or C- terminus of the peptide. Additions can be of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,

17, 18, 19, or 20 amino acid resides. Such additions may constitute amino acid sequences that are present in SARS-CoV-2 in their entirety or in part. In a preferred embodiment, additions of amino acid sequences that are present in SARS-CoV-2 N protein are of 15 amino acids or less. Such additions may also constitute amino acid sequences which are not present in SARS-CoV- 2 N protein. Addition of sequences which are not present in SARS-CoV-2 N protein include, but are not limited to, small charged sequences (e.g., lysine-lysine-lysine) and sequences that enable the formation of branched structures (e.g., lysine or methionine). In a preferred embodiment, additions of amino acid sequences that are not present in SARS-CoV-2 N protein are of 5 amino acids or less. Residue additions can be either classical or non-classical amino acids or a mixture thereof.

In some embodiments, a variant of the N protein includes a modification by amino acid deletions to the N-terminus, C-terminus, and/or middle of the peptide. In some preferred embodiments, deletions are to the N-terminus or C-terminus of the peptide. Deletions can be of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid resides. In a preferred embodiment, deletions of amino acid sequences are of 10 amino acids or less. For example, in one embodiment, the peptide includes a deletion of the sequence FYYLGTGP (SEQ ID NO: 4) (corresponding to residues 110-117 of the sequence shown in SEQ ID NO: 3) in an N protein.

> Full length N protein, wild-type

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALT Q

HGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGT GPE

AGLPYGANKDGIIVWATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEG SRGG

SQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSG KG

QQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQG TD

YKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLN KHIDA

YKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADS TQA

(SEQ ID NO: 3)

A) N-terminal domain (NTD)-modified full length N protein

In one aspect the invention relates to a peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the first 175, 174, 173,

172, 171 , 170, 169, 168, 167, 166, 165, 164, 163, 162, 161 , 160, 159, 158, 157, 156, 155, 154,

153, 152, 151 , 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135,

134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of SEQ ID NO: 1 are modified, as compared to SEQ ID NO: 1.

In another aspect, the invention relates to a peptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence shown in SEQ ID NO: 5 (also shown in FIG. 3B).

In some embodiments, the peptide includes a deletion of the sequence FYYLGTGP (SEQ ID NO: 4) (corresponding to residues 110-117 of the sequence shown in SEQ ID NO: 3) in an N protein. Another exemplary peptide sequence includes:

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTAL

TQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWY -

EAGLPYGANKDGIIVWATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE GS

RGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESK

MSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGD

QELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPN F

KDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDF SK

QLQQSMSSADSTQA (SEQ ID NO: 3)

Further examples of a variant peptide includes a peptide having at least 80%, 81 %,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1 , wherein the residues FYYLGTGP (SEQ ID NO: 4) at positions 110-117 according to SEQ ID NO: 1 include substitutions, such as alanine substitutions.

In another aspect, the invention relates to a fragment of any one of the peptides described herein, including a fragment of a peptide having the sequence SEQ ID NO: 1 .

In another aspect, the invention relates to a nucleic acid molecule that encodes any one of the peptides described herein.

In some embodiments, the peptide further includes a linker sequence. In some embodiments, the linker sequence is GRS. In some embodiments, the peptide further includes a 3C protease cleavage sequence. In some embodiments, the peptide further includes a peptide sequence tag. B) CTD-dimer N protein

In one aspect the invention relates to a peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 2. In one embodiment, the first 150, 149, 148,

147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129,

128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114,

113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of

SEQ ID NO: 2 are modified, as compared to SEQ ID NO: 2.

In another aspect, the invention relates to a peptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 6, also shown in FIG. 6.

In another aspect, the invention relates to a fragment of any one of the peptides described herein, including a fragment of a peptide having the sequence SEQ ID NO: 2.

In another aspect, the invention relates to a nucleic acid molecule that encodes any one of the peptides described herein.

In some embodiments, the peptide further includes a linker sequence. In some embodiments, the linker sequence is GRS. In some embodiments, the peptide further includes a 3C protease cleavage sequence. In some embodiments, the peptide further includes a peptide sequence tag.

PEPTIDE VARIANTS

In another embodiment, a peptide or variant thereof described herein may be further modified by covalent attachment to another moiety. The covalent attachment between the peptide and moiety may be direct or indirect (e.g. through a linker molecule) by methods known in the art. In some embodiments, the moiety is a bead. In some embodiments, the moiety to which the peptide is attached includes a carrier molecule (e.g. bovine serum albumin or human serum albumin). In another embodiment, the moiety is a red blood cell. In another specific embodiment, the moiety is a latex particle.

Linker. In some embodiments, the peptide further includes a linker sequence. Suitable linkers may include a peptide sequence including alternating sets of glycine and serine residues, and may include an glutamic acid or lysine residue. In preferred embodiments, the linker sequence is Gly-Arg-Ser.

Protease cleavage site. In some embodiments, the methods of detecting described herein includes contacting a known quantity of SARS protease in solution to a peptide or variant thereof described herein further including a detectable marker and cleavage site for SARS protease, wherein SARS protease activity is monitored by measuring the intensity of the marker on the cleaved product. Accordingly, in some preferred embodiments, the peptide further includes a 3C protease cleavage sequence.

Peptide sequence tag. In some embodiments, the peptide further includes a peptide sequence tag. The peptides described herein may be produced by recombinant expression or chemical synthesis. Host cells suitable for recombinant expression include expression in bacterial, mammalian, insect, yeast, etc. Recombinant expression may be used to produce any peptide or variant thereof described herein.

Recombinant production of the peptide or variant thereof described herein may be facilitated by the addition of a peptide tag to be expressed as a fusion protein comprising the peptide or variant thereof described herein and the peptide tag. Exemplary peptide tags include a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag„ chitin- binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination antitermination factor (NusA), E. coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). A preferred peptide tag includes His-tag and GST. After purification, the peptide tag may be removed from the expressed fusion protein by methods known in the art.

METHODS OF DETECTING

Any one peptide or variant thereof described herein may be used in assays to detect whether a human has had a previous SARS-CoV-2 infection and in assays to detect the presence of a SARS-CoV-2 specific antibody in a biological sample. For example, the peptides described herein may be used in assays to identify individuals exposed to SARS-CoV-2 and to identify biological samples containing SARS-CoV-2 antigens or antibodies to SARS-CoV-2. In a preferred embodiment, the methods described herein detects all immunoglobulin classes, including IgM and IgG. Positive sera could be present in individuals with a current or previous infection.

In a preferred embodiment, the methods described herein includes a multiplexed assay with an N protein from SARS-CoV-2 and from seasonal coronaviruses to allow differentiation of seroconversion against the pandemic of seasonal strains.

The methods described herein may include use of solid supports, or immune- precipitation. In some embodiments, the method includes use of a labeled antibody or polypeptide. The label may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or a dye molecule.

In some embodiments, the methods described herein may include an element to amplify a signal from a resultant immune complex. Such elements are known in the art, such as, for example, an enzyme, radioisotope, fluorophore, bioluminescent molecule, chemiluminescent molecule, biotin, avidin, streptavidin or the like.

In some embodiments, the peptide or variant thereof described herein is bound to a solid matrix or support to facilitate separation of the sample from the peptide or variant thereof described herein after incubation. Exemplary solid supports that may be used include nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, microchips, high or low density biochips, recombinant immunoassays, microfluidity devices, micromagnetic beads, activated beads, and Protein A beads. The solid support containing the peptide or variant thereof described herein is typically washed after separating it from the test sample, and prior to detection of a bound antibody.

In another embodiment, the biological sample is contacted with a combination of proteins in addition to a peptide or variant thereof described herein. a) Detecting a previous SARS-CoV-2 infection by contacting a sample with an N-terminal domain-modified nucleocapsid polypeptide or variant thereof

In one aspect, the invention relates to a method of detecting a previous SARS-CoV-2 infection in a biological sample. The method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the sample.

In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 1 . In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has a sequence, wherein the first 175, 174, 173, 172, 171 , 170, 169, 168, 167, 166, 165, 164, 163, 162, 161 , 160, 159, 158, 157, 156, 155, 154, 153, 152, 151 , 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of SEQ ID NO: 1 are modified, as compared to SEQ ID NO: 1.

In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 5 (also shown in FIG. 3B). b) Detecting a previous SARS-CoV-2 infection by contacting a sample with a C-terminal domain dimer nucleocapsid peptide or variant thereof

In another aspect, the invention relates to a method of detecting a previous SARS-CoV- 2 infection in a biological sample. The method includes a) contacting the sample with a C- terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the sample.

In some embodiments, the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 2. In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the C-terminal domain dimer nucleocapsid peptide has a sequence, wherein the first 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of SEQ ID NO: 2 are modified, as compared to SEQ ID NO: 2.

In some embodiments, the C-terminal domain dimer nucleocapsid peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 6, also shown in FIG. 6. c) Detecting a SARS-CoV-2 antibody by contacting a sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof

In another aspect, the invention relates to a method of detecting a SARS-CoV-2 antibody in a biological sample. The method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 antibody in the sample.

In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 1 . In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has a sequence, wherein the first 175, 174, 173, 172, 171 , 170, 169, 168, 167, 166, 165, 164, 163, 162, 161 , 160, 159, 158, 157, 156, 155, 154, 153, 152, 151 , 150, 149, 148, 147,

146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128,

127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113,

112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of SEQ

ID NO: 1 are modified, as compared to SEQ ID NO: 1.

In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 5 (also shown in FIG. 3B). d) Detecting a SARS-CoV-2 antibody by contacting a sample with a C-terminal domain dimer nucleocapsid peptide or variant thereof

In yet another aspect, the invention relates to a method of detecting a SARS-CoV-2 antibody in a biological sample. The method includes a) contacting the sample with a C- terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 antibody in the sample.

In some embodiments, the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 2. In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the C-terminal domain dimer nucleocapsid peptide has a sequence, wherein the first 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136,

135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of SEQ ID NO: 2 are modified, as compared to SEQ ID NO: 2.

In some embodiments, the C-terminal domain dimer nucleocapsid peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 6, also shown in FIG. 6.

BIOLOGICAL SAMPLE

The biological sample may be from any mammal, including humans, non-primates (e.g., a bat, pangolin, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) and non-human primates (e.g., mammals including cynomolgus monkeys, chimpanzees, etc.).

In some embodiments, the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV-2 spike polypeptide has been administered.

In some embodiments, the sample is from a human or animal prior to administration of an immunogenic composition eliciting an immune response against a SARS-CoV-2 spike polypeptide.

In some embodiments, the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV-2 spike polypeptide had not been administered.

In some embodiments, the sample is from a human or animal who has not been exposed to SARS-CoV-2 virus.

In some embodiments, the sample is from a human or animal who has been exposed to SARS-CoV-2 virus and is or has recovered from a SARS-CoV-2 infection, such as, for example, convalescent serum.

In some embodiments, the sample is from a human or animal that has been administered with at least one dose of an immunogenic composition, e.g., a vaccine, against SARS-CoV-2 virus.

In some embodiments, the sample is from a human or animal that has been administered with at least two doses of an immunogenic composition, e.g., a vaccine, against SARS-CoV-2 virus.

In some embodiments, the polypeptide or variant thereof is attached to a solid support prior to contact with the sample.

In some embodiments, the sample is selected from any one of blood, serum, plasma, saliva, urine, mucus, fecal matter, and tissue extract. EXAMPLES

The following Examples illustrate embodiments of the invention.

EXAMPLE 1: Strategy for a non-vaccine antigen-specific antibody binding assay (NVA) to distinguish a history of SARS-CoV-2 infection from a history of other coronavirus infections in recipients of a COVID-19 vaccine candidates

To detect a history of SARS-CoV-2 infection serologically in a subject who has been immunized with a vaccine that includes or expresses an antigen that is derived from the SARS-CoV-2 spike (S) glycoprotein, we seek a SARS-CoV-2 protein other than S that is highly immunogenic and well enough behaved biochemically to be an easily produced target antigen for immunoassays. The assay should distinguish a history of infection with SARS-CoV-2 from a history of infection with common seasonal coronaviruses. Without being bound by theory or mechanism, it may not be necessary for the assay to distinguish a history of SARS-CoV-2 infection from a history of infection with the closely related SARS-CoV or MERS-CoV; SARS-CoV no longer circulates in humans; and the MERS-CoV now causes only small clusters of human infection after zoonotic spread from camels.

The nucleocapsid (N) of SARS-CoV-2 is the most abundant protein of coronaviruses. N coats the viral RNA and forms a helical nucleocapsid. As might be expected from an abundant protein that is in complex with RNA and forms a highly multivalent, regular array, N is highly immunogenic and, therefore, is often chosen as a target antigen for diagnostic assays.

Table 2. Percentage amino acid identity of coronavirus spike and nudeocapsid proteins to SARS-CoV-2 proteins.

N has 3 domains, the NTD (N terminal domain), a SR rich linker, and the CTD (C-terminal domain; Figure 1). The domain structure is conserved among the different coronavirus N’s, but the homology is low (Table 2 above).

Figure 2. Alignment of the N-terminal domain conserved region.

The C-terminal domain is relatively variable between coronavirus strains and is responsible for dimerization. The N-terminal domain interacts with RNA and is more conserved, and there is a stretch of amino acids in the NTD that is very conserved (Figure 2). Due to these conserved regions, it is possible that infections by any human coronavirus could elicit antibodies that cross- react with SARS-CoV-2 N as has been shown in the past for SARS.

Therefore, we have designed and are expressing 3 constructs derived from N for use as SARS- CoV-2 — specific immunoassay targets

1) Full length N protein: WT protein with a 3C protease cleavage site plus a twin-strep tag and a His-8 tag

2) NTD-modified full length N protein: This protein will be similar to the previous one but the sequence FYYLGTGP (SEQ ID NO: 4) will be substituted by FAYAGAG (SEQ ID NO: 29), to disrupt the conserved epitope without altering tertiary structure. Other substitutions for FYYLGTGP (SEQ ID NO: 4) may be used. This design is guided by the known structure of the NTD (Figure 4). a. Exemplary NTD-modified full length N protein sequence includes:

>NTD-modified FL N protein

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWF

TALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRW

YFAYAGAGAEAGLPYGANKDGIIVWATEGALNTPKDHIGTRNPANNAAIVLQLPQGT

TLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAAL

ALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAF

GRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGT

WLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQ

KKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO: 3)

3) CTD domain: This construct includes only the CTD of N since this domain is less conserved. This construct will include the same tags as the previous versions. Its design is guided by the known structure of the CTD (Figure 5). a. Exemplary CTD-dimer N protein sequence includes:

>CTD-dimer N protein

MNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIG MEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADE TQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO: 7)

Figure 4. Structure guided design of a modified N-terminal domain of SARS-CoV-2 N to reduce cross-reactivity with other coronaviruses. (Structural coordinates for the SARS-CoV-2 N- terminal domain from PDB ID: 6M3M; S. Kang et al. , Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. 2020).

The assay is being developed on an ELISA or a Luminex platform and will be tested initially with human non-immune and convalescent sera for SARS-CoV-2. To mitigate against the possibility that an assay based on SARS-CoV-2 N alone shows insufficient specificity, for the N proteins of seasonal coronavirus strains are being synthesized. A multiplexed assay with N’s of the pandemic and of seasonal coronavirus strains should allow the differentiation of seroconversion against the pandemic distinct from common seasonal strains.

The SARS-CoV N polypeptides may be detected using enzyme-linked immunosorbent assay (ELISA), enzyme-based histochemical assays, using fluorescent, radioactive, and/or luminescent systems.

Figure 5. Structure of the C-terminal domain of SARS-CoV N. PDB ID: 2GIB. I. M. Yu, M. L. Oldham, J. Zhang, J. Chen, Crystal structure of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona- and arteriviridae. J Biol Chem 281 , 17134-17139 (2006).

EXAMPLE 2. A single-plex SARS CoV-2 full-length (FL) Nucleoprotein Ig Direct Luminex Immunoassay was developed. Magnetic beads coated with SARS-CoV-2 Nucleocapsid protein are contacted with respective serum dilutions, and binding of antibodies, if any from the respective serum dilutions, to the SARS-CoV-2 Nucleocapsid protein are detected using PE goat anti-human specific antibodies, and analyzed in a BioPlex reader. The serum samples may include any one of a standard, convalescent serum, and test serum. The incubation magnetic beads coated with SARS-CoV-2 Nucleocapsid protein and the serum dilutions occurs at room temperature for about 120 minutes, whereby an antibody, if any, in the respective serum samples bind to the SARS-CoV-2 Nucleocapsid protein. PE-goat anti-human Ig secondary antibodies are added for detection of anti-N antibodies in the sera samples and incubated with the sera samples at room temperature for about 90 minutes. Results (positive or negative) from the single-plex SARS CoV-2 assay described herein were identical to at least 91- 100% of the results observed from the ELECSYS (Roche) immunoassay intended for qualitative detection of antibodies to SARS-CoV-2 in human serum and plasma.

Table 1 :

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