Corona nucleocapsid antigen for antibody immunoassay

Abstract

The present invention relates to coronavirus antigens comprising a coronavirus nucleocapsid specific amino acid sequence, compositions and kits comprising them and methods of production thereof. Further included are methods of using said coronavirus antigens for detecting anti-coronavirus antibodies in a sample, and methods of differentiating between immune responses in a patient caused by a natural coronavirus infection or by vaccination against coronavirus.

Description

[0001] This invention relates to coronavirus antigens comprising a coronavirus nucleocapsid-specific amino acid sequence, compositions, kits comprising the same, and methods for manufacturing the same. It also includes methods for using said coronavirus antigens to detect anti-coronavirus antibodies in samples, and methods for differentially diagnosing immune responses in patients induced by natural coronavirus infection or by coronavirus vaccination. Background Technology

[0002] SARS-CoV-2, formerly known as nCoV-19 (novel coronavirus 2019), is the pathogen of the 2019 coronavirus disease (COVID-19), which triggered a pandemic in early 2020, leading to severe restrictions on public life and significant economic impacts worldwide. Diagnostic tests that allow for the detection of patients with acute infection became rapidly available. However, the number of available tests could not meet the high demand during the pandemic. As a result, many patients outside clinics and hospitals did not receive testing, as available tests were primarily reserved for those with very critical conditions. Statistics show that four out of five patients infected with SARS-CoV-2 experienced only mild symptoms, such as a mild sore throat, dry cough, or mild fever. Therefore, it remains unclear how many people have been or are still infected, and how many have recovered from the infection.

[0003] To assess the extent of the current pandemic, accurately evaluating the infection rate and true mortality rate of SARS-2 would be extremely helpful. Furthermore, it would allow for the lifting of public lockdowns for patients who have known to have recovered from the disease and acquired immunity, and provide assistance to patients who still require services such as clinic services and hospitalization.

[0004] Therefore, there is an urgent need for immunological tests capable of detecting antibodies against the SARS-CoV-2 virus in patients. Such antibody tests would allow for the identification of patients previously affected by infection, who may experience mild progression of the disease to the point that they are unaware of it. Thus, such tests would allow for the first reliable assessment of the true infection rate across different groups and the general population. Furthermore, such tests would allow for the assessment of whether vaccines developed against SARS-CoV-2 infection truly and effectively stimulate an immune response in patients, and are therefore absolutely necessary in evaluating the success of vaccination campaigns.

[0005] However, there is still no available automated high-throughput assay to detect anti-SARS-CoV-2 antibodies in patients with the required sensitivity and specificity. With currently approved antibody tests, only one-third of infected patients are correctly diagnosed, while two-thirds are falsely reported. One of the main problems here is equipping the test with antigens that can be recognized with high sensitivity and specificity by anti-SARS-CoV-2 antibodies.

[0006] Since the first report of SARS in 2002 / 2003, several coronavirus antigens have been known in the field. The spike protein of the coronavirus, especially its receptor-binding domain (RBD), is considered the most promising candidate because it has previously been shown to have high immunoreactivity (Wang et al. (Clin Chem (2003) 49(12), 1989-1996; and He et al. (J. Clin. Microbiol. (2004) 42(11), 5309-5314), i.e., in SARS infection). During the humoral immune response following CoV-2 infection, a strong antibody response to the receptor-binding domain (RBD) is generated. Therefore, the receptor-binding domain can also serve as a primary antigen in current assay development (Amanat et al., medRxiv, March 18, 2020). In this recent manuscript, the authors describe the use of the CoV-2 receptor-binding domain as a capture antigen in ELISA formats. However, sensitivity data were determined based on only four positive sera (from three COVID-19 patients), and specificity data relied on only 59 negative sera. The sample volume analyzed was too small to demonstrate statistically significant sensitivity and specificity. Furthermore, antibody assays in ELISA formats typically require time-consuming and laborious manual steps and are generally excluded from high-throughput applications due to the limited availability of the assays.

[0007] Contrary to the prevailing bias in the art that antigens derived from the spike protein are the most promising for developing coronavirus antibody assays, this invention relates to an immunological assay for reliably detecting anti-SARS-CoV-2 antibodies using the nucleocapsid protein of the SARS-CoV-2 virus as an antigen. Surprisingly, the inventors have demonstrated that by using the nucleocapsid protein of SARS-CoV-2 as an antigen, high sensitivity and specificity can be achieved in the resulting immunoassay, allowing for the development of the much-needed and eagerly anticipated automated high-throughput coronavirus antibody assay. Summary of the Invention

[0008] In a first aspect, the present invention relates to a coronavirus antigen suitable for detecting anti-coronavirus antibodies in isolated biological samples, comprising a coronavirus nucleocapsid-specific amino acid sequence, particularly the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 or a coronavirus nucleocapsid-specific amino acid sequence having 95% sequence homology with the amino acid sequence of SEQ ID NO: 1. Specifically, the polypeptide does not contain other coronavirus-specific amino acid sequences.

[0009] In a second aspect, the present invention relates to compositions comprising the coronavirus antigen of the first aspect of the present invention.

[0010] In a third aspect, the present invention relates to a method for producing a coronavirus antigen specific to the nucleocapsid of a coronavirus, the method comprising the following steps:

[0011] a) Culture host cells transformed with an expression vector, particularly *E. coli* cells, wherein the expression vector comprises a recombinant DNA molecule operably linked to an antigen encoding the first aspect of the invention, particularly a recombinant DNA molecule comprising the sequence according to SEQ ID NO: 3.

[0012] b) Expressing the polypeptide, and

[0013] c) Purify the polypeptide.

[0014] In a fourth aspect, the present invention relates to a method for detecting antibodies specific to coronaviruses in isolated samples, wherein the coronavirus antigen of the first aspect of the invention, the composition of the second aspect of the invention, or the coronavirus antigen obtained by the method of the third aspect of the invention is used as a capture reagent and / or binding coupler for said anti-coronavirus antibodies.

[0015] In a fifth aspect, the present invention relates to a method for detecting antibodies specific to coronaviruses in isolated samples, the method comprising:

[0016] a) An immune reaction mixture is formed by mixing a body fluid sample with the coronavirus antigen of the first aspect of the present invention, the composition of the second aspect of the present invention, or the coronavirus antigen obtained by the method of the third aspect of the present invention.

[0017] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0018] c) Detect the presence and / or concentration of any of the said immune response products.

[0019] In a sixth aspect, the present invention relates to a method for identifying whether a patient has been previously exposed to coronavirus infection, comprising:

[0020] a) An immune reaction mixture is formed by mixing a patient's bodily fluid sample with the coronavirus antigen of the first aspect of the present invention, the composition of the second aspect of the present invention, or the coronavirus antigen obtained by the method of the third aspect of the present invention.

[0021] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0022] c) Detect the presence and / or absence of any of the said immune response products, wherein the presence of an immune response product indicates that the patient has been exposed to coronavirus infection in the past.

[0023] In a seventh aspect, the present invention relates to a method for differential diagnosis between an immune response induced by natural coronavirus infection and an immune response induced by vaccination, wherein the vaccination is based on an antigen derived from an S protein, E protein, or M protein, comprising...

[0024] a) An immune reaction mixture is formed by mixing a patient's bodily fluid sample with the coronavirus antigen of the first aspect of the present invention, a composition comprising the coronavirus antigen of the first aspect of the present invention, or a coronavirus antigen obtained by the method of the third aspect of the present invention.

[0025] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0026] c) Detect the presence and / or absence of any of the said immune response products, wherein the presence of an immune response product indicates that the immune response in the patient is caused by natural coronavirus infection, and wherein the absence of an immune response product indicates that the immune response in the patient is caused by vaccination with a spike protein-derived antigen.

[0027] In an eighth aspect, the present invention relates to the use of the coronavirus antigen of the first aspect of the invention, the composition of the second aspect of the invention, or the coronavirus antigen obtained by the method of the third aspect in a high-throughput in vitro diagnostic test for detecting anti-coronavirus antibodies.

[0028] In a ninth aspect, the present invention relates to a kit for detecting anti-coronavirus antibodies, the kit comprising the coronavirus antigen of the first aspect of the invention, the composition of the second aspect of the invention, or the coronavirus antigen obtained by the method of the third aspect of the invention. Attached Figure Description

[0029] Figure 1 Based on the following alignment of known coronavirus nucleocapsid sequences with UniProt ID NO, Gene Bank Acc NO, and their respective SEQ ID NO:

[0030] Severe Acute Respiratory Syndrome Coronavirus 2N (SARS-CoV-2), β-CoV: UniProt ID P0DTC9; GeneBank Acc.: MN908947; SEQ ID NO: 16 Severe Acute Respiratory Syndrome Coronavirus N (SARS-CoV), β-CoV: UniProt ID P59595; GeneBank Acc.: AY278741; SEQ ID NO: 17

[0031] Middle East Respiratory Syndrome-Associated Coronavirus N (MERS-CoV), β-CoV: UniProt ID T2BBK0; GeneBank Acc.: KF600632 (SEQ ID NO18)

[0032] Human coronavirus NL63 N (HCoV-NL63), α-CoV: UniProt ID Q6Q1R8; Gene Bank Acc: AY567487; SEQ ID NO: 19

[0033] Human coronavirus 229E N (HCoV-229E), α-CoV: UniProt ID P15130; Gene Bank Acc: X51325; SEQ ID NO: 20

[0034] Human coronavirus OC43 N (HCoV-OC43), β-CoV: UniProt ID P33469; Gene Bank Acc.: AY585228; SEQ ID NO: 21

[0035] Human coronavirus HKU1 N (HCoV-HKU1), β-CoV: UniProt ID Q5MQC6; Gene Bank Acc.: AY597011; SEQ ID NO: 22

[0036] Figure 2 Sequence comparison: (A) Sequence identity (%) between the SARS CoV-2 nucleocapsid amino acid sequence and the nucleocapsid sequences of different coronaviruses; (B) Sequence homology (%) between the SARS CoV-2 nucleocapsid amino acid sequence and the nucleocapsid sequences of different coronaviruses.

[0037] Figure 3 Illustration of the EcSlyD-EcSlyD-CoV-2N(1-419) antigen

[0038] Figure 4a Comparison of the immunoreactivity of antigens derived from the SARS-CoV-2 S, E, and M proteins.

[0039] Figure 4b Comparison of different antigens derived from the SARS CoV-2 nucleocapsid protein

[0040] Figure 5 Comparison of immunoreactivity of full-length nucleocapsids with no, one, or two SlyD chaperone proteins fused to them.

[0041] Figure 6The effect of bead pretreatment of ruthenium conjugates (as an additional step in the production process) on assay performance.

[0042] Figure 7 Sensitivity of SARS-CoV-2 assays; A) Preliminary results from samples obtained from 129 confirmed SARS-CoV-2 patients; and B) Further results, including a total of 214 confirmed SARS-CoV-2 patients; C) Additional results from an additional 292 confirmed SARS-CoV-2 patients.

[0043] Figure 8 Specificity of SARS-CoV-2 assays; A) Results from the first set of measurement samples from 5192 patients and 80 potentially cross-reactive samples; B) Results from the second set of measurement samples from 5261 patients; C) Results from all patients (10453 in total). Common cold and coronavirus cross-reactive samples were not routinely diagnosed or from blood donors and were therefore excluded from the overall specificity calculation.

[0044] Figure 9 Correlation between the assay performance of venous serum samples and capillary blood samples Figure 10 Comparison of the immunoreactivity of antigens containing SARS-CoV-2 nucleocapsid sequences fused with two SlyD- or two SlpA-chaperone proteins.

[0045] Figure 11: Reactivity of the N-terminal domain of nucleocapsid proteins from SARS-CoV-2, OC43, NL63, 229E, and HKU1. Measurements were performed in DAGS format on a cobas e411 automated analyzer. The concentrations of the biotin conjugate (R1) and ruthenium conjugate (R2) were each 100 ng / ml. Signal readings (counts) were normalized relative to the mean of their respective negative values ​​to produce signal dynamics (s / n).

[0046] Figure 12 Schematic diagram of four single-point mutation variants of the SARS-CoV-2 nucleocapsid antigen.

[0047] Figure 13 WT of SARS-CoV-2 nucleocapsid antigen compared with signal recovery of 3MUT or 8MUT single-point mutant variants

[0048] sequence list

[0049] SEQ ID NO: 1: Amino acid sequence of the nucleocapsid of coronavirus SARS CoV-2

[0050] SEQ ID NO: 2: Amino acid sequence of the SARS-CoV-2 nucleocapsid fused with a SlyD chaperone protein.

[0051] SEQ ID NO: 3: Amino acid sequence of the SARS-CoV-2 nucleocapsid fused with two SlyD chaperone proteins

[0052] SEQ ID NO: 4: Nucleotide sequence of the nucleocapsid of coronavirus SARS CoV-2

[0053] SEQ ID NO: 5: Nucleotide sequence of the SARS-CoV-2 nucleocapsid fused with a SlyD chaperone protein.

[0054] SEQ ID NO: 6: Nucleotide sequence of the SARS-CoV-2 nucleocapsid fused with two SlyD chaperone proteins.

[0055] SEQ ID NO: 7: Connector peptide

[0056] SEQ ID NO: 8: Amino acid sequence of the SARS CoV-2-N 3MUT variant

[0057] SEQ ID NO: 9: Amino acid sequence of the EcSlyD-EcSlyD-SARS CoV-2-N 3MUT variant

[0058] SEQ ID NO: 10: Amino acid sequence of SARS CoV-2-N8MUT variant

[0059] SEQ ID NO: 11: Amino acid sequence of the EcSlyD-EcSlyD-SARS CoV-2-N8MUT variant

[0060] SEQ ID NO: 12: Amino acid sequence of SARS CoV-2-N 12MUT variant

[0061] SEQ ID NO: 13: Amino acid sequence of the EcSlyD-EcSlyD-SARS CoV-2-N12MUT variant

[0062] SEQ ID NO: 14: Amino acid sequence of SARS CoV-2-N15MUT variant

[0063] SEQ ID NO: 15: Amino acid sequence of the EcSlyD-EcSlyD-SARS CoV-2-N15MUT variant

[0064] SEQ ID NO: 16: Amino acid sequence of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), β-CoV: UniProt ID P0DTC9; Gene Bank Acc.: MN908947

[0065] SEQ ID NO: 17: Amino acid sequence of severe acute respiratory syndrome coronavirus (SARS CoV), β-CoV: UniProt ID P59595; Gene Bank Acc.: AY278741

[0066] SEQ ID NO: 18: Amino acid sequence of Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), β-CoV: UniProt ID T2BBK0; Gene Bank Acc.: KF600632

[0067] SEQ ID NO: 19: Amino acid sequence of human coronavirus NL63 (HCoV-NL63), α-CoV: UniProt IDQ6Q1R8; Gene Bank Acc: AY567487

[0068] SEQ ID NO: 20: Human coronavirus 229E (HCoV-229E), α-CoV amino acid sequence: UniProt IDP15130; Gene Bank Acc: X51325

[0069] SEQ ID NO: 21: Amino acid sequence of human coronavirus OC43 (HCoV-OC43), β-CoV: UniProt IDP33469; Gene Bank Acc.: AY585228

[0070] SEQ ID NO: 22: Amino acid sequence of human coronavirus HKU1 (HCoV-HKU1), β-CoV: UniProt IDQ5MQC6; Gene Bank Acc.: AY597011 Detailed Implementation

[0071] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these methods, schemes, and reagents can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0072] Several documents are referenced throughout this specification. Each of these references (including all patents, patent applications, scientific publications, manufacturer's instructions, user manuals, etc.), whether cited above or below, is incorporated herein by reference in its entirety. In the event of any conflict between the definitions or teachings of such incorporated references and those cited in this specification, the text of this specification shall prevail.

[0073] Elements of the invention will now be described. These elements are listed along with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention solely to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any arrangement and combination of the elements described herein should be considered as disclosed in the specification of this application.

[0074] definition

[0075] The word “comprise” and its variations such as “comprises” and “comprising” should be understood to imply the inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0076] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” “the,” and “the” include the plural referents.

[0077] Concentration, amount, and other numerical data may be expressed or presented in a “range” format herein. It should be understood that this range format is used solely for convenience and brevity, and therefore should be flexibly interpreted to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered by the range, as if each value and subrange were explicitly listed. For example, the numerical range “150 mg to 600 mg” should be interpreted to include not only the explicitly listed value of 150 mg to 600 mg, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 580 mg, 590 mg, 600 mg, and subranges such as from 150 to 200, from 150 to 250, from 250 to 300, from 350 to 600, etc. The same principle applies to ranges listing only a single value. Furthermore, this interpretation applies regardless of the breadth of the range or characteristic described.

[0078] When used in conjunction with numerical values, the term “about” means to cover a range of values ​​that has a lower limit of 5% less than the indicated value and an upper limit of 5% greater than the indicated value.

[0079] The “symptoms” of a disease refer to noticeable symptoms in the tissues, organs, or organisms affected by the disease, and include, but are not limited to, pain, weakness, tenderness, tension, stiffness, and spasms in the tissues, organs, or individuals. The “signals” or “signals” of a disease include, but are not limited to, changes or alterations in specific indicators (such as biomarkers or molecular markers) (e.g., presence, absence, increase, decrease, or decrease) or the development, presence, or worsening of symptoms. Symptoms of pain include, but are not limited to, discomfort that may manifest as persistent or varying degrees of burning, throbbing, itching, or stinging pain.

[0080] The terms “disease” and “illness” are used interchangeably in this document to refer to an abnormal condition, particularly an abnormal medical condition, such as ailment or injury, in which an tissue, organ, or individual is no longer able to perform its function effectively. Usually, but not always, a disease is associated with specific symptoms or signs that indicate its presence. Therefore, the presence of such symptoms or signs indicates that an tissue, organ, or individual is diseased. Changes in these symptoms or signs indicate the progression of the disease. The progression of a disease is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a “worsening” or “improving” condition. A “worsening” condition is characterized by a decrease in the ability of an tissue, organ, or organism to perform its function effectively, while a “improving” condition is typically characterized by an increase in the ability of an tissue, organ, or individual to perform its function effectively. Examples of diseases include, but are not limited to, infectious diseases, inflammatory diseases, skin diseases, endocrine disorders, intestinal diseases, neurological disorders, joint diseases, hereditary disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0081] The term "coronavirus" refers to a group of related viruses that cause diseases in mammals and birds. In humans, coronaviruses cause respiratory infections ranging from mild to fatal. Mild illness includes some cases of the common cold, while more deadly variants can lead to SARS, MERS, and COVID-19. Coronaviruses contain a single-stranded, positive-sense RNA genome.

[0082] The viral envelope is formed by a lipid bilayer, anchoring membrane (M), envelope (E), and spike (S) structural proteins. Within the envelope, multiple copies of the nucleocapsid (N) protein form the nucleocapsid, which binds to the positive-sense single-stranded RNA genome in a continuous beaded conformation. Its genome includes Orfs 1a and 1b, encoding a multiprotein replicase / transcriptase, followed by sequences encoding the spike (S)-enveloping protein, envelope (E)-protein, membrane (M)-protein, and nucleocapsid (N)-protein. Scattered between these reading frames are reading frames of accessory proteins that differ between different viral strains.

[0083] Several human coronaviruses are known, four of which cause fairly mild symptoms in patients:

[0084] Human coronavirus NL63 (HCoV-NL63), α-CoV

[0085] Human coronavirus 229E (HCoV-229E), α-CoV:

[0086] Human coronavirus HKU1 (HCoV-HKU1), β-CoV:

[0087] Human coronavirus OC43 (HCoV-OC43), β-CoV:

[0088] Three human coronaviruses can produce potentially severe symptoms:

[0089] Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), β-CoV

[0090] Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), β-CoV

[0091] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), β-CoV

[0092] SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 is highly contagious in humans, and the World Health Organization (WHO) has designated the ongoing COVID-19 pandemic as a Public Health Emergency of International Concern. Symptoms include high fever, sore throat, dry cough, and fatigue. In severe cases, it can develop into pneumonia.

[0093] The term "natural coronavirus" refers to coronaviruses that occur in nature, i.e., any coronaviruses disclosed above. It should be understood that natural coronaviruses contain all the proteins and nucleic acid molecules present in naturally occurring viruses. Unlike natural coronaviruses, "viral fragments," "virus-like particles," or coronavirus-specific antigens contain only some, but not all, of the proteins and nucleic acid molecules present in naturally occurring viruses. Therefore, such "viral fragments," "virus-like particles," or coronavirus-specific antigens are not infectious but can still elicit an immune response in patients. Therefore, vaccination with coronavirus-specific viral fragments, coronavirus-specific virus-like particles, or coronavirus-specific antigens will produce antibodies against these viral fragments, virus-like particles, or antigens in patients.

[0094] As used herein, “patient” means any mammal, fish, reptile, or bird that may benefit from the diagnosis, prognosis, or treatment described herein. In particular, “patient” is selected from the group consisting of: laboratory animals (e.g., mice, rats, rabbits, or zebrafish), livestock (including, for example, guinea pigs, rabbits, horses, donkeys, cattle, sheep, goats, pigs, chickens, camels, cats, dogs, tortoises, tortoises, snakes, lizards, or goldfish), or primates (including chimpanzees, bonobos, gorillas, and humans). Particularly preferred is that “patient” is a human.

[0095] The terms “sample” or “target sample” are used interchangeably herein and refer to a portion or section of a tissue, organ, or individual, typically smaller than such tissue, organ, or individual, intended to represent the whole tissue, organ, or individual. At the time of analysis, the sample provides information about the state of the tissue or the health or disease status of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymph; or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. Analysis of the sample can be performed on a visual or chemical basis. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of the tissue, organ, or individual to allow for morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or changes in its quantity, concentration, or level. The sample is an in vitro sample, which will be analyzed in vitro and will not be transferred back into the body.

[0096] The terms “nucleic acid” and “nucleic acid molecule” are used synonymously herein and refer to single-stranded or double-stranded oligomers or polymers of deoxyribonucleotide or ribonucleotide bases, or both. A nucleotide monomer consists of a nucleobase, a pentose sugar (e.g., but not limited to ribose or 2′-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed by phosphodiester bonds between individual nucleotide monomers. In the context of this invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, but also includes synthetic forms of nucleic acids containing other bonds (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254: 1497-1500, 1991)). Typically, nucleic acids are single-stranded or double-stranded molecules and are composed of naturally occurring nucleotides. The description of a single-stranded nucleic acid also defines the sequence of the complementary strand (at least partially). Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Exemplary double-stranded nucleic acid molecules may have 3′ or 5′ overhangs, and therefore are not required or assumed to be completely double-stranded along their entire length. Nucleic acids can be obtained by biological, biochemical, or chemical synthetic methods or any methods well known in the art, including but not limited to amplification and reverse transcription of RNA. The term nucleic acid includes chromosomes or chromosome segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence contains or encodes a complementary sequence, except for any explicitly stated sequence.

[0097] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, that nucleic acid is "operably linked". For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence, or if a ribosome binding site is located to facilitate translation, then the ribosome binding site is operably linked to the coding sequence.

[0098] The term "complementarity" refers to a lock-and-key relationship between two structures. In nature, complementarity is a fundamental principle of DNA replication and transcription because it is a property shared between two DNA or RNA sequences, so that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequence will be complementary.

[0099] The term "sequence comparison" refers to the process of comparing a test sequence with one of its reference sequences. When using a sequence comparison algorithm, the test and reference sequences are input into a computer program, with subsequence coordinates specified if necessary, and sequence algorithm program parameters specified. Default program parameters are typically used, or alternative parameters can be specified. Based on these parameters, the sequence comparison algorithm then calculates the percentage of sequence identity or similarity of the test sequence relative to the reference sequence. In sequence alignment, the term "comparison window" refers to an extension of consecutive positions in the sequence that is compared to a reference extension of consecutive positions in a sequence with the same number of positions. The number of consecutive positions can range from 10 to 1000, meaning it can include 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 consecutive positions. Typically, the number of consecutive positions ranges from approximately 20 to 800, approximately 20 to 600, approximately 50 to 400, approximately 50 to approximately 200, and approximately 100 to approximately 150. The sequence alignment methods used for comparison are well known in the art. The best sequence alignment for comparison can be performed, for example, by Smith and Waterman’s local algorithm (Adv. Appl. Math. 2: 482, 1970), by Needleman and Wunsch’s homology alignment algorithm (J. Mol. Biol. 48: 443, 1970), by Pearson and Lipman’s search similarity method (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), by computerized execution of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 Addendum)). The algorithms suitable for determining sequence identity and sequence similarity percentages are BLAST and BLAST 2.0, described by Altschul et al. (Nuc. Acids Res. 25: 3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215: 403-10, 1990), respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).The algorithm involves first identifying high-scoring sequence pairs (HSPs) by recognizing short fields of length W in the query sequence, which match or satisfy a certain positive threshold score T when aligned with fields of the same length in the database sequence. T is called the neighbor field score threshold (Altschul et al., ibid.). These initial neighbor field hits serve as seeds to trigger a search for longer HSPs containing them. Field hits extend bidirectionally along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, parameters M (reward for matching residue pairs; always > 0) and N (penalty for mismatched residues; always < 0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Field hits cease extending in each direction when the cumulative alignment score decreases by an amount X from its maximum realized value; when the cumulative score reaches or falls below zero due to the accumulation of one or more negative residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W) of 11, an expected value (E) of 10, M = 5, and N = -4, and compares two strands. For amino acid sequences, the BLASTP program uses a default word length of 3, an expected value (E) of 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989) with an alignment (B) of 50, an expected value (E) of 10, M = 5, N = -4, and compares two strands. The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-87, 1993). One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the tested nucleic acid with a reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0100] The term "at least 90% sequence identity" is used herein in relation to comparisons of amino acid or nucleotide sequences. In the case of two or more nucleic acid or polypeptide amino acid sequences, the term "identical" means that the two or more sequences or subsequences are identical, i.e., contain the same nucleotide or amino acid sequence. The term "at least 90% sequence identity" specifically refers to having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding amino acid or nucleotide sequence.

[0101] The term "at least 90% sequence homology" is used herein in relation to comparisons of amino acid or nucleotide sequences. In addition to identical residues (sequence identity), the percentage of conserved residues with similar physicochemical properties (e.g., leucine and isoleucine) (similarity percentage) is generally used to "quantify homology." The term "at least 90% sequence homology" specifically refers to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with the corresponding amino acid or nucleotide sequence. Optionally, the amino acid sequence in question and the reference amino acid sequence exhibit the specified sequence identity or sequence homology over a continuous extension of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids or over the entire length of the reference amino acid sequence. Optionally, the nucleic acid sequence under discussion and the reference nucleic acid sequence exhibit specified sequence identity or sequence homology over consecutive extensions of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides or over the entire length of the reference nucleic acid sequence.

[0102] The term "recombinant DNA molecule" refers to a molecule prepared by combining two otherwise separate segments of a DNA sequence, achieved through the artificial manipulation of isolated polynucleotide fragments using genetic engineering techniques or chemical synthesis. In doing so, polynucleotide fragments with desired functions can be linked together to produce the desired functional combination. Recombinant DNA techniques for expressing proteins in prokaryotic or lower or higher eukaryotic host cells are well known in the art. They have been described, for example, by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual).

[0103] The terms “vector” and “plasmid” are used interchangeably herein and refer to a protein or polynucleotide or a mixture thereof that can be introduced into or that can introduce the proteins and / or nucleic acids contained therein into a cell. Examples of plasmids include, but are not limited to, plasmids, phages, viruses, or artificial chromosomes.

[0104] The term "amino acid" generally refers to any monomeric unit comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, mercapto, selenyl, sulfonyl, alkyl, aryl, acyl, ketyl, azide, hydroxyl, hydrazine, cyano, halogenated, acylhydrazine, alkenyl, alkynyl, ether, borate ester, borate, dioxophosphoryl, phosphonyl, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids containing photoactivated crosslinking agents, metal-bound amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids containing metals, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, amino acids modified with other carbohydrates, amino acids containing polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids containing carbon-linked sugars, redox-active amino acids, amino acids containing aminothioic acids, and amino acids containing one or more toxic moieties. As used herein, the term “amino acid” includes the following twenty naturally or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0105] The terms “measurement,” “measuring,” “detecting,” or “detection” preferably include qualitative, semi-quantitative, or quantitative measurements. The term “detection of presence” is a descriptive measurement indicating the presence or absence of something without making any statement about the quantity (e.g., yes or no statement). The term “detection of quantity” refers to a quantitative measurement where an absolute number (ng) is detected. The term “detection of concentration” refers to a quantitative measurement where the quantity is determined based on a given volume (e.g., ng / mL).

[0106] As used herein, the term "immunoglobulin (Ig)" refers to the glycoproteins of the immunoglobulin superfamily that confer immunity. "Surface immunoglobulins" attach to the membrane of effector cells via their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, major histocompatibility complex (MHC) proteins of classes I and II, β-2 microglobulin (approximately 2M), CD3, CD4, and CDS.

[0107] Generally, as used herein, the term "antibody" refers to a secretory immunoglobulin that lacks a transmembrane region and is therefore released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, represented by the Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibody, i.e., these chains are present in IgA, IgD, IgE, IgG, and IgM antibodies, each playing a different role and guiding an appropriate immune response against different types of antigens. Different heavy chains vary in size and composition; and can comprise approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is present in mucosal regions (such as the digestive, respiratory, and urogenital tracts) as well as in saliva, tears, and breast milk, and prevents pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4: 389-417). IgD primarily functions as an antigen receptor on B cells unexposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE participates in allergic reactions by triggering the release of histamine from mast cells and basophils through its binding to allergens. IgE is also involved in parasite protection (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype capable of crossing the placenta to provide passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). There are four distinct IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum, with IgG1 being the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG (approximately 4%). The biological characteristics of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in both monomeric and secretory pentamer forms, exhibiting very high affinity. Before sufficient IgG is produced, IgM participates in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity (Geisberger et al. (2006) Immunology 118:429-437).Antibodies exist not only as monomers but also as dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM in teleosts), or pentamers of five Ig units (e.g., IgM in mammals). Antibodies typically consist of four polypeptide chains, comprising two identical heavy chains and two identical light chains linked by disulfide bonds and resembling a "Y"-shaped macromolecule. Each chain includes numerous immunoglobulin domains, some of which are constant domains and others are variable domains. An immunoglobulin domain consists of a two-layer sandwich structure of 7 to 9 antiparallel Ig chains arranged in two lamellae. Typically, the heavy chain of an antibody includes four Ig domains, three of which are constant domains (CH domains: CHI, CH2, CH3) and one is a variable domain (VH). The light chain typically includes one constant Ig domain (CL) and one variable Ig domain (VL). For example, the human IgG heavy chain consists of four Ig domains linked from the N-terminus to the C-terminus in the sequence VwCH1-CH2-CH3 (also known as VwCy1-Cy2-Cy3), while the human IgG light chain consists of two immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VL-CL, which are either κ- or α-type (VK-CK or VA.-CA.). For example, the constant chain of human IgG comprises 447 amino acids. Throughout this specification and claims, the amino acid positions in the immunoglobulins are numbered according to the “EU index”, see Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th edition. Department of Health and Human Service, National Institutes of Health, Bethesda, MD. “See Kabat’s EU index” refers to the residue number of the human IgG 1EU antibody. Therefore, the CH domain in the IgG context is as follows: "CH1" refers to amino acid positions 118-220 according to the EU index of Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index of Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index of Kabat.

[0108] The term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (Kd). Affinity can be measured by methods commonly known in the art, including, but not limited to: surface plasmon resonance-based assays (such as the BIAcore assay described in PCT application publication WO2005 / 012359); enzyme-linked immunosorbent assays (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind antigens rapidly and tend to maintain a longer binding time. Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of this invention.

[0109] The term "antigen (Ag)" is a molecule or molecular structure that binds to an antigen-specific antibody (Ab) or a B-cell antigen receptor (BCR). The presence of an antigen in the body typically triggers an immune response. In the body, when cells of the immune system come into contact with an antigen, they specifically produce each antibody to match the antigen; this allows for precise recognition or matching of the antigen and initiation of a customized response. In most cases, an antibody reacts and binds to only one specific antigen; however, in some cases, an antibody may cross-react and bind to more than one antigen. Antigens are typically proteins, peptides (amino acid chains), and polysaccharides (monosaccharide / simple sugar chains) or combinations thereof.

[0110] In diagnostic testing, antigens are commonly used in serological tests to assess whether a patient has been exposed to a pathogen (such as a virus or bacteria) and has developed antibodies against that pathogen. These antigens are typically recombinant and can be linear peptides designed to represent natural antigens or more complex folded molecules.

[0111] To more closely approximate natural antigens and achieve high epitope density, monomeric antigens can be polymerized via chemical cross-linking to generate antigens. A large number of homo- and hetero-bifunctional cross-linking agents are readily available and well-known in the art. However, there are some serious drawbacks to using chemically induced antigen polymerization as a designator in serological assays. For example, partially inserting the cross-linking agent into the antigen can impair antigenicity by interfering with native-like conformations or masking key epitopes. Furthermore, the introduction of non-natural tertiary contacts can interfere with the reversibility of protein folding / unfolding and may be a source of interference problems that must be overcome through anti-interference strategies in the immunoassay mixture.

[0112] A newer technique involves fusing the antigen of interest with an oligochaete protein to deliver a high epitope density to the antigen. The advantages of this technique lie in its high reproducibility and the triple function of the oligochaete protein fusion coupler: first, the chaete protein enhances the expression rate of the fusion peptide in host cells (e.g., *E. coli*); second, the chaete protein promotes the refolding process of the target antigen and improves its overall solubility; and third, it can repeatedly assemble the target antigen into an ordered oligomeric structure.

[0113] The term "chaperone protein" is well known in the art and refers to a protein folding aid that helps in folding and maintaining the structural integrity of other proteins. Examples of folding aids are described in detail in WO 03 / 000877. For example, chaperone proteins of the peptidyl prolyl isomerase class, such as those of the FKBP family, can be used for fusion with antigenic variants. Examples of suitable FKBP chaperone proteins as fusion partners are FkpA (aa 26-270, UniProt ID P45523), SlyD (1-165, UniProt ID P0A9K9), and SlpA (2-149, UniProt ID P0AEM0). Another suitable chaperone protein as a fusion partner is Skp (21-161, UniProt ID P0AEU7), a trimeric chaperone protein from the periplasm of *E. coli*, which does not belong to the FKBP family. The complete sequence of the chaperone protein is not always required. Functional fragments of chaperone proteins that still possess the desired capabilities and functions (so-called binding capacity modules) can also be used (see WO 98 / 13496).

[0114] Antigens may further include “effective groups,” such as a “tag” or “label.” The term “tag” refers to those effector groups that provide an antigen with the ability to bind to or be bound by other molecules. Examples of tags include, but are not limited to, His tags, which are attached to an antigen sequence to allow for its purification. Tags may also include a partner of a bioaffinity binding pair, which allows the antigen to be bound by a second partner of the binding pair. The term “bioaffinity binding pair” refers to two partner molecules (i.e., two partners in a pair) that have a strong affinity for each other. Examples of partners of bioaffinity binding pairs are a) biotin or biotin analogs / avidin or streptoavidin; b) haptens / anti-haptens antibodies or antibody fragments (e.g., digoxigenin / anti-digoxigenin antibody); c) glycosides / lectins; d) complementary oligonucleotide sequences (e.g., complementary LNA sequences); and generally e) ligands / receptors.

[0115] The term "label" refers to those effector groups that allow the detection of antigens. Labels include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, or chemical labels. Exemplary suitable labels include fluorescent dyes, luminescent or electrochemiluminescent complexes (e.g., ruthenium or iridium complexes), electron-dense reagents, and enzyme labels.

[0116] As used herein, "particle" refers to a small, localized object to which physical properties such as volume, mass, or average size can be attributed. Particles can therefore be symmetrical, spherical, substantially spherical, or spherical, or irregular, asymmetrical in shape or form. Particle size can vary. The term "microparticle" refers to particles with diameters in the nanometer and micrometer range.

[0117] The microparticles as defined above may comprise or be composed of any suitable material known to those skilled in the art; for example, they may comprise or be composed of inorganic or organic materials, or substantially of them. Typically, they may comprise metals or metal alloys, or organic materials, or comprise or be composed of carbohydrate elements, or comprise or be composed of them. Examples of envisioned materials for microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silica, and ferromagnetic metals, alloys, or composites. In one embodiment, the microparticles are magnetic or ferromagnetic metals, alloys, or compositions. In another embodiment, the material may have specific properties, such as being hydrophobic or hydrophilic. Such microparticles are typically dispersed in aqueous solutions and retain a small negative surface charge, thereby keeping the microparticles separate and preventing nonspecific aggregation.

[0118] In one embodiment of the invention, the microparticles are paramagnetic microparticles, and in the measurement method according to this disclosure, the separation of such microparticles is facilitated by magnetic force. A magnetic force is applied to pull the paramagnetic or magnetic particles from the solution / suspension and retain them as needed, while the liquid in the solution / suspension can be removed and the particles can be washed, for example.

[0119] A “kit” is any article (e.g., package or container) containing at least one reagent of the present invention, such as a pharmaceutical product for treating a condition or a probe for specifically detecting a biomarker gene or protein. Kits are preferably marketed, distributed, or sold as a unit for performing the methods of the present invention. Typically, a kit may further include separate carriers to tightly accommodate one or more container devices such as vials, tubes, etc., particularly each container device containing one of the independent elements used in the methods of the first aspect. Kits may further include one or more other containers containing other materials, including but not limited to buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. Labels may be present on the containers to indicate the use of the composition for a specific application and may also indicate guidelines for in vivo or in vitro use. Computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., an optical disc) or directly on a computer or data processing device. Furthermore, kits may contain standard amounts of the biomarker for calibration purposes, as described elsewhere herein.

[0120] "Packaging insert" refers to the instruction leaflet typically included in the commercial packaging of a therapeutic product or medicine, which contains information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be used in conjunction with the packaged product, and / or warnings related to the use of such therapeutic products or medicines.

[0121] Example

[0122] Currently available ELISA-based immunoassays for detecting antibodies against SARS-CoV-2 in patient samples use spike protein-derived antigens as the immunoreaction reagent. However, we have found that these assays lack specificity, leading to a considerable number of false positive results. Surprisingly, by limiting the antigen to the coronavirus nucleocapsid, as explained further below, the number of erroneous samples can be significantly reduced while maintaining the high sensitivity of the assay.

[0123] Furthermore, all current vaccination strategies focus on the development of spike protein-based vaccines. Detecting anti-SARS-CoV-2 antibodies in samples from vaccinated patients using spike protein-derived antigens can determine whether vaccination was successful and whether patients have developed anti-spike protein antibodies. However, because the long-term effects of vaccination and natural SARS-CoV-2 infection are unclear and will affect patients, it is important to be able to distinguish between patients exposed to natural SARS-CoV-2 infection and those who have previously been vaccinated. Therefore, there is an urgent need for an anti-SARS-CoV-2 antibody assay that can detect not only spike protein antibodies but also antibodies against other viral proteins.

[0124] Therefore, in a first aspect, the present invention thus relates to a coronavirus antigen suitable for detecting anti-coronavirus antibodies in isolated biological samples, the coronavirus antigen comprising a coronavirus nucleocapsid-specific amino acid sequence or a variant thereof according to SEQ ID NO: 1. In embodiments, the coronavirus antigen detects anti-coronavirus antibodies in isolated biological samples, the coronavirus antigen comprising a coronavirus nucleocapsid-specific amino acid sequence or a variant thereof according to SEQ ID NO: 1.

[0125] In the examples, the antigen does not contain other coronavirus-specific amino acid sequences.

[0126] In this embodiment, the coronavirus antigen is immunoreactive, meaning that antibodies present in the biological sample bind to the antigen. Therefore, any peptides derived from the coronavirus nucleocapsid that are not bound by antibodies are excluded.

[0127] like Figure 1 and Figure 2 As shown, the amino acid sequence of SARS-CoV-2 exhibits approximately 93% sequence homology and approximately 90% sequence identity with its closest relative, SARS-CoV. As shown, the sequence identity and homology with other coronaviruses remain much lower. Therefore, due to limited sequence identity and homology, coronavirus antigens containing the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 are specific for the detection of SARS-CoV and SARS-CoV-2.

[0128] In the embodiments, the coronavirus is SARS-CoV or SARS-CoV-2 virus, particularly SARS-CoV-2 virus. In a specific embodiment, the coronavirus nucleocapsid is a SARS-CoV-2 specific nucleocapsid. In particular, the coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 is specific for the detection of SARS-CoV-2.

[0129] In the embodiments, for antibodies or subsets of antibodies generated against corresponding nucleocapsid antigens of other coronaviruses, the coronavirus antigen does not exhibit immune cross-reactivity, i.e., it only shows strongly reduced or completely eliminated immunoreactivity. Specifically, the coronavirus antigen does not exhibit immune cross-reactivity with corresponding nucleocapsid antigens of coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. Specifically, the coronavirus antigen does not exhibit immune cross-reactivity with corresponding nucleocapsid antigens of coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0130] In this embodiment, the coronavirus antigen is soluble. Therefore, the coronavirus antigen is suitable for in vitro assays aimed at detecting antibodies against the antigen in isolated biological samples.

[0131] Therefore, the coronavirus antigen is suitable for in vitro assays aimed at detecting anti-coronavirus antibodies with high sensitivity and specificity. In examples, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific examples, the sensitivity is >99% or >99.5%. In specific examples, the sensitivity is 100%. In examples, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific examples, the specificity is >99% or >99.5%. In specific examples, the specificity is 99.8%. In specific examples, the sensitivity is 100% and the specificity is 99.8%.

[0132] In some embodiments, the coronavirus antigen is suitable for detecting or testing antibodies against the coronavirus in fluid samples. In some embodiments, the sample is a human sample, particularly a human fluid sample. In some embodiments, the sample is a human blood or urine sample. In some embodiments, the sample is a human whole blood, plasma, or serum sample.

[0133] In the embodiments, the coronavirus antigen is a linear antigen or its native state. In a particular embodiment, the coronavirus nucleocapsid-specific amino acid sequence contained in the coronavirus antigen is folded in its native state.

[0134] In the embodiments, variants of the coronal nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 are included. These variants are readily generated by those skilled in the art through conserved or homologous substitutions of the disclosed amino acid sequence (e.g., substitution of cysteine ​​for alanine or serine). In the embodiments, the variants exhibit modifications to their amino acid sequences, particularly selected from the group consisting of amino acid exchanges, deletions, or insertions compared to the amino acid sequence of SEQ ID NO: 1.

[0135] In the embodiments, 1 to 10 amino acids are deleted or inserted at one or both ends of the C-terminus or N-terminus, and in one embodiment, 1 to 5 amino acids are inserted. In particular, the variant may be an isotype exhibiting the most common protein isotype. In one embodiment, such a substantially similar protein has at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence homology with SEQ ID NO: 1.

[0136] In the embodiments, the coronal nucleocapsid variant comprises an amino acid sequence according to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

[0137] In the embodiments, the variants include post-translational modifications, particularly selected from the group consisting of glycosylation or phosphorylation.

[0138] It should be understood that such variants are classified as coronavirus nucleocapsid variants, meaning they are capable of binding to and detecting anti-coronavirus antibodies present in isolated samples.

[0139] In the embodiments, the overall three-dimensional structure of the coronal nucleocapsid remains unchanged, so epitopes that were previously (i.e., in the wild type) available for antibody binding remain accessible in the variant.

[0140] In the embodiments, the coronavirus antigen further comprises at least one chaperone protein. Therefore, the coronavirus antigen comprises the coronavirus nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 as described above or below, and the amino acid sequence of the chaperone protein.

[0141] In a particular embodiment, the coronavirus antigen comprises two chaperone proteins. In this embodiment, the chaperone proteins are selected from the group consisting of SlyD, SlpA, FkpA, and Skp. In a particular embodiment, the chaperone protein is SlyD, specifically having the amino acid sequence given in UniProt ID P0A9K9.

[0142] In a specific embodiment, the coronavirus antigen comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and a SlyD chaperone protein. In a specific embodiment, the coronavirus antigen comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and two SlyD chaperone proteins.

[0143] The fusion of the two chaperone proteins results in higher solubility of the resulting antigen.

[0144] In embodiments, the chaperone protein is fused to a coronal nucleocapsid-specific amino acid sequence at the N- and / or C-terminus of the nucleocapsid, particularly at the N-terminus. Therefore, in certain embodiments, the coronal antigen comprises one SlyD chaperone protein at the N-terminus of the coronal nucleocapsid-specific amino acid sequence. In certain embodiments, the coronal antigen comprises two SlyD chaperone proteins at the N-terminus of the coronal nucleocapsid-specific amino acid sequence. In embodiments, the coronal antigen comprises one SlyD chaperone protein at the N-terminus of the coronal nucleocapsid-specific amino acid sequence and one SlyD chaperone protein at the C-terminus of the coronal nucleocapsid-specific amino acid sequence.

[0145] In embodiments, the coronavirus antigen further comprises a linker sequence. These sequences are not specific to antibodies against coronaviruses and are not recognized in in vitro diagnostic immunoassays. In particular, the coronavirus antigen comprises a linker sequence between a coronavirus nucleocapsid sequence and one or more chaperone proteins. In some embodiments, the linker is a Gly-rich linker. In some embodiments, the linker has a sequence as shown in SEQ ID NO: 7.

[0146] In one embodiment, the coronavirus antigen comprises the amino acid sequence according to SEQ ID NO: 2. In another embodiment, the coronavirus antigen does not comprise any other amino acid sequence. In a particular embodiment, the coronavirus antigen consists of the amino acid sequence according to SEQ ID NO: 2.

[0147] In one embodiment, the coronavirus antigen comprises the amino acid sequence according to SEQ ID NO: 3. In another embodiment, the coronavirus antigen does not comprise any other amino acid sequence. In a particular embodiment, the coronavirus antigen consists of SEQ ID NO: 3.

[0148] In some embodiments, the coronavirus antigen comprises an amino acid sequence according to SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. In some embodiments, the coronavirus antigen does not comprise any other amino acid sequence. In certain embodiments, the coronavirus antigen is composed of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

[0149] It should be understood that the coronavirus antigen composed of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 does not contain any additional amino acid sequence, but may still contain other chemical molecules, such as markers and / or tags.

[0150] In a specific embodiment, the sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is at least 96%, at least 97%, at least 98%, or at least 99%. In a specific embodiment, the sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is at least 98%.

[0151] In a specific embodiment, the sequence homology with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is at least 96%, at least 97%, at least 98%, or at least 99%. In a specific embodiment, the homology with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is at least 98%.

[0152] In embodiments, the coronavirus antigen further comprises a tag or label. Thus, the coronavirus antigen comprises the coronavirus nucleocapsid-specific amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 as described above or below, and a tag and / or label, and optionally, the amino acid sequence of one or more chaperone proteins.

[0153] In certain embodiments, the tag allows the coronavirus antigen to bind directly or indirectly to a solid phase. In certain embodiments, the tag is a partner of a bioaffinity binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxigenin, haptens, or complementary oligonucleotide sequences (especially complementary LNA sequences). In certain embodiments, the tag is biotin.

[0154] In certain embodiments, the labeling allows for the detection of coronavirus antigens. In certain embodiments, a coronavirus-specific nucleocapsid sequence is labeled. In embodiments where at least one chaperone protein is present in the antigen, the coronavirus-specific nucleocapsid sequence is labeled, or at least one chaperone protein is labeled, or both are labeled. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen at a stoichiometry of 1:1 to 15:1. In certain embodiments, the stoichiometry is 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0155] In a second aspect, the present invention relates to a composition comprising a coronavirus antigen suitable for detecting antibodies against coronaviruses in isolated biological samples, comprising a coronavirus nucleocapsid-specific amino acid sequence or a variant thereof according to SEQ ID NO: 1. In embodiments, the coronavirus antigen detects antibodies against coronaviruses in isolated biological samples, the coronavirus antigen comprising a coronavirus nucleocapsid-specific amino acid sequence or a variant thereof according to SEQ ID NO: 1.

[0156] In the embodiments, the coronavirus antigen does not contain other coronavirus-specific amino acid sequences.

[0157] In this embodiment, the coronavirus antigen is immunoreactive, meaning that antibodies present in the biological sample bind to the antigen. Therefore, any peptides derived from the coronavirus nucleocapsid that are not bound by antibodies are excluded.

[0158] like Figure 1 and Figure 2 As shown, the amino acid sequence of SARS-CoV-2 exhibits approximately 93% sequence homology and approximately 90% sequence identity with its closest relative, SARS-CoV. As shown, the sequence identity and homology with other coronaviruses remain much lower. Therefore, due to limited sequence identity and homology, coronavirus antigens containing the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 are specific for the detection of SARS-CoV and SARS-CoV-2.

[0159] In the embodiments, the coronavirus is SARS-CoV or SARS-CoV-2 virus, particularly SARS-CoV-2 virus. In a specific embodiment, the coronavirus nucleocapsid is a SARS-CoV-2 specific nucleocapsid. In particular, the coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 is specific for the detection of SARS-CoV-2.

[0160] In the embodiments, the coronavirus antigen does not exhibit immune cross-reactivity with antibodies or subsets of antibodies generated against corresponding nucleocapsid antigens of other coronaviruses, i.e., it only shows strongly reduced or completely eliminated immunoreactivity. Specifically, the coronavirus antigen does not exhibit immune cross-reactivity with corresponding nucleocapsid antigens of coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. Specifically, the coronavirus antigen does not exhibit immune cross-reactivity with corresponding nucleocapsid antigens of coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0161] In this embodiment, the coronavirus antigen is soluble. Therefore, the coronavirus antigen is suitable for in vitro assays aimed at detecting antibodies against the antigen in isolated biological samples.

[0162] Therefore, the coronavirus antigen is suitable for in vitro assays aimed at detecting anti-coronavirus antibodies with high sensitivity and specificity. In examples, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific examples, the sensitivity is >99% or >99.5%. In specific examples, the sensitivity is 100%. In examples, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific examples, the specificity is >99% or >99.5%. In specific examples, the specificity is 99.8%. In specific examples, the sensitivity is 100% and the specificity is 99.8%.

[0163] In some embodiments, the coronavirus antigen is suitable for detecting or testing antibodies against the coronavirus in fluid samples. In some embodiments, the sample is a human sample, particularly a human fluid sample. In some embodiments, the sample is a human blood or urine sample. In some embodiments, the sample is a human whole blood, plasma, or serum sample.

[0164] In the embodiments, the coronavirus antigen is a linear antigen or its native state. In a particular embodiment, the coronavirus nucleocapsid-specific amino acid sequence contained in the coronavirus antigen is folded in its native state.

[0165] In the embodiments, variants of the coronal nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 are included. These variants can be readily generated by those skilled in the art through conserved or homologous substitutions of the disclosed amino acid sequence (e.g., substitution of cysteine ​​for alanine or serine). In the embodiments, the variants exhibit modifications to their amino acid sequences, particularly selected from the group consisting of amino acid exchanges, deletions, or insertions compared to the amino acid sequence of SEQ ID NO: 1.

[0166] In the embodiments, 1 to 10 amino acids are deleted or inserted at one or both ends of the C-terminus or N-terminus, and in one embodiment, 1 to 5 amino acids are inserted. In particular, the variant may be an isotype exhibiting the most common protein isotype. In one embodiment, such a substantially similar protein has at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence homology with SEQ ID NO: 1.

[0167] In the embodiments, the coronal nucleocapsid variant comprises an amino acid sequence according to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

[0168] In the embodiments, the variants include post-translational modifications, particularly selected from the group consisting of glycosylation or phosphorylation.

[0169] It should be understood that such variants are classified as coronavirus nucleocapsid variants, meaning they are capable of binding to and detecting anti-coronavirus antibodies present in isolated samples.

[0170] In the embodiments, the overall three-dimensional structure of the coronal nucleocapsid remains unchanged, so epitopes that were previously (i.e., in the wild type) available for antibody binding remain accessible in the variant.

[0171] In the embodiments, the coronavirus antigen further comprises at least one chaperone protein. Therefore, the coronavirus antigen comprises the coronavirus nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 as described above or below, and the amino acid sequence of the chaperone protein.

[0172] In a particular embodiment, the coronavirus antigen comprises two chaperone proteins. In this embodiment, the chaperone proteins are selected from the group consisting of SlyD, SlpA, FkpA, and Skp. In a particular embodiment, the chaperone protein is SlyD, specifically having the amino acid sequence given in UniProt ID P0A9K9.

[0173] In a specific embodiment, the coronavirus antigen comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and a SlyD chaperone protein. In a specific embodiment, the coronavirus antigen comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and two SlyD chaperone proteins.

[0174] The fusion of the two chaperone proteins results in higher solubility of the resulting antigen.

[0175] In embodiments, the chaperone protein is fused to a coronal nucleocapsid-specific amino acid sequence at the N- and / or C-terminus of the nucleocapsid, particularly at the N-terminus. Therefore, in certain embodiments, the coronal antigen comprises one SlyD chaperone protein at the N-terminus of the coronal nucleocapsid-specific amino acid sequence. In certain embodiments, the coronal antigen comprises two SlyD chaperone proteins at the N-terminus of the coronal nucleocapsid-specific amino acid sequence. In embodiments, the coronal antigen comprises one SlyD chaperone protein at the N-terminus of the coronal nucleocapsid-specific amino acid sequence and one SlyD chaperone protein at the C-terminus of the coronal nucleocapsid-specific amino acid sequence.

[0176] In embodiments, the coronavirus antigen further comprises a linker sequence. These sequences are not specific to antibodies against coronaviruses and are not recognized in in vitro diagnostic immunoassays. Specifically, the coronavirus antigen comprises a linker sequence between a coronavirus nucleocapsid sequence and one or more chaperone proteins. In some embodiments, the linker is a Gly-rich linker. In some embodiments, the linker has a sequence as shown in SEQ ID NO: 7.

[0177] In one embodiment, the coronavirus antigen comprises the amino acid sequence according to SEQ ID NO: 2. In another embodiment, the coronavirus antigen does not comprise any other amino acid sequence. In a particular embodiment, the coronavirus antigen consists of the amino acid sequence according to SEQ ID NO: 2.

[0178] In one embodiment, the coronavirus antigen comprises the amino acid sequence according to SEQ ID NO: 3. In another embodiment, the coronavirus antigen does not comprise any other amino acid sequence. In a particular embodiment, the coronavirus antigen consists of SEQ ID NO: 3.

[0179] In some embodiments, the coronavirus antigen comprises an amino acid sequence according to SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. In some embodiments, the coronavirus antigen does not comprise any other amino acid sequence. In certain embodiments, the coronavirus antigen is composed of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

[0180] It should be understood that the coronavirus antigen composed of SEQ ID NO:2 or SEQ ID NO:3 does not contain any additional amino acid sequence, but may still contain other chemical molecules, such as markers and / or tags.

[0181] In a specific embodiment, the sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is at least 96%, at least 97%, at least 98%, or at least 99%. In a specific embodiment, the sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is at least 98%.

[0182] In a specific embodiment, the sequence homology with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is at least 96%, at least 97%, at least 98%, or at least 99%. In a specific embodiment, the homology with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is at least 98%.

[0183] In embodiments, the coronavirus antigen further comprises a tag or label. In certain embodiments, a coronavirus-specific nucleocapsid sequence is tagged. In embodiments where at least one chaperone protein is present in the antigen, either the coronavirus-specific nucleocapsid sequence is tagged or at least one chaperone protein is tagged, or both are tagged.

[0184] Therefore, the coronavirus antigen comprises the coronavirus nucleocapsid-specific amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 as described above or below, and a tag and / or label, and optionally the amino acid sequence of one or more chaperone proteins.

[0185] In certain embodiments, the tag allows the antigen to bind directly or indirectly to a solid phase. In certain embodiments, the tag is a partner of a bioaffinity binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxigenin, haptens, or complementary oligonucleotide sequences (especially complementary LNA sequences). In certain embodiments, the tag is biotin.

[0186] In certain embodiments, the label allows for the detection of the antigen. In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in a stoichiometric ratio of 1:1 to 15:1. In certain embodiments, the stoichiometry is 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0187] In some embodiments, the composition comprises one or more additional coronavirus antigens. In some specific embodiments, the composition comprises one, two, or three additional antigens. In some specific embodiments, the composition comprises one or more additional coronavirus antigens, which contain amino acid sequences of the E protein, M protein, and / or S protein or portions thereof. In some specific embodiments, the composition comprises additional coronavirus antigens, which contain amino acid sequences of the S protein or portions thereof (e.g., the receptor-binding domain of the S protein).

[0188] In certain embodiments, the additional coronavirus antigen is immunoreactive, meaning that antibodies present in the biological sample bind to the antigen. Therefore, any coronavirus-derived peptides that are not bound by anti-coronavirus antibodies are excluded.

[0189] In the embodiments, for antibodies or subsets of antibodies generated against corresponding antigens of other coronaviruses, the coronavirus antigens do not exhibit immune cross-reactivity, i.e., they only show strongly reduced or completely eliminated immunoreactivity. Specifically, other coronavirus antigens do not exhibit immune cross-reactivity with corresponding antigens of coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. In particular, other coronavirus antigens do not exhibit immune cross-reactivity with corresponding antigens of coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0190] In this embodiment, the additional coronavirus antigen is soluble. Therefore, this antigen is suitable for in vitro assays designed to detect antibodies against said antigen in isolated biological samples.

[0191] In a third aspect, the present invention relates to a method for producing a coronavirus antigen specific to the nucleocapsid of a coronavirus, the method comprising the following steps:

[0192] a) Culture host cells transformed with an expression vector containing a recombinant DNA molecule operatively linked to the coronavirus antigen as described above for the first aspect of the invention.

[0193] b) Expressing the polypeptide, and

[0194] c) Purify the polypeptide.

[0195] Optionally, as an additional step d), functional dissolution is required to bring the coronal nucleocapsid antigen into a soluble and immunoreactive conformation using refolding techniques known in the art.

[0196] In a specific embodiment, the host cell is an *E. coli* cell, a CHO cell, or a HEK cell. In a specific embodiment, the host cell is an *E. coli* cell.

[0197] In embodiments where the antigen comprises a coronavirus nucleocapsid and one or more chaperone proteins, the recombinant DNA molecule according to the invention may further comprise a sequence encoding a linker peptide having 5 to 100 amino acid residues between the coronavirus antigens. Such a linker sequence may, for example, have a proteolytic cleavage site. In one embodiment, it is possible to add a non-coronavirus-specific linker or peptide fusion amino acid sequence to the coronavirus nucleocapsid because these sequences are not specific to antibodies against coronaviruses and will not be recognized in in vitro diagnostic immunoassays.

[0198] In a particular embodiment, the recombinant DNA molecule comprises the sequence according to SEQ ID NO: 4.

[0199] In a particular embodiment, the recombinant DNA molecule comprises the sequence according to SEQ ID NO: 5.

[0200] In a particular embodiment, the recombinant DNA molecule comprises the sequence according to SEQ ID NO: 6.

[0201] In a fourth aspect, the present invention relates to a method for detecting antibodies specific to coronaviruses in isolated biological samples, wherein a coronavirus antigen according to the first aspect of the invention, a composition according to the second aspect of the invention, or a coronavirus antigen obtained by the method according to the third aspect of the invention is used as a capture reagent and / or binding partner of the anti-coronavirus antibody.

[0202] In a fifth aspect, the present invention relates to a method for detecting antibodies specific to coronaviruses in isolated biological samples, the method comprising:

[0203] a) An immune reaction mixture is formed by mixing isolated biological samples with coronavirus antigens or compositions containing coronavirus antigens.

[0204] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the isolated biological sample to react with the coronavirus antigen to form an immune reaction product; and

[0205] c) Detect the presence, amount and / or concentration of any of the said immune response products.

[0206] In some embodiments, the method is an in vitro method. In embodiments, the method exhibits high sensitivity and specificity. In embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific embodiments, the sensitivity is >99% or >99.5%. In specific embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific embodiments, the specificity is >99% or >99.5%. In specific embodiments, the specificity is 99.8%. In specific embodiments, the sensitivity is 100% and the specificity is 99.8%.

[0207] In the embodiments, the antibodies detected by the method of the present invention are anti-coronavirus antibodies of IgG, IgM or IgA subclass or all three subclasses in the same immunoassay.

[0208] In the embodiments, the detected antibodies target the nucleocapsid of coronaviruses, particularly antibodies targeting the nucleocapsid of SARS-CoV or SARS-CoV-2 viruses. In a specific embodiment, the detected antibodies target the nucleocapsid of the SARS-CoV-2 virus.

[0209] In some embodiments, the biological sample from which the isolated coronavirus-specific antibodies are detected is a human sample, particularly a human fluid sample. In certain embodiments, the sample is a human blood or urine sample. In certain embodiments, the sample is a human whole blood, plasma, or serum sample. In certain embodiments, the sample is a venous or capillary human whole blood, plasma, or serum sample.

[0210] In this embodiment, the coronavirus antigen mixed with the isolated biological sample in step a) comprises the coronavirus nucleocapsid-specific amino acid sequence or a variant thereof according to SEQ ID NO: 1. In this embodiment, the coronavirus antigen does not contain other coronavirus-specific amino acid sequences.

[0211] In this embodiment, the coronavirus antigen is immunoreactive, meaning that antibodies present in the biological sample bind to the antigen. Therefore, any peptides derived from the coronavirus nucleocapsid that are not bound by antibodies are excluded.

[0212] like Figure 1 and Figure 2As shown, the amino acid sequence of SARS-CoV-2 exhibits approximately 93% sequence homology and approximately 90% sequence identity with its closest relative, SARS-CoV. As shown, the sequence identity and homology with other coronaviruses remain much lower. Therefore, due to limited sequence identity and homology, coronavirus antigens containing the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 are specific for the detection of SARS-CoV and SARS-CoV-2.

[0213] In the embodiments, the coronavirus is SARS-CoV or SARS-CoV-2 virus, particularly SARS-CoV-2 virus. In a specific embodiment, the coronavirus nucleocapsid is a SARS-CoV-2 specific nucleocapsid. In particular, the coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 is specific for the detection of SARS-CoV-2.

[0214] In the embodiments, the coronavirus antigen does not exhibit immune cross-reactivity with antibodies or subsets of antibodies generated against corresponding nucleocapsid antigens of other coronaviruses, i.e., it only shows strongly reduced or completely eliminated immunoreactivity. Specifically, the coronavirus antigen does not exhibit immune cross-reactivity with corresponding nucleocapsid antigens of coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. Specifically, the coronavirus antigen does not exhibit immune cross-reactivity with corresponding nucleocapsid antigens of coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0215] In this embodiment, the coronavirus antigen is soluble. Therefore, the coronavirus antigen is suitable for in vitro assays aimed at detecting antibodies against the antigen in isolated biological samples.

[0216] Therefore, the coronavirus antigen is suitable for in vitro assays aimed at detecting anti-coronavirus antibodies with high sensitivity and specificity. In examples, the sensitivity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific examples, the sensitivity is >99% or >99.5%. In specific examples, the sensitivity is 100%. In examples, the specificity is >95%, >96%, >97%, >98%, >99%, >99.5%. In specific examples, the specificity is >99% or >99.5%. In specific examples, the specificity is 99.8%. In specific examples, the sensitivity is 100% and the specificity is 99.8%.

[0217] In this embodiment, the coronavirus antigen is soluble. Therefore, this antigen is suitable for use in vitro methods.

[0218] In the embodiments, the coronavirus antigen is a linear antigen or its native state. In a particular embodiment, the coronavirus nucleocapsid-specific amino acid sequence contained in the antigen is folded in its native state.

[0219] In the embodiments, variants of the coronal nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 are included. These variants can be readily generated by those skilled in the art through conserved or homologous substitutions of the disclosed amino acid sequence (e.g., substitution of cysteine ​​for alanine or serine). In the embodiments, the variants exhibit modifications to their amino acid sequences, particularly selected from the group consisting of amino acid exchanges, deletions, or insertions compared to the amino acid sequence of SEQ ID NO: 1.

[0220] In the embodiments, 1 to 10 amino acids are deleted or inserted at one or both ends of the C-terminus or N-terminus, and in one embodiment, 1 to 5 amino acids are inserted. In particular, the variant may be an isotype exhibiting the most common protein isotype. In one embodiment, such a substantially similar protein has at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence homology with SEQ ID NO: 1.

[0221] In the embodiments, the coronal nucleocapsid variant comprises an amino acid sequence according to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

[0222] In the embodiments, the variants include post-translational modifications, particularly selected from the group consisting of glycosylation or phosphorylation.

[0223] It should be understood that such variants are classified as coronavirus nucleocapsid variants, meaning they are capable of binding to and detecting anti-coronavirus antibodies present in isolated samples.

[0224] In the embodiments, the overall three-dimensional structure of the coronal nucleocapsid remains unchanged, so epitopes that were previously (i.e., in the wild type) available for antibody binding remain accessible in the variant.

[0225] In the embodiments, the coronavirus antigen further comprises at least one chaperone protein. Therefore, the coronavirus antigen comprises the coronavirus nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 as described above or below, and the amino acid sequence of the chaperone protein.

[0226] In a particular embodiment, the coronavirus antigen comprises two chaperone proteins. In this embodiment, the chaperone proteins are selected from the group consisting of SlyD, SlpA, FkpA, and Skp. In a particular embodiment, the chaperone protein is SlyD, specifically having the amino acid sequence given in UniProt ID P0A9K9.

[0227] In a specific embodiment, the coronavirus antigen comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and a SlyD chaperone protein. In a specific embodiment, the coronavirus antigen comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and two SlyD chaperone proteins.

[0228] The fusion of the two chaperone proteins results in higher solubility of the resulting antigen.

[0229] In embodiments, the chaperone protein is fused to a coronal nucleocapsid-specific amino acid sequence at the N- and / or C-terminus of the nucleocapsid, particularly at the N-terminus. Therefore, in certain embodiments, the coronal antigen comprises one SlyD chaperone protein at the N-terminus of the coronal nucleocapsid-specific amino acid sequence. In certain embodiments, the coronal antigen comprises two SlyD chaperone proteins at the N-terminus of the coronal nucleocapsid-specific amino acid sequence. In embodiments, the coronal antigen comprises one SlyD chaperone protein at the N-terminus of the coronal nucleocapsid-specific amino acid sequence and one SlyD chaperone protein at the C-terminus of the coronal nucleocapsid-specific amino acid sequence.

[0230] In embodiments, the coronavirus antigen further comprises a linker sequence. These sequences are not specific to antibodies against coronaviruses and are not recognized in in vitro diagnostic immunoassays. Specifically, the coronavirus antigen comprises a linker sequence between a coronavirus nucleocapsid sequence and one or more chaperone proteins. In some embodiments, the linker is a Gly-rich linker. In some embodiments, the linker has a sequence as shown in SEQ ID NO: 7.

[0231] In one embodiment, the coronavirus antigen comprises the amino acid sequence according to SEQ ID NO: 2. In another embodiment, the coronavirus antigen does not comprise any other amino acid sequence. In a particular embodiment, the coronavirus antigen consists of the amino acid sequence according to SEQ ID NO: 2.

[0232] In one embodiment, the coronavirus antigen comprises the amino acid sequence according to SEQ ID NO: 3. In another embodiment, the coronavirus antigen does not comprise any other amino acid sequence. In a particular embodiment, the coronavirus antigen consists of SEQ ID NO: 3.

[0233] In some embodiments, the coronavirus antigen comprises an amino acid sequence according to SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. In some embodiments, the coronavirus antigen does not comprise any other amino acid sequence. In certain embodiments, the coronavirus antigen is composed of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

[0234] It should be understood that the coronavirus antigen composed of SEQ ID NO:2 or SEQ ID NO:3 does not contain any additional amino acid sequence, but may still contain other chemical molecules, such as markers and / or tags.

[0235] In a specific embodiment, the sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is at least 96%, at least 97%, at least 98%, or at least 99%. In a specific embodiment, the sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is at least 98%.

[0236] In a specific embodiment, the sequence homology with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is at least 96%, at least 97%, at least 98%, or at least 99%. In a specific embodiment, the homology with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 is at least 98%.

[0237] In embodiments, the coronavirus antigen further comprises a tag or label. Thus, the coronavirus antigen comprises the coronavirus nucleocapsid-specific amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 as described above or below, and a tag and / or label, and optionally, the amino acid sequence of one or more chaperone proteins.

[0238] In certain embodiments, the tag allows the antigen to bind directly or indirectly to a solid phase. In certain embodiments, the tag is a partner of a bioaffinity binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxigenin, haptens, or complementary oligonucleotide sequences (especially complementary LNA sequences). In certain embodiments, the tag is biotin.

[0239] In certain embodiments, labeling allows for the detection of coronavirus antigens. In certain embodiments, a coronavirus-specific nucleocapsid sequence is labeled. In embodiments where at least one chaperone protein is present in the antigen, either the coronavirus-specific nucleocapsid sequence is labeled, or at least one chaperone protein is labeled, or both are labeled.

[0240] In certain embodiments, the label is an electrochemiluminescent ruthenium or iridium complex. In certain embodiments, the electrochemiluminescent ruthenium complex is a negatively charged electrochemiluminescent ruthenium complex. In certain embodiments, the label is a negatively charged electrochemiluminescent ruthenium complex present in the antigen in a stoichiometry of 1:1 to 15:1. In certain embodiments, the stoichiometry is 2:1, 2.5:1, 3:1, 5:1, 10:1, or 15:1.

[0241] In embodiments, the method includes the additional step of adding a solid phase to the immunoreaction mixture. In embodiments, the solid phase is a solid-phase extraction (SPE) column or beads. In certain embodiments, the solid phase comprises or is composed of particles. In embodiments, the particles are non-magnetic, magnetic, or paramagnetic. In embodiments, the particles are coated. The coating may vary depending on the intended use (i.e., according to the intended capture molecules). Which coating is suitable for which analyte is well known to those skilled in the art. Beads may be made of a variety of different materials. Beads may have various sizes and include surfaces with or without pores.

[0242] In certain embodiments, the particles are microparticles. In some embodiments, the microparticles have a diameter of 50 nanometers to 20 micrometers. In some embodiments, the microparticles have a diameter between 100 nm and 10 μm. In some embodiments, the microparticles have a diameter of 200 nm to 5 μm, particularly 750 nm to 5 μm, and particularly 750 nm to 2 μm. In certain embodiments, the microparticles are magnetic or paramagnetic. In particular, the microparticles are paramagnetic.

[0243] In the embodiments, the solid phase is added before the sample is added to the antigen or after the formation of the immune reaction mixture. Therefore, the addition of the solid phase can be performed in step a), in step b), or after step b) of the method.

[0244] In the embodiments, the method performed is an immunoassay for detecting anti-coronavirus antibodies in isolated biological samples. Immunoassays for detecting antibodies are well known in the art, as are the methods, practical applications, and procedures for performing such assays. The coronavirus nucleocapsid antigen according to the invention can be used to improve the assay for detecting anti-coronavirus antibodies, regardless of the label used and regardless of the detection modality (e.g., radioisotope assay, enzyme immunoassay, electrochemiluminescence assay, etc.) or assay principle (e.g., test strip assay, sandwich assay, indirect test concept, or homogeneous assay, etc.).

[0245] In this embodiment, the method performed is an immunoassay that detects anti-coronavirus antibodies in a separated sample based on the so-called dual-antigen sandwich concept (DAGS). This assay concept is sometimes also referred to as the dual-antigen bridging concept because it bridges two antigens via an antibody analyte. In such an assay, the ability of an antibody to bind to at least two different molecules of a given antigen and their two (IgG, IgE), four (IgA), or ten (IgM) complementary sites is utilized.

[0246] In an embodiment, an immunoassay for determining anti-coronavirus antibodies according to the DAGS format is performed by incubating a sample containing anti-coronavirus antibodies with two different coronavirus antigens (i.e., a first (“capture”) coronavirus antigen and a second coronavirus (“detect”) antigen), each of which specifically binds to the anti-coronavirus antibody.

[0247] In the embodiments, the structures of the "capture antigen" and the "detection antigen" are immune-cross-reactive. A fundamental requirement for performing this method is the presence of one or more relevant epitopes on both antigens. Therefore, both antigens contain the coronal nucleocapsid-specific amino acid sequence as described above or below. In the embodiments, the two antigens contain the same or different fusion moieties (e.g., SlyD fused to the coronal nucleocapsid-specific antigen labeled as being immobilized, and FkpA fused to the coronal nucleocapsid-specific antigen to be detected), because such variations significantly mitigate the problem of non-specific binding, thereby reducing the risk of false positive results.

[0248] In embodiments, the first antigen may bind directly or indirectly to a solid phase and typically carries an effector group, which is part of a bioaffinity binding pair. In specific embodiments, the first antigen is conjugated with biotin, and the complementary solid phase is coated with avidin or streptoavidin. In embodiments, the second antigen carries a label that confers specific detectability to the antigen molecule, either alone or in complex with other molecules. In a particular embodiment, the second antigen is labeled with a ruthenium complex.

[0249] Therefore, in step b) of this method, an immune reaction mixture comprising a first antigen, a sample antibody, and a second antigen is formed.

[0250] This ternary complex, consisting of an analyte antibody sandwiched between two antigen molecules, is called an immune complex or immune reaction product.

[0251] In an embodiment, the method may include an additional step of separating the liquid phase from the solid phase.

[0252] Therefore, in the embodiments, the method for detecting antibodies specific to coronaviruses in isolated samples includes...

[0253] a) Add a first coronavirus antigen carrying an effector group as part of a bioaffinity binding pair to the sample, which can directly or indirectly bind to a solid phase, and a second coronavirus antigen carrying a detectable label, wherein the first and second coronavirus antigens specifically bind to the anticoronavirus antibody.

[0254] b) Forming an immune reaction mixture comprising a first antigen, a sample antibody, and a second antigen, wherein a solid phase carrying the corresponding effector groups of the said bioaffinity binding pair is added before, during, or after the formation of the immune reaction mixture.

[0255] c) Maintaining the immune reaction mixture for a period of time sufficient to allow anti-coronavirus antibodies against the coronavirus antigen in the body fluid sample to react with the coronavirus antigen and form immune reaction products.

[0256] d) Separating the liquid phase from the solid phase

[0257] e) Detect the presence of any of the said immunoreaction products in the solid or liquid phase or both.

[0258] Finally, the presence of any of the said immunoreaction products is detected in the solid phase, liquid phase, or both.

[0259] In the embodiments, the maximum total duration of the method for detecting coronavirus antibodies is less than one hour, i.e., less than 60 minutes; in one embodiment, less than 30 minutes; in another embodiment, less than 20 minutes; in one embodiment, between 15 and 30 minutes; and in another embodiment, between 15 and 20 minutes. The duration includes the reagents required for sample transfer and assay, incubation time, optional washing steps, detection steps, and the final output of results.

[0260] In a sixth aspect, the present invention relates to a method for identifying whether a patient has been previously exposed to coronavirus infection, comprising:

[0261] a) An immune reaction mixture is formed by mixing a patient's bodily fluid sample with the coronavirus antigen of the first aspect of the present invention, the composition of the second aspect of the present invention, or the coronavirus antigen obtained by the method of the third aspect of the present invention.

[0262] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0263] c) Detect the presence and / or absence of any of the said immune response products, wherein the presence of an immune response product indicates that the patient has been exposed to coronavirus infection in the past.

[0264] In this embodiment, the patient was exposed to coronavirus infection prior to the implementation of this method. Specifically, the patient was exposed to coronavirus infection for at least 5 days prior to the implementation of this method. Specifically, the patient was exposed to coronavirus infection for at least 10 days prior to the implementation of this method. Specifically, the patient was exposed to coronavirus infection for at least 14 days prior to the implementation of this method.

[0265] In a seventh aspect, the present invention relates to a method for differential diagnosis between an immune response induced by natural coronavirus infection and an immune response induced by vaccination, wherein the vaccination is based on an antigen derived from an S protein, E protein, or M protein, comprising...

[0266] a) An immune reaction mixture is formed by mixing a patient's bodily fluid sample with the coronavirus antigen of the first aspect of the present invention, a composition comprising the coronavirus antigen of the first aspect of the present invention, or a coronavirus antigen obtained by the method of the third aspect of the present invention.

[0267] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0268] c) Detect the presence and / or absence of any of the said immune response products, wherein the presence of an immune response product indicates that the immune response in the patient is due to natural coronavirus infection, and wherein the absence of an immune response product indicates that the immune response in the patient is due to inoculation with an antigen derived from the S protein, E protein, or M protein.

[0269] In one embodiment, the method allows for the differentiation between patients naturally infected with coronaviruses and patients vaccinated against coronaviruses, wherein the vaccinated patients are given an antigen derived from the coronavirus S protein, E protein, or M protein.

[0270] In the embodiments, patients infected with natural coronaviruses were infected with SARS-Cov-1 or SARS-Cov-2, especially SARS-Cov-2.

[0271] In the embodiments, the natural coronavirus contains a nucleocapsid protein.

[0272] In an eighth aspect, the present invention relates to the use of a coronavirus antigen according to the first aspect of the invention, a composition according to the second aspect of the invention, or a coronavirus antigen obtained by the method according to the third aspect of the invention in a high-throughput in vitro diagnostic assay for detecting anti-coronavirus antibodies. In a particular embodiment, the coronavirus antigen according to the first aspect of the invention, the composition according to the second aspect of the invention, or the coronavirus antigen obtained by the method according to the third aspect of the invention is used in a method according to the fourth or fifth aspect of the invention.

[0273] In a ninth aspect, the present invention relates to a kit for detecting anti-coronavirus antibodies, the kit comprising a coronavirus antigen according to a first aspect of the invention, a composition according to a second aspect of the invention, or a coronavirus antigen obtained by a method according to a third aspect of the invention.

[0274] In embodiments, the kit contains a coronavirus antigen according to the first aspect of the invention, a composition according to the second aspect of the invention, or a coronavirus antigen obtained by the method according to the third aspect of the invention, in a separate container or in a separate compartment of a single container unit. In a particular embodiment, the contained coronavirus antigen is covalently coupled to biotin.

[0275] In the embodiments, the kit further contains microparticles, particularly microparticles coated with avidin or streptomycin, in a separate container or in a separate compartment of a single container unit.

[0276] In other embodiments, the present invention relates to the following items:

[0277] 1. A coronavirus antigen suitable for detecting antibodies against coronaviruses in isolated biological samples, comprising a coronavirus nucleocapsid-specific amino acid sequence or a variant thereof according to SEQ ID NO: 1, wherein said polypeptide does not contain other coronavirus-specific amino acid sequences.

[0278] 2. The coronavirus antigen described in Project 1, wherein the coronavirus is CoV-1 or CoV-2 virus, particularly CoV-2 virus.

[0279] 3. The coronavirus antigen described in item 1 or 2, wherein the antigen further comprises at least one chaperone protein, particularly two chaperone proteins.

[0280] 4. The coronavirus antigen described in Project 3, wherein the chaperone protein is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

[0281] 5. The coronavirus antigen described in items 2 to 4, wherein the chaperone protein is fused to a coronavirus nucleocapsid-specific amino acid sequence at the N- and / or C-terminus of the nucleocapsid.

[0282] 6. The coronavirus antigen described in items 1 to 5, wherein the polypeptide comprises a coronavirus nucleocapsid-specific amino acid sequence according to SEQ ID NO: 1 and two SlyD chaperone proteins.

[0283] 7. The coronavirus antigen described in any one of items 1 to 6 is soluble and immunoreactive.

[0284] 8. The coronavirus antigen of any one of claims 1 to 7, wherein the SARS-CoV-2 coronavirus nucleocapsid variant comprises an amino acid sequence according to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

[0285] 9. The coronavirus antigen of any one of items 1 to 8, further comprising a label, particularly a label that allows detection of the antigen (particularly Ru, particularly negatively charged Ru), and / or a label that binds the antigen directly or indirectly to a solid phase (particularly an effector group as part of a bioaffinity binding pair, particularly biotin).

[0286] 10. A composition comprising the coronavirus antigen described in any one of items 1 to 9.

[0287] 11. The composition of item 10, comprising additional coronavirus antigens, particularly coronavirus antigens containing an amino acid sequence of E protein, M protein and / or S protein or a portion thereof.

[0288] 12. A method for producing a coronavirus antigen specific to the nucleocapsid of a coronavirus, the method comprising the steps of:

[0289] a) Culture host cells transformed with an expression vector, particularly *E. coli* cells, wherein the expression vector comprises a recombinant DNA molecule operably linked to encoding a polypeptide according to any one of items 1 to 9, particularly a recombinant DNA molecule comprising the sequence according to SEQ ID NO: 3.

[0290] b) Expressing the polypeptide, and

[0291] c) Purify the polypeptide.

[0292] 13. A method for detecting antibodies specific to coronaviruses in an isolated sample, wherein a coronavirus antigen according to any one of items 1 to 9, a composition according to items 10 to 11, or a coronavirus antigen obtained by the method according to item 12 is used as a capture agent and / or binding coupler for the anti-coronavirus antibody.

[0293] 14. A method for detecting antibodies specific to coronaviruses in an isolated sample, the method comprising:

[0294] a) To form an immune reaction mixture by mixing a body fluid sample with a coronavirus antigen according to any one of items 1 to 9, a composition according to items 10 to 11, or a coronavirus antigen obtained by the method described in item 12.

[0295] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0296] c) Detect the presence and / or concentration of any of the said immune response products.

[0297] 15. The method for detecting antibodies specific to coronaviruses in an isolated sample according to item 14, wherein the immune reaction is performed in a double-antigen sandwich configuration, comprising:

[0298] a) Add a first coronavirus antigen carrying an effector group as part of a bioaffinity binding pair to the sample, which can directly or indirectly bind to a solid phase, and a second coronavirus antigen carrying a detectable label, wherein the first and second coronavirus antigens specifically bind to the anticoronavirus antibody.

[0299] b) Forming an immune reaction mixture comprising a first antigen, a sample antibody, and a second antigen, wherein a solid phase carrying the corresponding effector groups of the said bioaffinity binding pair is added before, during, or after the formation of the immune reaction mixture.

[0300] c) Maintaining the immune reaction mixture for a period of time sufficient to allow anti-coronavirus antibodies against the coronavirus antigen in the body fluid sample to react with the coronavirus antigen and form immune reaction products.

[0301] d) Separating the liquid phase from the solid phase

[0302] e) Detect the presence of any of the said immunoreaction products in the solid or liquid phase or both.

[0303] 16. The method for detecting antibodies specific to coronaviruses in isolated samples according to any one of items 13 to 15, wherein the detected antibodies are IgA, IgG or IgM antibodies, particularly IgG antibodies.

[0304] 17. A method for identifying whether a patient has been previously exposed to coronavirus infection, comprising:

[0305] a) An immune reaction mixture is formed by mixing a patient's bodily fluid sample with any one of the coronavirus antigens described in items 1 to 9, the compositions described in items 10 to 11, or the coronavirus antigen obtained by the method described in item 12.

[0306] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0307] c) Detect the presence and / or absence of any of the said immune response products.

[0308] The presence of immune reaction products indicates that the patient had been exposed to coronavirus infection in the past.

[0309] 18. A method for differential diagnosis between an immune response induced by natural coronavirus infection and an immune response induced by vaccination, wherein the vaccination is based on an antigen derived from an S protein, E protein, or M protein, the method comprising:

[0310] a) An immune reaction mixture is formed by mixing a patient's bodily fluid sample with the coronavirus antigen of the first aspect of the present invention, a composition comprising the coronavirus antigen of the first aspect of the present invention, or a coronavirus antigen obtained by the method of the third aspect of the present invention.

[0311] b) Maintaining the immune reaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to react with the coronavirus antigen to form an immune reaction product; and

[0312] c) Detect the presence and / or absence of any of the said immune response products.

[0313] The presence of immune response products indicates that the immune response in the patient is due to natural coronavirus infection, and the absence of immune response products indicates that the immune response in the patient is due to vaccination with spike protein-derived antigens.

[0314] 19. The use of the coronavirus antigen according to any one of items 1 to 9, the composition of items 10 to 11, or the coronavirus antigen obtained by the method of item 12 in a high-throughput in vitro diagnostic assay for detecting anti-coronavirus antibodies.

[0315] 20. Use of the coronavirus antigen according to any one of items 1 to 9, the composition described in items 10 to 11, or the method described in item 12 in the methods of items 13 to 18.

[0316] 21. A kit for detecting antibodies against coronaviruses, comprising the coronavirus antigen according to any one of items 1 to 9, the composition described in items 10 to 11, or the coronavirus antigen obtained by the method described in item 12.

[0317] 22. The kit according to item 18, which contains at least microparticles coated with avidin or streptoavidin in a separate container or in a separate compartment of a single container unit, and a coronavirus antigen covalently coupled to biotin according to any one of items 1 to 9, the composition of items 10 to 11, or the coronavirus antigen obtained by the method of item 12.

[0318] 23. The kit according to item 13, which contains at least microparticles coated with avidin or streptoavidin in a separate container or in a separate compartment of a single container unit, and a μ-capture binding coupler covalently coupled to biotin.

[0319] The following examples and accompanying drawings are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the illustrated procedures without departing from the spirit of the invention.

[0320] Example

[0321] Example 1: Cloning and purification of coronal nucleocapsid antigen

[0322] Cloning of expression boxes

[0323] Based on the pET24a expression plasmid from Novagen (Madison, WI, USA), the expression cassette encoding the fusion protein was obtained essentially as described (Scholz, C. et al., J. Mol. Biol. (2005) 345, 1229-1241). The nucleocapsid antigen sequence from SARS coronavirus 2 (SARS CoV-2) was retrieved from GenBank no. MN90847.3. The synthetic gene encoding nucleocapsid antigen aa 1-419 (i.e., the full-length version of the nucleocapsid or N protein) with a glycine-rich linker region fused to the N-terminal frame was purchased from Eurofins (Regensburg, Germany). Since the native amino acid sequence of the coronavirus N protein does not contain any cysteine ​​residues, amino acid substitutions were not required to prevent undesirable side effects such as oxidation or intermolecular disulfide bond bridging. The BamHI and XhoI restriction sites were located at the 5′ and 3′ ends of the N coding region, respectively. Encoding one or two EcSlyD units (SwissProt accession number) that are linked by a glycine-rich linker region and contain part of another linker region at the C-terminus. P0A9K9Another synthetic gene (residues 1-165) was also purchased from Eurofins. The NdeI and BamHI restriction sites are located at the 5′ and 3′ ends of the cassette, respectively. The genes and restriction sites were designed to allow for simple fusion of the chaperone protein portion EcSlyD-EcSlyD and the N antigen portion within the cassette. To avoid unintentional recombination and increase the genetic stability of the expression cassette in the *E. coli* host, the nucleotide sequence encoding the EcSlyD unit is as degenerate as the nucleotide sequence encoding the extension linker region. That is, different codon combinations are used to encode the same amino acid sequence.

[0324] The pET24a vector was digested with NdeI and XhoI and inserted into a cassette containing a tandem SlyD fused to the coronal nucleocapsid (1-419) frame. Accordingly, expression cassettes were constructed containing *E. coli* SlpA (2-149, SwissProt ID P0AEM0), *E. coli* Skp (21-161, SwissProt ID P0AEU7), or *E. coli* FkpA (26-270, SwissProt ID P45523), as well as an expression cassette containing a nucleocapsid fragment from SARS coronavirus 2. All recombinant fusion peptide variants contained a C-terminal hexahistine tag to facilitate Ni-NTA-assisted purification and refolding. QuikChange (Stratagene, LaJolla, CA, USA) and standard PCR techniques were used to generate point mutation, deletion, insertion, and extension variants or restriction sites in their respective expression cassettes.

[0325] Figure 3 A schematic diagram of the nucleocapsid antigen N1-419 is shown, which has two SlyD chaperone protein units fused to its N-terminus within the framework. To indicate the E. coli origin of the SlyD fusion chaperone, the depicted fusion polypeptide has been named EcSlyD-EcSlyD-CoV-2N(1-419).

[0326] Sequencing of the inserted fragment of the obtained plasmid revealed that it encodes the required fusion protein. The complete amino acid sequences of the antigen variants CoV-2N(1-419), EcSlyD-CoV-2N(1-419), and EcSlyD-EcSlyD-CoV-2N(1-419) are shown in SEQ ID NO. 1, 2, and 3, respectively. The amino acid sequence of the linker L is shown in SEQ ID NO: 7.

[0327] Purification of recombinant protein containing nucleocapsid from SARS coronavirus 2

[0328] All nucleocapsid antigen variants were purified using almost identical protocols. *E. coli* BLR(DE3) cells containing a specific pET24a expression plasmid were grown at 37°C in LB medium with kanamycin (30 μg / ml) to OD0.05. 600 The concentration was 1.5, and cytoplasmic overexpression was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside. Three hours after induction, cells were harvested by centrifugation (5000g for 20 minutes), frozen, and stored at -20°C. For cell lysis, the frozen precipitate was resuspended in frozen 50 mM sodium phosphate pH 8.0, 7.0 M GdmCl, and 5 mM imidazole, and the suspension was stirred on ice for 2 hours to complete cell lysis. After centrifugation and filtration (0.45 μm / 0.2 μm), the crude lysate was applied to a Ni-NTA column equilibrated with lysis buffer including 5.0 mM TCEP. The subsequent washing step was tailored to the corresponding target protein and ranged from 5 to 15 mM imidazole (in 50 mM sodium phosphate pH 8.0, 7.0 M GdmCl, and 5.0 mM TCEP). At least 10-15 volumes of wash buffer were applied. Then, the GdmCl solution was replaced with 50 mM potassium phosphate (pH 8.0), 100 mM KCl, 10 mM imidazole, and 5.0 mM TCEP to induce conformational refolding of the matrix-binding protein. To avoid reactivation of the co-purified protease, a mixture of protease inhibitors was added to the refolding buffer. EDTA-free (Roche). A total of 15–20 column volumes of refolding buffer were applied for the overnight reaction. TCEP was then removed by washing with 3–5 column volumes of 50 mM potassium phosphate (pH 8.0), 100 mM KCl, and 10 mM imidazole. Both were mixed without EDTA inhibitors. Subsequently, the imidazole concentration (still in 50 mM potassium phosphate pH 8.0, 100 mM KCl) was increased to 30–50 mM (depending on the respective target protein) to remove non-specifically bound protein contaminants. Native proteins were then eluted with 250 mM imidazole in the same buffer. The purity of the protein-containing fractions was assessed by Tricine-SDS-PAGE and pooled. Finally, the proteins were subjected to size exclusion chromatography (Superdex HiLoad, Amersham Pharmacia), and the protein-containing fractions were pooled and concentrated to 10–20 mg / mL in an Amicon unit (YM10).

[0329] After coupling purification and refolding protocols, approximately 10-15 mg of protein can be obtained from 1 g of E. coli wet cells, depending on the respective target protein (N protein without chaperone ~10 mg / g; EcSlyD-N(1-419) ~12 mg / g; EcSlyD-EcSlyD-N(1-419) ~15 mg / ml).

[0330] Example 2: Spectral Measurement

[0331] Protein concentration was measured using a Uvikon XL dual-beam spectrophotometer. The molar extinction coefficient (ε) was determined using the procedure described in Pace (1995), Protein Sci. 4, 2411-2423. 280 The molar extinction coefficient (ε) for different fusion peptides M280 Listed in Table 1.

[0332] Table 1: Protein parameters of the SARS coronavirus 2 nucleocapsid fusion peptide variants generated and used in this study. All parameters refer to their respective protein monomers.

[0333]

[0334] The SARS-CoV-2 N clone without the chaperone protein was the full-length version (1-419), but after overproduction in E. coli, the N-terminal methionine was co-translationally cleaved by N-methionylaminopeptidase. Therefore, data for the mature (cleaved) SARS-CoV-2 nucleocapsid version (2-419) are presented in Table 1. The amino acid sequences of the coronavirus variants are shown in SEQ ID NO: 1, 2, and 3, respectively.

[0335] Example 3: Conjugation of biotin-tagged and ruthenium complex-labeled nucleocapsid antigens

[0336] The lysine ε-amino groups of the fusion peptides were modified with biotin- and ruthenium-labeled molecules activated with N-hydroxysuccinimide at protein concentrations of 10–30 mg / mL. The label / protein ratios ranged from 1:1 to 10:1 (mol:mol), depending on the respective fusion protein. The reaction buffer consisted of 150 mM potassium phosphate (pH 8.0), 100 mM KCl, and 0.5 mM EDTA. The reaction was carried out at room temperature for 15 minutes and stopped by adding buffered L-lysine to a final concentration of 10 mM. To avoid hydrolytic deactivation of the tags, the respective stock solutions were prepared in anhydrous DMSO (seccosolv quality, Merck, Germany). All the fusion proteins studied tolerated DMSO at concentrations up to 25% in the reaction buffer well. After coupling, unreacted free labels were removed by passing the crude protein conjugate through a gel filtration column (Superdex 200 HiLoad).

[0337] Example 4: Immunoreactivity (i.e., antigenicity) of different nucleocapsid antigen variants in anti-SARS CoV-2 immunoassay

[0338] In automatic Immunoreactivity (i.e. antigenicity) of peptide fusion variants of the cobas e 411 analyzer (Roche Diagnostics GmbH) for evaluating the coronal nucleocapsid antigen. It is a registered trademark of Roche Group. Measurement is performed using a double-antigen sandwich method.

[0339] Signal detection in the cobas automated analyzer is based on electrochemiluminescence. A biotin conjugate (i.e., the antigen-capturing compound) is immobilized on the surface of streptavidin-coated magnetic beads, while the detected antigen carries a complex ruthenium cation (converting between redox states 2+ and 3+) as the signal component. In the presence of a specific immunoglobulin analyte, the luminescent ruthenium complex bridges to the solid phase and emits 620 nm light upon excitation by a platinum electrode. The signal is output in units of arbitrary luminescence intensity.

[0340] Recombinant coronavirus nucleocapsid antigen was evaluated using a dual-antigen sandwich (DAGS) immunoassay. For this purpose, recombinant coronavirus N antigen was used as both a biotin and a ruthenium conjugate to detect anti-coronary nucleocapsid antibodies in human serum.

[0341] Nucleocapsid protein N is one of the immunodominant antigens of coronaviruses, and as disclosed in this patent application, soluble variants of N are valuable tools for detecting coronavirus infection. In all measurements, EcSkp-EcSlyD-EcSlyD (EP2893021(B1)) or chemically polymerized and unlabeled EcSlyD-EcSlyD is used in large quantities (5-30 μg / ml) in the reaction buffer as an anti-interference substance to avoid immune cross-reactivity via chaperone protein fusion units.

[0342] Specifically, this study carefully examined three nucleocapsid variants of SARS coronavirus 2: a full-length N(1-419) without any fusion partner, a full-length N(1-419) fused with one SlyD chaperone protein, and a full-length N(1-419) fused with two SlyD chaperone protein units. To detect both anti-SARS CoV-2N IgM and IgG molecules, EcSlyD-EcSlyD-N(1-419)-biotin and EcSlyD-EcSlyD-N-ruthenium were used in R1 (reagent buffer 1) and R2 (reagent buffer 2), respectively. The concentrations of the antigen-binding compounds in R1 and R2 were each ~100 ng / ml (unless otherwise specified). In analytical gel filtration assays, we found that EcSlyD-EcSlyD-N(1-419) formed soluble and regular oligomers with an epitope density sufficient to bind and detect type M immunoglobulins.

[0343] In addition, The EcSlyD fusion peptide, representing a putative immunodominant fragment of the coronavirus antigen, was evaluated in the assay. Notably, the antigenicity of fragments from the spike protein (617-649, 338-516), E protein (8-65, 45-75), M protein (1-32, 132-163, 100-222), and N protein (151-178, 374-404) was examined. All of these chaperone protein fusions were cloned, purified, biotinylated, and ruthenium-modified, almost identical to the description of the N variant. These fragments were chosen because literature suggested that the corresponding sequences from SARS-CoV-1 were immunoreactive. In fact, for SARS-CoV-1, the immunodominant epitopes of the coronavirus spike protein (He et al., J. Immunol. (2004); 173: 4050-4057), coronavirus M protein (J. Clin. Microbiol. (2005); 43(8): 3718-3726) and coronavirus N protein (J. Clin. Microbiol. (2004) 42(2): 5309-5314) have been described.

[0344] Unfortunately, human coronavirus seroconversion kits—an indispensable tool for developing improved in vitro diagnostic assays—are not yet commercially available. To assess the antigenic properties of different nucleocapsid variants in the early stages of SARS-CoV-2 infection, we had to re-obtain surplus serum from clinics and hospitals.

[0345] In the first experiment, the immunoreactivity of all coronavirus antigen candidates was assessed using the DAGS format described above. For this purpose, biotinylated and ruthenium-coated variants of the candidate antigens under study were incubated with the samples before the addition of streptoacid-coated beads. Figure 4a and Figure 4b The data clearly show that the recombinant fusion peptide containing the coronavirus protein fragment does not exhibit any immunoreactivity: even at concentrations as high as 500 ng / ml, spike protein fragments 617-649 showed absolutely no reaction with the five sera tested in the anti-coronavirus positive group (see [link to study]). Figure 4a The detected signals were within the system-inherent background of approximately 500 counts, ruling out the presence of an immunodominant epitope in the spike protein (617-649). The same was true for another fragment from the spike protein (338-516), which contains the so-called receptor-binding domain and is considered one of the most immunodominant regions in the coronavirus proteome. Furthermore, the recombinant-derived RBD variant EcSlyD-spike protein (338-516) showed no reactivity, a stark contrast to previous reports on the antigenicity of this domain. E protein variants (45-75) and (8-65)—both fused with the solubility-enhancing E. coli SlyD protein—also showed no reactivity, as did fragments 1-32, 132-163, and 100-222 of the coronavirus M protein. The results for the 100-222 region of the M protein were significant because this is the intracellular domain portion of the M protein, meaning this fragment has a certain probability of adopting a native-like conformation and thus presenting a conformational epitope. However, the intracellular domain of the M protein showed no reactivity whatsoever. N-fragments 1 51-178 and 374-404 were also unreactive. Figure 4b In contrast, the full-length nucleocapsid antigen (second to last column) showed weak but significant immunoreactivity despite a very high background signal. When the SlyD unit was fused to the nucleocapsid at the N-terminus, the solubility of the resulting fusion polypeptide was significantly enhanced, and the background signal decreased from ~490,000 counts to 120,000 counts. Figure 4b (Last column). As a result, the signal-to-noise ratio was significantly increased, and anti-coronavirus positive and negative sera could be distinguished well. Nevertheless, the background signal remained very high, but this could be mitigated by reducing the antigen concentration in the assay.

[0346] Figure 4bThe results show that the fusion of a SlyD unit with the SARS CoV-2 nucleocapsid antigen delivers solubility to its target protein and improves its physicochemical properties, producing an immunoreactive coronavirus antigen that is highly suitable for detecting anti-coronavirus antibodies.

[0347] In the next step, we explored whether the fusion of another SlyD unit would further improve the physicochemical characterization of the nucleocapsid antigen.

[0348] Figure 5 The CoV-2 nucleocapsid antigen was shown in chaperone-free form, as well as fused with one SlyD unit and fused with two SlyD units. Evaluation. To ensure fair comparison, variants were applied at the same molar concentration. Notably, the addition of one SlyD chaperone protein unit significantly reduced the background signal, thereby improving the signal-to-noise ratio. When a second SlyD chaperone protein unit was added to the coronavirus nucleocapsid antigen, the background signal was further improved, and the signal-to-noise ratio increased further. In short, the solubility of the coronavirus N protein greatly benefits from the fusion of chaperone proteins such as SlyD. Figure 5 The comparison clearly shows that even signal recovery was significantly improved when two SlyD units were added to N instead of just one. Long-term stability is a critical issue and prerequisite for any antigen used in immunoassays. Signal recovery and the actual signal-to-noise ratio recovery should not be significantly affected when the antigen is incubated under thermal stress conditions such as 35°C. Table 5 also shows that the fusion of two SlyD chaperone protein units with the coronavirus N antigen improved overall signal recovery and made N usable for... The DAGS format was used for reliable detection of anti-coronavirus antibodies. After overnight incubation at 35°C, the signal-to-noise ratio recovery of the EcSlyD-EcSlyD-CoV-2-N conjugate was significantly higher than that of the CoV-2N conjugate without a chaperone protein. We found that this also applies to the SlpA (SlyD-like protein A)-N fusion protein. E. coli SlpA is a close relative of E. coli SlyD and has very favorable properties in terms of thermal stability (see Example 7 below).

[0349] Further optimization was performed on the anti-interference additives, buffer composition, and antigen concentration in R1 (= Reagent 1; biotin conjugate) and R2 (= Reagent 2; ruthenium conjugate), as well as the adsorption pretreatment of the ruthenium conjugate with beads. Figure 6 Ultimately A compatible nucleocapsid antigen paved the way, with excellent background values ​​(i.e., very low signal in negative sera) and a good signal-to-noise ratio (s / n), which helps to distinguish between positive and negative anti-coronavirus sera well.

[0350] In summary, we conclude that the coronavirus protein fragments touted as immunodominant epitopes, whether linear (e.g., spike protein 617-649) or conformational (e.g., the receptor-binding domain RBD contained within the spike protein), do not exhibit significant antigenicity in our hands. When evaluated in an automated analyzer, we found no antigenicity using promising coronavirus protein fragments, but only using the full-length nucleocapsid antigen from CoV-2. However, the native form of the N protein could not be used due to excessively high background signal. Assay. The fusion of two SlyD chaperone protein units with the N antigen resolves this deficiency and makes the N antigen suitable for assays. High-throughput applications on the platform.

[0351] Example 5: Sensitivity and specificity of the anti-SARS-CoV-2 immunoassay as described above

[0352] Initially, we further examined 129 patients identified as infected with SARS-CoV-2 by PCR analysis using our nucleocapsid antigen-based prototype antibody immunoassay. Serum samples were collected at different time intervals after a positive PCR test and analyzed using the aforementioned antibody assay to clarify the presence of any anti-CoV-2 antibodies in the samples. Results were categorized into three groups: less than 7 days after a positive PCR, 7 to 13 days after the initial PCR result, and 14 days and longer.

[0353] Six days after a positive PCR test, 74% of patients were identified as positive for SARS-CoV-2. Seven to 13 days after a positive PCR test, 95% of patients were identified as positive for SARS-CoV-2. Fourteen days after a positive PCR test, our assay detected 100% positivity in all patients. Results also showed... Figure 7 A) in.

[0354] In another experiment, a total of 204 samples from 69 symptomatic patients with PCR-confirmed SARS-CoV-2 infection were tested using the Elecsys anti-SARS-CoV-2 assay described above. One or more serial samples from these patients were collected after PCR confirmation at different time points. Results also showed… Figure 7 B) in.

[0355] In the third experiment, an additional 292 samples from 61 symptomatic patients with PCR-confirmed SARS-CoV-2 infection were tested using the Elecsys anti-SARS-CoV-2 detection method described above. One or more serial samples from these patients were collected after PCR confirmation at different time points. Results also showed... Figure 7In C), a sample is non-reactive after 14 days but becomes reactive after 16 days. Therefore, for this dataset, the sensitivity is 100% after 16 days.

[0356] For specificity testing, 1591 routine diagnostic serum and plasma samples collected prior to December 2019 (“pre-pandemic samples”) were initially analyzed using the antibody assay described above. All samples were classified as SARS-CoV-2 antibody negative due to their donation dates. Of the 1591 samples, only 2 were identified as having anti-SARS-CoV-2 reactivity. Therefore, the antibody assay described above has a specificity of 99.87%.

[0357] In a separate experiment, more patient samples were analyzed. The first group of samples, from 5272 patients, was analyzed, including the initial 1591 samples mentioned above. The following samples are attached.

[0358] • 3420 samples from patients undergoing routine diagnosis

[0359] · 1772 samples from blood donors

[0360] • 40 samples from patients diagnosed with the common cold, and

[0361] • 40 potentially cross-reactive samples from patients who had previously been infected with coronaviruses HKU1, NL63, 229E, or OC43, as confirmed by PCR.

[0362] All samples were obtained before December 2019 and tested using the Elecsys anti-SARS-CoV-2 assay as described above. Ten false positive samples were detected. The overall specificity obtained in the first sample group was 99.81%. The lower confidence limit was 99.65%. Results are shown in Figure 8 In A.

[0363] In the second group, an additional 5,261 samples from patients were analyzed. The following samples were included in the specificity study.

[0364] • 2376 samples from patients undergoing routine diagnosis

[0365] · 2,885 samples from blood donors

[0366] In addition, samples from 4,696 dialysis patients were analyzed.

[0367] The overall specificity obtained in the second sample group was 99.79%. The 95% confidence limit was 99.63%. The results showed... Figure 8 B in.

[0368] The combined results of the measurements of the first and second groups of samples (a total of 10,453) are shown in the following figures. Figure 8 C.

[0369] Example 6: Capillary blood is a suitable sample type for the above-mentioned anti-SARS-CoV-2 immunoassay.

[0370] To analyze whether capillary blood was suitable as the sample type for the aforementioned anti-SARS-CoV-2 immunoassay, capillary blood samples were compared with serum samples prepared from venous blood. The effects of three different anticoagulants (Li heparin plasma, K2-EDTA plasma, and CAT serum) were also analyzed. For Li heparin plasma and K2-EDTA plasma, 10 samples were tested, with 5 positive and 5 negative. For CAT serum, 7 samples were tested, with 5 positive and 2 negative. The results are summarized in Tables 2, 3, and 4 below, and are respectively... Figure 9 A, B, and C.

[0371] Table 2: Correlation between venous serum samples and capillary Li-heparin plasma samples

[0372]

[0373] Table 3: Correlation between venous serum samples and capillary K2-EDTA plasma samples

[0374]

[0375] Table 4: Correlation between venous serum samples and capillary CAT serum samples

[0376]

[0377] To account for variations in capillary blood sample volume, venous whole blood (collected without clot activators or anticoagulants) was transferred in different volumes to capillary collection tubes containing anticoagulants (300 μl, 400 μl, 600 μl, 800 μl = reference), centrifuged, and tested using an Elecsys Anti-SARS-CoV-2 analyzer on a cobas e analyzer. One negative sample and one spiked positive sample were tested. The results are shown in Table 5 below.

[0378] Table 5: Effect of Sample Amount Variation

[0379]

[0380] Example 7: Fusion of nucleocapsid antigen with alternative chaperone protein

[0381] Using the same method as described in Examples 1 to 3 above, the nucleocapsid sequence from SARS coronavirus 2 (SARS CoV-2) was also fused with a substitute chaperone protein (i.e., SlpA). The resulting fusion polypeptide was conjugated with a biotin tag or a ruthenium complex label. Immunoreactivity was tested as described in Example 4 above and compared with the reactivity of the above SlyD-antigen construct. The results are shown in... Figure 10 .

[0382] Example 8: Differential diagnosis of SARS-CoV-2 compared with common cold coronaviruses 229E, OC43, NL63 and HKU1

[0383] In addition to the nucleocapsid antigen from SARS-CoV-2, it is also worthwhile to possess nucleocapsid homologs from six other well-known human pathogenic coronaviruses: 229E, OC43, SARS-CoV-1, NL63, HKU1, and MERS (listed in the order they appear in the scientific literature). The so-called common cold coronaviruses 229E, OC43, NL63, and HKU1 still circulate in populations worldwide and are pathogens of cold-like illnesses—especially during winter (Human coronavirus circulation in the United States 2014–2017, J. Clin. Virol. 101 (2018), 52–56). Having their respective antigens should facilitate both interference-resistant methods for anti-SARS-CoV-2 immunoassays and the differential diagnosis of suspected sera under investigation. For example, when serum that yields a false positive for SARS CoV-2 is reacted with rec.EcSlyD-EcSlyD-N constructs from 229E and NL63 (both alpha-coronaviruses), it must be ruled out that antibodies generated during relatively harmless alpha-coronavirus infection may indeed cross-react with the rec.EcSlyD-EcSlyD-CoV-2-N designator used in anti-SARS CoV-2 antibody tests, thus falsely indicating SARS CoV-2 infection. A true positive result should be confirmed or ruled out, respectively, by a specific blocking assay (i.e., adding unlabeled common cold coronavirus N antigen to the sample being examined) or by differential diagnosis using a labeled N variant from a common cold coronavirus that reacts with the SARS CoV-2-reactive sample.

[0384] Therefore, we cloned, expressed, and purified (in *E. coli* Bl21) rec.EcSlyD-EcSlyD fusion protein versions of the N antigens from 229E, OC43, SARS-CoV-1, NL63, HKU1, and MERS, as described for rec.EcSlyD-EcSlyD-N from SARS-CoV-2. All N variants, except for OC43 and HKU1, were readily available from *E. coli* in high yields and were soluble and stable in our possession. Protein data and yields are summarized in Table 6.

[0385] Table 6: Protein signatures of the EcSlyD-EcSlyD-N antigen variants that were cloned, expressed, and examined in this study. EcSlyD-EcSlyD fusion proteins were constructed from the nucleocapsid proteins of seven known human pathogenic coronaviruses and purified substantially as described in the Examples section.

[0386]

[0387] Furthermore, following essentially the same purification protocol described for the full-length N version rec.EcSlyD-EcSlyD-CoV-2-N, we cloned, expressed, and purified the so-called N-terminal domain (NTD) of the N protein from SARS-CoV-2, 229E, OC43, NL63, and HKU1 from the E. coli BL21 overproducer. Unlike the full-length N protein, the NTD does not form dimers or tetramers but is strictly monomeric. Therefore, the NTD is particularly suitable for detecting type G immunoglobulins. However, type M immunoglobulins are not recognized by the strictly monomeric NTD when the antigen is used as a capture and detection molecule in the form of a double antigen sandwich (DAGS). Physiologically, the NTD binds to and accommodates polyanionic single-stranded viral RNA polymers within coronavirus particles. We can demonstrate that the solubility of the NTD is significantly improved compared to the full-length N protein, and its thermally induced unfolding is completely reversible—in stark contrast to the full-length N antigen. Melting profiles monitored by near-UV CD spectroscopy revealed highly favorable folding behavior of rec.EcSIyD-N_NTD, as the near-UV CD signal was fully recovered after thermal unfolding / refolding cycles of 20°C–80°C–20°C (data not shown), indicating the unfolded state and high solubility of potential folding intermediates. The reversibility of thermally induced unfolding is a very fortunate and desirable feature of the protein, and the absence of any aggregation tendency characterizes the NTD as an excellent antigen for immunoassays. Protein data and yields for various NTD constructs are shown in Table 7.

[0388] Table 7: Protein characteristics of cloned, expressed, and purified EcSlyD-N_NTD antigen variants

[0389]

[0390] As described, all rec.EcSlyD-N_NTD variants were biotinylated and ruthenium-conjugated. Each pair of biotin and ruthenium conjugates... The assessment showed excellent background signal and was highly effective in differentiating between positive and negative sera. Figure 11a In section +b, the reactivity of NTDs from SARS-CoV-2, OC43, NL63, 229E, and HKU1 with human serum is described. Serum was partially pre-characterized by recomLine lateral flow assay for SARS-CoV-2 IgG [Avidity] RUO (trade number 7374, Mikrogen GmbH, Neuried, Germany) because reliable data on the actual seropositivity rate of common cold coronavirus antibodies were not yet available. In short, we wanted to ensure that at least one of the sera studied was negative for each of the four common cold coronaviruses. Figure 11 shows that we did not observe any immune reactivity against the novel pathogen SARS-CoV-2 in the pre-pandemic common cold coronavirus group beginning in 2019. Figure 11a +b, column 1). All CCC sera were anti-SARS CoV-2 negative, producing electrochemiluminescent signals (450 to 600 counts) close to the system's intrinsic background. As for the anti-SARS CoV-2 positive group (starting from 2020), the signal of SARS CoV-2 NTD was significantly reduced relative to the full-length version of SARS CoV-2-N. This was expected because the NTD (46–176) lacks the complete C-terminal portion of the molecule (177–419), and therefore lacks many native epitopes. Furthermore, due to the strictly monomeric nature of the NTD, many anticoronavirus antibodies in polyclonal patient sera that might target N in conformational folded dimer and higher oligomeric forms are no longer able to recognize and bind to their target molecules. However, the rather poor signal levels we observed with SARS CoV-2-NTD still appear to be sufficient to reliably distinguish between positive and negative sera ( Figure 11a +b, column 1). This finding also applies to common cold coronaviruses (CCC) NTDs from OC43 and HKU1 (+b, column 1). Figure 11a +b, columns 2 and 5). From column 2, it can be inferred that the prevalence of antibodies against OC43 appears to be fairly moderate. For this β-coronavirus, we... The evaluation revealed numerous sera with background signals close to the system's intrinsic background. This suggests that the OC43 antigen generally exhibits excellent solubility, particularly the OC43 antigen-ruthenium conjugate. Notably, we also identified sera with high signal levels and fairly good signal dynamics, capable of effectively distinguishing between positive and negative sera. Regarding NL63 and 229E, we were unable to find true negative sera with signals within the system's intrinsic background range in the first test. All tested sera appeared to be anti-CoV positive for both NL63 and 229E. Figure 11a +b, columns 3 and 4), and the signal dynamics were very high. True positivity in serum was confirmed by reference measurements in which human serum was replaced with buffer and universal diluent at the sample sites, respectively. In this experimental setup, both NL63 and 229E showed significantly low background signal in the 400–650 count range (NTD). Figure 11b (The three "buffer" rows below). This finding is important in two ways: First, it rules out specific or non-specific association reactions between biotinylated and ruthenium-containing NL63 and 229E NTD molecules in the assay, which would lead to a sharp increase in signal. Second, it confirms that the NL63 and 229E NTD-ruthenium conjugate is highly soluble and... The assay does not bind to the bead surface coated with streptoavidin. In summary, the data indicate that the high signal intensity of NL63 and 229E measured in human serum is a valid result, highlighting the very high prevalence of antibodies against coronaviruses NL63 and 229E—far higher than OC43. Image completed using data from HKU1. Figure 11a +b, column 5). The prevalence of this coronavirus strain also appears to be quite high, but we did find truly negative sera for HKU1 compared to NL63 and 229E, with a signal close to the system's inherent background. Based on our very preliminary data and a small number of tested sera, it is easy to infer a preliminary prevalence ranking of common cold coronaviruses OC43 < HKU1 << NL63, 229E in the analysis group.

[0391] Our results may be due to chance due to the small amount of serum tested, but it appears that alpha coronavirus 229E and NL63 have been circulating in the analyzed cohorts, particularly during the previous winter, while OC43 infection appears to be rather rare.

[0392] In summary, the expression, purification, and modification (i.e., biotinylation and ruthenylation) of the N-terminal domains of the N antigens from coronaviruses SARS-CoV-2, OC43, NL63, 229E, and HKU1 enabled us to establish a simple serological differentiation between related common cold coronaviruses. Given the relentless global spread of SARS-CoV-2 during this unprecedented pandemic, our approach may be an attractive option for a simple differential diagnosis, distinguishing potentially life-threatening SARS-CoV-2 infection from the harmless cold caused by one of the four well-known common cold viruses OC43, NL63, 229E, and HKU1.

[0393] Example 8: Mutations in wild-type SARS-CoV-2 nucleocapsid antigen

[0394] Due to the increasing number of newly emerging SARS-CoV-2 mutant variants, we generated four mutant variants containing 3, 8, 12, or 15 single-point mutations (see...). Figure 12 Each of them is expressed as being fused to the two EcSlyD units as described above via the connector of SEQ ID NO: 7.

[0395] 3MUT: SEQ ID NO: 8

[0396] EcSlyD-EcSlyD-SARS CoV-2-N 3MUT: SEQ ID NO: 9

[0397] 8MUT: SEQ ID NO: 10

[0398] EcSlyD-EcSlyD-SARS CoV-2-N 8MUT: SEQ ID NO: 11

[0399] 12MUT: SEQ ID NO: 12

[0400] EcSlyD-EcSlyD-SARS CoV-2-N12MUT: SEQ ID NO: 13

[0401] 15MUT: SEQ ID NO: 14

[0402] EcSlyD-EcSlyD-SARS CoV-2-N15MUT: SEQ ID NO: 15

[0403] The introduced single-point mutation corresponds to a naturally occurring mutation in the SARS-CoV-2 variant currently circulating in the population. The most common circulating mutations are:

[0404] B.1.1.7 (UK): D3L, S235F

[0405] B.1.525 (UK / Nigeria): D3, A12G, T205I

[0406] COH.20G / 677H (Ohio): P67S, P199L, D377Y

[0407] B.1.351 (South Africa): T205I

[0408] P.1(B.1.1.28.1): P80R, R203K

[0409] P.2 (Brazil): A119S

[0410] P.3 (Philippines): R203K, G204R

[0411] N.9 (Brazil): I292T

[0412] EPI_ISL_1360318 (India): R203M

[0413] exist Figure 12 In the diagram, individual amino acid exchanges contained in the four mutant variants are indicated by patterned bars and specified amino acid exchange indicators.

[0414] Other SARS-CoV-2 nucleocapsid single-point mutations were also introduced; these mutations are less frequently found in the population than the ones mentioned above, and... Figure 12 These are indicated by black bars. These are based on... http: / / cov-glue.cvr.gla.ac.uk / # / home The CoV-GLU database published on (updated February 24, 2021, 17:11:05 GMT) selected the most common mutations found in infected individuals worldwide.

[0415] Two variants containing either three (3MUT) or eight (8MUT) single-point mutations were evaluated to assess the impact of selected mutations within the nucleocapsid protein on detection performance. Therefore, we used a labeled nucleocapsid variant in the dual-antigen sandwich (DAGS) immunoassay format as described above.

[0416] Serum samples from 50 individuals were tested in parallel with wild-type EcSlyD-EcSlyD-nucleocapsid fusion protein or protein variants containing three (3 MUT) or eight (8 MUT) single-point mutations. Mean COI recovery for the variants was calculated and compared with wild-type reactivity (see Table 8 and...). Figure 13 ).

[0417] Table 8: Recovery of WT compared to 3MUT and 8MUT

[0418] n＝50 WT 3MUT 8MUT Minimum value 100％ 88％ 71％ Maximum value 100％ 108％ 123％ average value 100％ 95％ 85％ SD 0％ 4％ 12％ CV 0％ 4％ 14％

[0419] The introduction of three single-point mutations in the nucleocapsid protein sequence resulted in a very slight decrease in reactivity (5%) in all tested samples. Since these point mutations correspond to amino acid substitutions found in the B.1.1.7 (D3L, S235F) and B.1.351 (T205I) variants of SARS-CoV-2, we conclude that the Elecsys anti-SARS-CoV-2 assay provides valid results when used in conjunction with antiserum from individuals infected with one of the widest UK or South African variants. Even exchanges of up to eight amino acids within the protein sequence still resulted in an 85% recovery of mean COI and a higher signal change compared to the wild-type sequence. Importantly, the closer the signal is to the cutoff (COI = 1.0), the smaller the difference in reactivity between the variant and the wild-type nucleocapsid sequence, ensuring that sample classification as reactive or non-reactive is not affected by one of the variants. In short, although three (D3L, T205I, S235F) and eight (P67S, D103Y, S194L, G204R, A220V, M234I, H300Y, A376T) amino acid residues were substituted within the nucleocapsid antigen, respectively, we achieved N-based Almost wild-type reactivity was observed in antibody assays. From these observations, we conclude that positive antiserum from individuals infected with one of the known SARS-CoV-2 variants will detect positive regardless.

[0420] Furthermore, we found that the 3MUT, 8MUT, and 15MUT variants retained their native conformation (i.e., they exhibited native-like folding) because their elution behavior in analytical gel filtration (via a Superdex 200 column) was equivalent to that of the wild-type nucleocapsid antigen. In cases where the introduced mutations resulted in partial or global unfolding, a significant increase in molecular size was expected. Our observation was that the N variants with 3, 8, and 15 mutations, respectively, maintained their global folding and exhibited nearly identical elution behavior to the wild-type N protein. sequence list <110> Roche Diagnostics Operations, Inc. Roche Diagnostics GmbH F. Hoffmann-La Roche AG <120> Coronal nucleocapsid antigen used for antibody immunoassay <130> P36075-WO <150> EP 20171154.6 <151> 2020-04-23 <150> US 16 / 856162 <151> April 23, 2020 <150> EP 20173315.1 <151> May 6, 2020 <150> US 16 / 867750 <151> May 6, 2020 <150> EP 20178739.7 <151> June 8, 2020 <160> 22 <170> BiSSAP 1.3.6 <210> 1 <211> 419 <212> PRT <213> Coronaviridae <400> 1 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 2 <211> 607 <212> PRT <213> Artificial sequence <220> <223> EcSlyD‑CoV‑2‑N(1‑419) <400> 2 Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg 1 5 10 15 Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu 20 25 30 Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala 35 40 45 Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala 50 55 60 Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro 65 70 75 80 Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe 85 90 95 Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val 100 105 110 Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln 115 120 125 Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu 130 135 140 Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His 145 150 155 160 Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Met Ser Asp Asn 180 185 190 Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro 195 200 205 Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg 210 215 220 Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp 225 230 235 240 Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg 245 250 255 Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile 260 265 270 Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys 275 280 285 Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly 290 295 300 Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp 305 310 315 320 Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr 325 330 335 Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly 340 345 350 Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser 355 360 365 Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn 370 375 380 Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly 385 390 395 400 Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn 405 410 415 Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln 420 425 430 Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln 435 440 445 Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg 450 455 460 Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile 465 470 475 480 Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala 485 490 495 Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val 500 505 510 Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp 515 520 525 Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His 530 535 540 Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys 545 550 555 560 Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys 565 570 575 Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser 580 585 590 Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr Gln Ala 595 600 605 <210> 3 <211> 794 <212> PRT <213> Artificial sequence <220> <223> EcSlyD-EcSlyD-CoV-2-N(1-419) <400> 3 Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg 1 5 10 15 Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu 20 25 30 Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala 35 40 45 Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala 50 55 60 Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro 65 70 75 80 Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe 85 90 95 Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val 100 105 110 Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln 115 120 125 Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu 130 135 140 Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His 145 150 155 160 Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Val Ala Lys 180 185 190 Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg Thr Glu Asp Gly Val 195 200 205 Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu Asp Tyr Leu His Gly 210 215 220 His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala Leu Glu Gly His Glu 225 230 235 240 Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala Asn Asp Ala Tyr Gly 245 250 255 Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met 260 265 270 Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp 275 280 285 Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val 290 295 300 Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Lys Phe Asn 305 310 315 320 Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu Glu Glu Leu Ala His 325 330 335 Gly His Val His Gly Ala His Asp His His His Asp His Asp His Asp 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 355 360 365 Gly Gly Gly Ser Gly Gly Gly Met Ser Asp Asn Gly Pro Gln Asn Gln 370 375 380 Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly 385 390 395 400 Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg 405 410 415 Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr 420 425 430 Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro 435 440 445 Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg 450 455 460 Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser 465 470 475 480 Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu 485 490 495 Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly 500 505 510 Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn 515 520 525 Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys 530 535 540 Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg 545 550 555 560 Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser 565 570 575 Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala 580 585 590 Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys 595 600 605 Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys 610 615 620 Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr 625 630 635 640 Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln 645 650 655 Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp 660 665 670 Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala 675 680 685 Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr 690 695 700 Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn 705 710 715 720 Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys 725 730 735 Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp 740 745 750 Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr 755 760 765 Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln 770 775 780 Ser Met Ser Ser Ala Asp Ser Thr Gln Ala 785 790 <210> 4 <211> 1284 <212> DNA <213> Coronaviridae <400> 4 atgagcgaca atggtccgca aaaccagcgt aatgcaccgc gcatcacgtt tggcggtccg 60 tcagactcca ccggcagcaa ccagaatggc gaacgcagtg gtgcacgctc gaaacaacgt 120 cgtccccagg gtctgccgaa caataccgcg tcatggttta cggccttgac acaacatggg 180 aaagaggatc tgaaatttcc gcgtggtcag ggcgttccga tcaacacgaa ctcttcgcct 240 gatgaccaga ttggctatta tcgccgtgct actcgccgca ttcgcggtgg agatggtaaa 300 atgaaggatt tgagtccccg gtggtacttc tactatctgg gaactggacc agaggcgggc 360 ttaccgtatg gcgccaacaa agatgggatc atttgggtag ctacggaagg tgcgcttaac 420 accccgaaag accacattgg gacgcgcaat ccagcgaaca atgctgcgat tgtcctgcag 480 ttaccccaag ggaccacgct gccaaaaggc ttctatgccg aaggctcacg tggcggctct 540 caagcgagta gtcgcagctc atcgcgcagc cgcaactcta gccggaattc aaccccaggt 600 agctctcgcg gcaccagtcc agcccgtatg gctggtaatg gaggcgatgc agctttagcc 660 ctcctgcttc tcgatcggct taaccagctg gagagcaaaa tgtcgggtaa agggcagcaa 720 cagcagggtc agaccgttac gaaaaaatcc gcagcagaag cgtccaaaaa gccgcgtcag 780 aaacgcacag ccaccaaagc gtataacgtg actcaagctt tcggacgtcg tggtccggaa 840 caaacccagg ggaatttcgg tgaccaagaa ctgattcgcc aaggcaccga ttacaaacat 900 tggccgcaga ttgcgcagtt tgcaccctct gcaagcgcct ttttcggcat gagccgcatt 960 ggcatggaag tcactccgtc gggcacatgg ctgacctaca cgggtgcgat caagctggat 1020 gataaagacc cgaatttcaa agatcaggtg atcctgctga acaaacacat cgatgcctac 1080 aaaacctttc ctccgaccga accgaaaaag gacaagaaaa agaaagcaga cgagacacaa 1140 gcgctgcctc agcgtcagaa gaaacagcag acggtgaccc tgttacctgc cgcggatttg 1200 gatgactttt cgaaacagct ccaacagtcc atgagttccg ccgatagcac tcaggcgctc 1260 gagcaccacc accaccacca ctga 1284 <210> 5 <211> 1848 <212> DNA <213> Artificial Sequence <220> <223> EcSlyD-CoV-2N (1-419) <400> 5 atgaaagtag caaaagacct ggtggtcagc ctggcctatc aggtacgtac agaagacggt 60 gtgttggttg atgagtctcc ggtgagtgcg ccgctggact acctgcatgg tcacggttcc 120 ctgatctctg gcctggaaac ggcgctggaa ggtcatgaag ttggcgacaa atttgatgtc 180 gctgttggcg cgaacgacgc ttacggtcag tacgacgaaa acctggtgca acgtgttcct 240 aaagacgtat ttatgggcgt tgatgaactg caggtaggta tgcgtttcct ggctgaaacc 300 gaccagggtc cggtaccggt tgaaatcact gcggttgaag acgatcacgt cgtggttgat 360 ggtaaccaca tgctggccgg tcagaacctg aaattcaacg ttgaagttgt ggcgattcgc 420 gaagcgactg aagaagaact ggctcatggt cacgttcacg gcgcgcacga tcaccaccac gatcacgacc acgacggtgg cggttccggc ggtggctctg gtggcggatc cggtggcggt 540 tccggcggtg gctctggtgg cggtatgagc gacaatggtc cgcaaaacca gcgtaatgca ccgcgcatca cgtttggcgg tccgtcagac tccaccggca gcaaccaga tggcgaacgc 660 agtggtgcac gctcgaaaca acgtcgtccc cagggtctgc cgaacaatac cgcgtcatgg tttacggcct tgacacaaca tgggaaagag gatctgaaat ttccgcgtgg tcagggcgtt ccgatcaaca cgaactcttc gcctgatgac cagattggct attatcgccg tgctactcgc cgcattcgcg gtggagatgg taaaatgaag gatttgagtc cccggtggta cttctactat ctgggaactg gaccagaggc gggcttaccg tatggcgcca acaaagatgg gatcatttgg gtagctacgg aaggtgcgct taacaccccg aaagaccaca ttgggacgcg caatccagcg aacaatgctg cgattgtcct gcagttaccc caagggacca cgctgccaaa aggcttctat gccgaaggct cacgtggcgg ctctcaagcg hold gctcatcgcg cagccgcaac tctagccgga attcaacccc aggtagctct cgcggcacca gtccagcccg tatggctggt 1200 aatggaggcg atgcagcttt agccctcctg cttctcgatc ggcttaacca gctggagagc 1260 aaaatgtcgg gtaaagggca gcaacagcag ggtcagaccg ttacgaaaaa atccgcagca 1320 gaagcgtcca aaaagccgcg tcagaaacgc acagccacca aagcgtataa cgtgactcaa 1380 gctttcggac gtcgtggtcc ggaacaaacc caggggaatt tcggtgacca agaactgatt 1440 cgccaaggca ccgattacaa acattggccg cagattgcgc agtttgcacc ctctgcaagc 1500 gcctttttcg gcatgagccg cattggcatg gaagtcactc cgtcgggcac atggctgacc 1560 tacacgggtg cgatcaagct ggatgataaa gacccgaatt tcaaagatca ggtgatcctg 1620 ctgaacaaac acatcgatgc ctacaaaacc tttcctccga ccgaaccgaa aaaggacaag 1680 aaaaagaaag cagacgagac acaagcgctg cctcagcgtc agaagaaaca gcagacggtg 1740 accctgttac ctgccgcgga tttggatgac ttttcgaaac agctccaaca gtccatgagt 1800 tccgccgata gcactcaggc gctcgagcac caccaccacc accactga 1848 <210> 6 <211> 2409 <212> DNA <213> Artificial sequence <220> <223> EcSlyD-EcSlyD-CoV-2-N(1-419) <400> 6 atgaaagtag caaaagacct ggtggtcagc ctggcctatc aggtacgtac agaagacggt 60 gtgttggttg atgagtctcc ggtgagtgcg ccgctggact acctgcatgg tcacggttcc 120 ctgatctctg gcctggaaac ggcgctggaa ggtcatgaag ttggcgacaa atttgatgtc 180 gctgttggcg cgaacgacgc ttacggtcag tacgacgaaa acctggtgca acgtgttcct 240 aaagacgtat ttatgggcgt tgatgaactg caggtaggta tgcgtttcct ggctgaaacc 300 gaccagggtc cggtaccggt tgaaatcact gcggttgaag acgatcacgt cgtggttgat 360 ggtaaccaca tgctggccgg tcagaacctg aaattcaacg ttgaagttgt ggcgattcgc 420 gaagcgactg aagaagaact ggctcatggt cacgttcacg gcgcgcacga tcaccaccac 480 gatcacgacc acgacggtgg cggttccggc ggtggctctg gtggcggaag cggcggaggc 540 tctgggggcg gatcaggcgg tggaaaggtc gcgaaagatc tcgtagtgag cctcgcttac 600 caagtgcgca ctgaggatgg ggttctggta gacgaatcac ccgtatcggc accgctcgat 660 tatttgcacg gccatggtag cctaattagt ggtttagaga cagcacttga gggacacgag 720 gtcggtgata agttcgacgt tgcagtggga gctaatgatg cctatgggca atatgatgag 780 aatctcgttc agcgcgtgcc gaaggatgtg ttcatgggtg tagacgagct ccaagtgggc 840 atgcggtttc ttgccgagac ggatcaaggc cctgtgccag tcgagattac cgcagtggag 900 gatgaccatg ttgtcgtgga cggaaatcac atgttagcgg gacaaaattt gaaatttaat 960 gtcgaggtcg tcgctatccg tgaggccacc gaagaagagc ttgcacacgg ccatgtccat 1020 ggtgcccatg accatcacca tgaccatgat catgatggcg gtgggtcggg tgggggaagt 1080 gggggtggat ccggtggcgg ttccggcggt ggctctggtg gcggtatgag cgacaatggt 1140 ccgcaaaacc agcgtaatgc accgcgcatc acgtttggcg gtccgtcaga ctccaccggc 1200 agcaaccaga atggcgaacg cagtggtgca cgctcgaaac aacgtcgtcc ccagggtctg 1260 ccgaacaata ccgcgtcatg gtttacggcc ttgacacaac atgggaaaga ggatctgaaa 1320 tttccgcgtg gtcagggcgt tccgatcaac acgaactctt cgcctgatga ccagattggc 1380 tattatcgcc gtgctactcg ccgcattcgc ggtggagatg gtaaaatgaa ggatttgagt 1440 ccccggtggt acttctacta tctgggaact ggaccagagg cgggcttacc gtatggcgcc 1500 aacaaagatg ggatcatttg ggtagctacg gaaggtgcgc ttaacacccc gaaagaccac 1560 attgggacgc gcaatccagc gaacaatgct gcgattgtcc tgcagttacc ccaagggacc 1620 acgctgccaa aaggcttcta tgccgaaggc tcacgtggcg gctctcaagc gagtagtcgc 1680 agctcatcgc gcagccgcaa ctctagccgg aattcaaccc caggtagctc tcgcggcacc 1740 agtccagccc gtatggctgg taatggaggc gatgcagctt tagccctcct gcttctcgat 1800 cggcttaacc agctggagag caaaatgtcg ggtaaagggc agcaacagca gggtcagacc 1860 gttacgaaaa aatccgcagc agaagcgtcc aaaaagccgc gtcagaaacg cacagccacc 1920 aaagcgtata acgtgactca agctttcgga cgtcgtggtc cggaacaaac ccaggggaat 1980 ttcggtgacc aagaactgat tcgccaaggc accgattaca aacattggcc gcagattgcg 2040 cagtttgcac cctctgcaag cgcctttttc ggcatgagcc gcattggcat ggaagtcact 2100 ccgtcgggca catggctgac ctacacgggt gcgatcaagc tggatgataa agacccgaat 2160 ttcaaagatc aggtgatcct gctgaacaaa cacatcgatg cctacaaaac ctttcctccg 2220 accgaaccga aaaaggacaa gaaaaagaaa gcagacgaga cacaagcgct gcctcagcgt 2280 cagaagaaac agcagacggt gaccctgtta cctgccgcgg atttggatga cttttcgaaa 2340 cagctccaac agtccatgag ttccgccgat agcactcagg cgctcgagca ccaccaccac 2400 caccactga 2409 <210> 7 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Linker sequence <400> 7 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly 20 <2><10> 8 <2><11> 420 <2><12> PRT <2><13> Artificial sequence <2><20> <2><23> 3 MUT <4><00> 8 Met Ser Leu Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Ile Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Phe Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Light Light Asp Light Light Light Light Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala Glu 420 <210> 9 <211> 795 <212> PRT <213> artificial sequence <220> <223> EcSlyD‑EcSlyD‑3 MUT <400> 9 Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg 1 5 10 15 Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu 20 25 30 Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala 35 40 45 Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala 50 55 60 Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro 65 70 75 80 Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe 85 90 95 Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val 100 105 110 Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln 115 120 125 Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu 130 135 140 Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His 145 150 155 160 Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Val Ala Lys 180 185 190 Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg Thr Glu Asp Gly Val 195 200 205 Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu Asp Tyr Leu His Gly 210 215 220 His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala Leu Glu Gly His Glu 225 230 235 240 Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala Asn Asp Ala Tyr Gly 245 250 255 Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met 260 265 270 Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp 275 280 285 Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val 290 295 300 Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Lys Phe Asn 305 310 315 320 Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu Glu Glu Leu Ala His 325 330 335 Gly His Val His Gly Ala His Asp His His His Asp His Asp His Asp 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 355 360 365 Gly Gly Gly Ser Gly Gly Gly Met Ser Leu Asn Gly Pro Gln Asn Gln 370 375 380 Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly 385 390 395 400 Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg 405 410 415 Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr 420 425 430 Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro 435 440 445 Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg 450 455 460 Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser 465 470 475 480 Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu 485 490 495 Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly 500 505 510 Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn 515 520 525 Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys 530 535 540 Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg 545 550 555 560 Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser 565 570 575 Ser Arg Gly Ile Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala 580 585 590 Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys 595 600 605 Met Phe Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys 610 615 620 Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr 625 630 635 640 Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln 645 650 655 Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp 660 665 670 Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala 675 680 685 Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr 690 695 700 Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn 705 710 715 720 Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys 725 730 735 Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp 740 745 750 Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr 755 760 765 Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln 770 775 780 Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Glu 785 790 795 <210> 10[[ID=3​​​​​​​​<223> 8 MUT <400> 10 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Ser Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Tyr Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Leu Arg Asn Ser Thr Pro Gly Ser Ser Arg Arg Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Val Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Ile Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys Tyr Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala Leu Glu 420 <210> 11 <211> 796 <212> PRT <213> Artificial Sequence <220> <223> EcSlyD‑EcSlyD‑CoV‑2‑N (1‑419, 8 mut) <400> 11 Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg 1 5 10 15 Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu 20 25 30 Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala 35 40 45 Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala 50 55 60 Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro 65 70 75 80 Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe 85 90 95 Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val 100 105 110 Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln 115 120 125 Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu 130 135 140 Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His 145 150 155 160 Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Val Ala Lys 180 185 190 Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg Thr Glu Asp Gly Val 195 200 205 Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu Asp Tyr Leu His Gly 210 215 220 His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala Leu Glu Gly His Glu 225 230 235 240 Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala Asn Asp Ala Tyr Gly 245 250 255 Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met 260 265 270 Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp 275 280 285 Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val 290 295 300 Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Lys Phe Asn 305 310 315 320 Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu Glu Glu Leu Ala His 325 330 335 Gly His Val His Gly Ala His Asp His His His Asp His Asp His Asp 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 355 360 365 Gly Gly Gly Ser Gly Gly Gly Met Ser Asp Asn Gly Pro Gln Asn Gln 370 375 380 Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly 385 390 395 400 Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg 405 410 415 Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr 420 425 430 Gln His Gly Lys Glu Asp Leu Lys Phe Ser Arg Gly Gln Gly Val Pro 435 440 445 Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg 450 455 460 Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Tyr Leu Ser 465 470 475 480 Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu 485 490 495 Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly 500 505 510 Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn 515 520 525 Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys 530 535 540 Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg 545 550 555 560 Ser Ser Ser Arg Ser Arg Asn Ser Leu Arg Asn Ser Thr Pro Gly Ser 565 570 575 Ser Arg Arg Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala 580 585 590 Ala Leu Val Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys 595 600 605 Ile Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys 610 615 620 Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr 625 630 635 640 Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln 645 650 655 Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp 660 665 670 Tyr Lys Tyr Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala 675 680 685 Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr 690 695 700 Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn 705 710 715 720 Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys 725 730 735 Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp 740 745 750 Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr 755 760 765 Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln 770 775 780 Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Leu Glu 785 790 795 <210> 12 <211> 421 <212> PRT <213> Artificial sequence <220> <223> 12MUT <400> 12 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Gly Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Arg 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Ser 85 90 95 Gly Asp Gly Lys Met Lys Tyr Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ser Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 Tyr Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Tyr Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Met Gly Ile Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Thr Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Ile Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Tyr Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala Leu Glu 420 <210> 13 <211> 796 <212> PRT <213> Artificial Sequence <220> <223> EcSlyD-EcSlyD-12MUT <400> 13 Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg 1 5 10 15 Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu 20 25 30 Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala 35 40 45 Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala 50 55 60 Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro 65 70 75 80 Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe 85 90 95 Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val 100 105 110 Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln 115 120 125 Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu 130 135 140 Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His 145 150 155 160 Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Val Ala Lys 180 185 190 Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg Thr Glu Asp Gly Val 195 200 205 Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu Asp Tyr Leu His Gly 210 215 220 His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala Leu Glu Gly His Glu 225 230 235 240 Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala Asn Asp Ala Tyr Gly 245 250 255 Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met 260 265 270 Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp 275 280 285 Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val 290 295 300 Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Lys Phe Asn 305 310 315 320 Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu Glu Glu Leu Ala His 325 330 335 Gly His Val His Gly Ala His Asp His His His Asp His Asp His Asp 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 355 360 365 Gly Gly Gly Ser Gly Gly Gly Met Ser Asp Asn Gly Pro Gln Asn Gln 370 375 380 Arg Asn Gly Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly 385 390 395 400 Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg 405 410 415 Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr 420 425 430 Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro 435 440 445 Ile Asn Thr Asn Ser Ser Arg Asp Asp Gln Ile Gly Tyr Tyr Arg Arg 450 455 460 Ala Thr Arg Arg Ile Arg Ser Gly Asp Gly Lys Met Lys Tyr Leu Ser 465 470 475 480 Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ser Gly Leu 485 490 495 Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly 500 505 510 Ala Leu Asn Thr Pro Lys Asp Tyr Ile Gly Thr Arg Asn Pro Ala Asn 515 520 525 Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys 530 535 540 Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Tyr Ser Arg 545 550 555 560 Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser 565 570 575 Ser Met Gly Ile Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala 580 585 590 Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys 595 600 605 Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys 610 615 620 Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr 625 630 635 640 Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln 645 650 655 Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Thr Arg Gln Gly Thr Asp 660 665 670 Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala 675 680 685 Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr 690 695 700 Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn 705 710 715 720 Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys 725 730 735 Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp 740 745 750 Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Ile Val Thr 755 760 765 Leu Leu Pro Ala Ala Asp Leu Tyr Asp Phe Ser Lys Gln Leu Gln Gln 770 775 780 Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Leu Glu 785 790 795 <210> 14 <211> 421 <212> PRT <213> Artificial sequence <220> <223> 15MUT <400> 14 Met Ser Leu Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Ser Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Leu Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Leu Arg Asn Ser Thr Leu Gly Ser Ser Lys Arg Ile Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Val Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Ile Phe Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Ser Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Thr Tyr Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Val Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala Leu Glu 420 <210> 15 <211> 796 <212> PRT <213> Artificial sequence <220> <223> EcSlyD-EcSlyD-15MUT <400> 15 Met Lys Val Ala Lys Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg 1 5 10 15 Thr Glu Asp Gly Val Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu 20 25 30 Asp Tyr Leu His Gly His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala 35 40 45 Leu Glu Gly His Glu Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala 50 55 60 Asn Asp Ala Tyr Gly Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro 65 70 75 80 Lys Asp Val Phe Met Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe 85 90 95 Leu Ala Glu Thr Asp Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val 100 105 110 Glu Asp Asp His Val Val Val Asp Gly Asn His Met Leu Ala Gly Gln 115 120 125 Asn Leu Lys Phe Asn Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu 130 135 140 Glu Glu Leu Ala His Gly His Val His Gly Ala His Asp His His His 145 150 155 160 Asp His Asp His Asp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Lys Val Ala Lys 180 185 190 Asp Leu Val Val Ser Leu Ala Tyr Gln Val Arg Thr Glu Asp Gly Val 195 200 205 Leu Val Asp Glu Ser Pro Val Ser Ala Pro Leu Asp Tyr Leu His Gly 210 215 220 His Gly Ser Leu Ile Ser Gly Leu Glu Thr Ala Leu Glu Gly His Glu 225 230 235 240 Val Gly Asp Lys Phe Asp Val Ala Val Gly Ala Asn Asp Ala Tyr Gly 245 250 255 Gln Tyr Asp Glu Asn Leu Val Gln Arg Val Pro Lys Asp Val Phe Met 260 265 270 Gly Val Asp Glu Leu Gln Val Gly Met Arg Phe Leu Ala Glu Thr Asp 275 280 285 Gln Gly Pro Val Pro Val Glu Ile Thr Ala Val Glu Asp Asp His Val 290 295 300 Val Val Asp Gly Asn His Met Leu Ala Gly Gln Asn Leu Lys Phe Asn 305 310 315 320 Val Glu Val Val Ala Ile Arg Glu Ala Thr Glu Glu Glu Leu Ala His 325 330 335 Gly His Val His Gly Ala His Asp His His His Asp His Asp His Asp 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 355 360 365 Gly Gly Gly Ser Gly Gly Gly Met Ser Leu Asn Gly Pro Gln Asn Gln 370 375 380 Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly 385 390 395 400 Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg 405 410 415 Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr 420 425 430 Gln His Gly Lys Glu Asp Leu Lys Phe Ser Arg Gly Gln Gly Val Pro 435 440 445 Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg 450 455 460 Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser 465 470 475 480 Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu 485 490 495 Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly 500 505 510 Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Leu Ala Asn 515 520 525 Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys 530 535 540 Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg 545 550 555 560 Ser Ser Ser Arg Ser Arg Asn Ser Leu Arg Asn Ser Thr Leu Gly Ser 565 570 575 Ser Lys Arg Ile Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala 580 585 590 Ala Leu Val Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys 595 600 605 Ile Phe Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys 610 615 620 Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr 625 630 635 640 Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln 645 650 655 Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp 660 665 670 Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala 675 680 685 Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr 690 695 700 Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn 705 710 715 720 Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys 725 730 735 Thr Phe Pro Ser Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Thr Tyr 740 745 750 Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr 755 760 765 Leu Leu Pro Ala Val Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln 770 775 780 Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Leu Glu 785 790 795 <210> 16 <211> 419 <212> PRT <213> Coronaviridae <400> 16 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 17 <211> 422 <212> PRT <213> Coronaviridae <400> 17 Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile 1 5 10 15 Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 20 25 30 Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn 35 40 45 Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 50 55 60 Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly 65 70 75 80 Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg 85 90 95 Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr 100 105 110 Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys 115 120 125 Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 130 135 140 Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu 145 150 155 160 Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly 165 170 175 Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 180 185 190 Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro 195 200 205 Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu 210 215 220 Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln 225 230 235 240 Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser 245 250 255 Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr 260 265 270 Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly 275 280 285 Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln 290 295 300 Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg 305 310 315 320 Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly 325 330 335 Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile 340 345 350 Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu 355 360 365 Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro 370 375 380 Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp 385 390 395 400 Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 405 410 415 Ala Asp Ser Thr Gln Ala 420 <210> 18 <211> 413 <212> PRT <213> Coronaviridae <400> 18 Met Ala Ser Pro Ala Ala Pro Arg Ala Val Ser Phe Ala Asp Asn Asn 1 5 10 15 Asp Ile Thr Asn Thr Asn Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys 20 25 30 Pro Arg Ala Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr 35 40 45 Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro 50 55 60 Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg 65 70 75 80 Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro 85 90 95 Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro 100 105 110 Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu His Gly Ala 115 120 125 Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser 130 135 140 Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe 145 150 155 160 His Ile Glu Gly Thr Gly Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser 165 170 175 Ser Val Ser Arg Asn Ser Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser 180 185 190 Gly Asn Ser Thr Arg Gly Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala 195 200 205 Val Gly Gly Asp Leu Leu Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala 210 215 220 Leu Glu Ser Gly Lys Val Lys Gln Ser Gln Pro Lys Val Ile Thr Lys 225 230 235 240 Lys Asp Ala Ala Ala Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser 245 250 255 Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly 260 265 270 Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr 275 280 285 Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser 290 295 300 Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp 305 310 315 320 Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys 325 330 335 Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu 340 345 350 Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln 355 360 365 Lys Ala Pro Lys Glu Glu Ser Thr Asp Gln Met Ser Glu Pro Pro Lys 370 375 380 Glu Gln Arg Val Gln Gly Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro 385 390 395 400 Ser Val Gln Pro Gly Pro Met Ile Asp Val Asn Thr Asp 405 410 <210> 19 <211> 377 <212> PRT <213> koronavirus <400> 19 Met Ala Ser Val Asn Trp Ala Asp Asp Arg Ala Ala Arg Lys Lys Phe 1 5 10 15 Pro Pro Pro Ser Phe Tyr Met Pro Leu Leu Val Ser Ser Asp Lys Ala 20 25 30 Pro Tyr Arg Val Ile Pro Arg Asn Leu Val Pro Ile Gly Lys Gly Asn 35 40 45 Lys Asp Glu Gln Ile Gly Tyr Trp Asn Val Gln Glu Arg Trp Arg Met 50 55 60 Arg Arg Gly Gln Arg Val Asp Leu Pro Pro Lys Val His Phe Tyr Tyr 65 70 75 80 Leu Gly Thr Gly Pro His Lys Asp Leu Lys Phe Arg Gln Arg Ser Asp 85 90 95 Gly Val Val Trp Val Ala Lys Glu Gly Ala Lys Thr Val Asn Thr Ser 100 105 110 Leu Gly Asn Arg Lys Arg Asn Gln Lys Pro Leu Glu Pro Lys Phe Ser 115 120 125 Ile Ala Leu Pro Pro Glu Leu Ser Val Val Glu Phe Glu Asp Arg Ser 130 135 140 Asn Asn Ser Ser Arg Ala Ser Ser Arg Ser Ser Thr Arg Asn Asn Ser 145 150 155 160 Arg Asp Ser Ser Arg Ser Thr Ser Arg Gln Gln Ser Arg Thr Arg Ser 165 170 175 Asp Ser Asn Gln Ser Ser Ser Asp Leu Val Ala Ala Val Thr Leu Ala 180 185 190 Leu Lys Asn Leu Gly Phe Asp Asn Gln Ser Lys Ser Pro Ser Ser Ser 195 200 205 Gly Thr Ser Thr Pro Lys Lys Pro Asn Lys Pro Leu Ser Gln Pro Arg 210 215 220 Ala Asp Lys Pro Ser Gln Leu Lys Lys Pro Arg Trp Lys Arg Val Pro 225 230 235 240 Thr Arg Glu Glu Asn Val Ile Gln Cys Phe Gly Pro Arg Asp Phe Asn 245 250 255 His Asn Met Gly Asp Ser Asp Leu Val Gln Asn Gly Val Asp Ala Lys 260 265 270 Gly Phe Pro Gln Leu Ala Glu Leu Ile Pro Asn Gln Ala Ala Leu Phe 275 280 285 Phe Asp Ser Glu Val Ser Thr Asp Glu Val Gly Asp Asn Val Gln Ile 290 295 300 Thr Tyr Thr Tyr Lys Met Leu Val Ala Lys Asp Asn Lys Asn Leu Pro 305 310 315 320 Lys Phe Ile Glu Gln Ile Ser Ala Phe Thr Lys Pro Ser Ser Ile Lys 325 330 335 Glu Met Gln Ser Gln Ser Ser His Val Ala Gln Asn Thr Val Leu Asn 340 345 350 Ala Ser Ile Pro Glu Ser Lys Pro Leu Ala Asp Asp Asp Ser Ala Ile 355 360 365 Ile Glu Ile Val Asn Glu Val Leu His 370 375 <210> 20 <211> 389 <212> PRT <213> Coronaviridae <400> 20 Met Ala Thr Val Lys Trp Ala Asp Ala Ser Glu Pro Gln Arg Gly Arg 1 5 10 15 Gln Gly Arg Ile Pro Tyr Ser Leu Tyr Ser Pro Leu Leu Val Asp Ser 20 25 30 Glu Gln Pro Trp Lys Val Ile Pro Arg Asn Leu Val Pro Ile Asn Lys 35 40 45 Lys Asp Lys Asn Lys Leu Ile Gly Tyr Trp Asn Val Gln Lys Arg Phe 50 55 60 Arg Thr Arg Lys Gly Lys Arg Val Asp Leu Ser Pro Lys Leu His Phe 65 70 75 80 Tyr Tyr Leu Gly Thr Gly Pro His Lys Asp Ala Lys Phe Arg Glu Arg 85 90 95 Val Glu Gly Val Val Trp Val Ala Val Asp Gly Ala Lys Thr Glu Pro 100 105 110 Thr Gly Tyr Gly Val Arg Arg Lys Asn Ser Glu Pro Glu Ile Pro His 115 120 125 Phe Asn Gln Lys Leu Pro Asn Gly Val Thr Val Val Glu Glu Pro Asp 130 135 140 Ser Arg Ala Pro Ser Arg Ser Gln Ser Arg Ser Gln Ser Arg Gly Arg 145 150 155 160 Gly Glu Ser Lys Pro Gln Ser Arg Asn Pro Ser Ser Asp Arg Asn His 165 170 175 Asn Ser Gln Asp Asp Ile Met Lys Ala Val Ala Ala Ala Leu Lys Ser 180 185 190 Leu Gly Phe Asp Lys Pro Gln Glu Lys Asp Lys Lys Ser Ala Lys Thr 195 200 205 Gly Thr Pro Lys Pro Ser Arg Asn Gln Ser Pro Ala Ser Ser Gln Thr 210 215 220 Ser Ala Lys Ser Leu Ala Arg Ser Gln Ser Ser Glu Thr Lys Glu Gln 225 230 235 240 Lys His Glu Met Gln Lys Pro Arg Trp Lys Arg Gln Pro Asn Asp Asp 245 250 255 Val Thr Ser Asn Val Thr Gln Cys Phe Gly Pro Arg Asp Leu Asp His 260 265 270 Asn Phe Gly Ser Ala Gly Val Val Ala Asn Gly Val Lys Ala Lys Gly 275 280 285 Tyr Pro Gln Phe Ala Glu Leu Val Pro Ser Thr Ala Ala Met Leu Phe 290 295 300 Asp Ser His Ile Val Ser Lys Glu Ser Gly Asn Thr Val Val Leu Thr 305 310 315 320 Phe Thr Thr Arg Val Thr Val Pro Lys Asp His Pro His Leu Gly Lys 325 330 335 Phe Leu Glu Glu Leu Asn Ala Phe Thr Arg Glu Met Gln Gln His Pro 340 345 350 Leu Leu Asn Pro Ser Ala Leu Glu Phe Asn Pro Ser Gln Thr Ser Pro 355 360 365 Ala Thr Ala Glu Pro Val Arg Asp Glu Val Ser Ile Glu Thr Asp Ile 370 375 380 Ile Asp Glu Val Asn 385 <210> 21 <211> 448 <212> PRT <213> Coronaviridae <400> 21 Met Ser Phe Thr Pro Gly Lys Gln Ser Ser Ser Arg Ala Ser Ser Gly 1 5 10 15 Asn Arg Ser Gly Asn Gly Ile Leu Lys Trp Ala Asp Gln Ser Asp Gln 20 25 30 Phe Arg Asn Val Gln Thr Arg Gly Arg Arg Ala Gln Pro Lys Gln Thr 35 40 45 Ala Thr Ser Gln Gln Pro Ser Gly Gly Asn Val Val Pro Tyr Tyr Ser 50 55 60 Trp Phe Ser Gly Ile Thr Gln Phe Gln Lys Gly Lys Glu Phe Glu Phe 65 70 75 80 Val Glu Gly Gln Gly Val Pro Ile Ala Pro Gly Val Pro Ala Thr Glu 85 90 95 Ala Lys Gly Tyr Trp Tyr Arg His Asn Arg Arg Ser Phe Lys Thr Ala 100 105 110 Asp Gly Asn Gln Arg Gln Leu Leu Pro Arg Trp Tyr Phe Tyr Tyr Leu 115 120 125 Gly Thr Gly Pro His Ala Lys Asp Gln Tyr Gly Thr Asp Ile Asp Gly 130 135 140 Val Tyr Trp Val Ala Ser Asn Gln Ala Asp Val Asn Thr Pro Ala Asp 145 150 155 160 Ile Val Asp Arg Asp Pro Ser Ser Asp Glu Ala Ile Pro Thr Arg Phe 165 170 175 Pro Pro Gly Thr Val Leu Pro Gln Gly Tyr Tyr Ile Glu Gly Ser Gly 180 185 190 Arg Ser Ala Pro Asn Ser Arg Ser Thr Ser Arg Thr Ser Ser Arg Ala 195 200 205 Ser Ser Ala Gly Ser Arg Ser Arg Ala Asn Ser Gly Asn Arg Thr Pro 210 215 220 Thr Ser Gly Val Thr Pro Asp Met Ala Asp Gln Ile Ala Ser Leu Val 225 230 235 240 Leu Ala Lys Leu Gly Lys Asp Ala Thr Lys Pro Gln Gln Val Thr Lys 245 250 255 His Thr Ala Lys Glu Val Arg Gln Lys Ile Leu Asn Lys Pro Arg Gln 260 265 270 Lys Arg Ser Pro Asn Lys Gln Cys Thr Val Gln Gln Cys Phe Gly Lys 275 280 285 Arg Gly Pro Asn Gln Asn Phe Gly Gly Gly Glu Met Leu Lys Leu Gly 290 295 300 Thr Ser Asp Pro Gln Phe Pro Ile Leu Ala Glu Leu Ala Pro Thr Ala 305 310 315 320 Gly Ala Phe Phe Phe Gly Ser Arg Leu Glu Leu Ala Lys Val Gln Asn 325 330 335 Leu Ser Gly Asn Pro Asp Glu Pro Gln Lys Asp Val Tyr Glu Leu Arg 340 345 350 Tyr Asn Gly Ala Ile Arg Phe Asp Ser Thr Leu Ser Gly Phe Glu Thr 355 360 365 Ile Met Lys Val Leu Asn Glu Asn Leu Asn Ala Tyr Gln Gln Gln Asp 370 375 380 Gly Met Met Asn Met Ser Pro Lys Pro Gln Arg Gln Arg Gly His Lys 385 390 395 400 Asn Gly Gln Gly Glu Asn Asp Asn Ile Ser Val Ala Val Pro Lys Ser 405 410 415 Arg Val Gln Gln Asn Lys Ser Arg Glu Leu Thr Ala Glu Asp Ile Ser 420 425 430 Leu Leu Lys Lys Met Asp Glu Pro Tyr Thr Glu Asp Thr Ser Glu Ile 435 440 445 <210> 22 <211> 441 <212> PRT <213> Coronaviridae <400> 22 Met Ser Tyr Thr Pro Gly His Tyr Ala Gly Ser Arg Ser Ser Ser Gly 1 5 10 15 Asn Arg Ser Gly Ile Leu Lys Lys Thr Ser Trp Ala Asp Gln Ser Glu 20 25 30 Arg Asn Tyr Gln Thr Phe Asn Arg Gly Arg Lys Thr Gln Pro Lys Phe 35 40 45 Thr Val Ser Thr Gln Pro Gln Gly Asn Thr Ile Pro His Tyr Ser Trp 50 55 60 Phe Ser Gly Ile Thr Gln Phe Gln Lys Gly Arg Asp Phe Lys Phe Ser 65 70 75 80 Asp Gly Gln Gly Val Pro Ile Ala Phe Gly Val Pro Pro Ser Glu Ala 85 90 95 Lys Gly Tyr Trp Tyr Arg His Ser Arg Arg Ser Phe Lys Thr Ala Asp 100 105 110 Gly Gln Gln Lys Gln Leu Leu Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly 115 120 125 Thr Gly Pro Tyr Ala Asn Ala Ser Tyr Gly Glu Ser Leu Glu Gly Val 130 135 140 Phe Trp Val Ala Asn His Gln Ala Asp Thr Ser Thr Pro Ser Asp Val 145 150 155 160 Ser Ser Arg Asp Pro Thr Thr Gln Glu Ala Ile Pro Thr Arg Phe Pro 165 170 175 Pro Gly Thr Ile Leu Pro Gln Gly Tyr Tyr Val Glu Gly Ser Gly Arg 180 185 190 Ser Ala Ser Asn Ser Arg Pro Gly Ser Arg Ser Gln Ser Arg Gly Pro 195 200 205 Asn Asn Arg Ser Leu Ser Arg Ser Asn Ser Asn Phe Arg His Ser Asp 210 215 220 Ser Ile Val Lys Pro Asp Met Ala Asp Glu Ile Ala Asn Leu Val Leu 225 230 235 240 Ala Lys Leu Gly Lys Asp Ser Lys Pro Gln Gln Val Thr Lys Gln Asn 245 250 255 Ala Lys Glu Ile Arg His Lys Ile Leu Thr Lys Pro Arg Gln Lys Arg 260 265 270 Thr Pro Asn Lys His Cys Asn Val Gln Gln Cys Phe Gly Lys Arg Gly 275 280 285 Pro Ser Gln Asn Phe Gly Asn Ala Glu Met Leu Lys Leu Gly Thr Asn 290 295 300 Asp Pro Gln Phe Pro Ile Leu Ala Glu Leu Ala Pro Thr Pro Gly Ala 305 310 315 320 Phe Phe Phe Gly Ser Lys Leu Asp Leu Val Lys Arg Asp Ser Glu Ala 325 330 335 Asp Ser Pro Val Lys Asp Val Phe Glu Leu His Tyr Ser Gly Ser Ile 340 345 350 Arg Phe Asp Ser Thr Leu Pro Gly Phe Glu Thr Ile Met Lys Val Leu 355 360 365 Glu Glu Asn Leu Asn Ala Tyr Val Asn Ser Asn Gln Asn Thr Asp Ser 370 375 380 Asp Ser Leu Ser Ser Lys Pro Gln Arg Lys Arg Gly Val Lys Gln Leu 385 390 395 400 Pro Glu Gln Phe Asp Ser Leu Asn Leu Ser Ala Gly Thr Gln His Ile 405 410 415 Ser Asn Asp Phe Thr Pro Glu Asp His Ser Leu Leu Ala Thr Leu Asp 420 425 430 Asp Pro Tyr Val Glu Asp Ser Val Ala 435 440

Claims

1. A coronavirus antigen suitable for detecting antibodies against a coronavirus in an isolated biological sample, wherein the coronavirus antigen further comprises at least one chaperone, wherein the coronavirus is a SARS-CoV-2 virus, and wherein the coronavirus antigen comprises an amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO:

11.

2. The coronavirus antigen according to claim 1, wherein the chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

3. A composition comprising at least one coronavirus antigen according to any one of claims 1 to 2.

4. A method of producing a coronavirus antigen specific for a coronavirus nucleocapsid, the method comprising the steps of: a) cultivating a host cell transformed with an expression vector comprising a recombinant DNA molecule operably linked encoding a coronavirus antigen according to any one of claims 1 to 2 b) expressing the coronavirus antigen, and c) purifying the coronavirus antigen.

5. The method according to claim 4, wherein the recombinant DNA molecule is a recombinant DNA molecule comprising a sequence according to SEQ ID NO:

6.

6. The method according to claim 4, wherein the host cell is an E. coli cell.

7. Use of a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3 or a coronavirus antigen obtained by a method according to any one of claims 4 to 6 for the manufacture of a kit for performing a method for detecting antibodies specific for a coronavirus in an isolated sample, wherein the coronavirus antigen according to any one of claims 1 to 2, the composition according to claim 3 or the coronavirus antigen obtained by a method according to any one of claims 4 to 6 is used as a capture reagent and / or binding partner for the antibodies specific for a coronavirus.

8. Use of a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3 or a coronavirus antigen obtained by a method according to any one of claims 4 to 6 for the manufacture of a kit for performing a method for detecting antibodies specific for a coronavirus in an isolated sample, the method comprising a) forming an immunoreaction mixture by mixing a body fluid sample with the coronavirus antigen according to any one of claims 1 to 2, the composition according to claim 3 or the coronavirus antigen obtained by a method according to any one of claims 4 to 6, b) maintaining the immunoreaction mixture for a period of time sufficient to allow antibodies against the coronavirus antigen present in the body fluid sample to immunoreact with the coronavirus antigen to form an immunoreaction product; and c) detecting the immunoreaction product. c) detecting the presence and / or concentration of any of said immunoreaction products.

9. Use of a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3, or a coronavirus antigen obtained by the method according to any one of claims 4 to 6, for the manufacture of a kit for performing a method of identifying whether a patient has been exposed to a coronavirus infection in the past, said method comprising a) forming an immunoreaction mixture by mixing a body fluid sample of said patient with a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3, or a coronavirus antigen obtained by the method according to any one of claims 4 to 6, b) maintaining said immunoreaction mixture for a period of time sufficient to allow antibodies against said coronavirus antigen present in said body fluid sample to immunoreact with said coronavirus antigen to form immunoreaction products; and c) detecting the presence and / or absence of any of said immunoreaction products, wherein the presence of an immunoreaction product is indicative of said patient having been exposed to a coronavirus infection in the past.

10. Use of a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3, or a coronavirus antigen obtained by the method according to any one of claims 4 to 6, for the manufacture of a kit for performing a method of differentiating between an immune response caused by a natural coronavirus infection and an immune response caused by a vaccination, wherein said vaccination is based on S protein, E protein or M protein derived antigens, said method comprising a) forming an immunoreaction mixture by mixing a body fluid sample with a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3, or a coronavirus antigen obtained by the method according to any one of claims 4 to 6, b) maintaining said immunoreaction mixture for a period of time sufficient to allow antibodies against said coronavirus antigen present in said body fluid sample to immunoreact with said coronavirus antigen to form immunoreaction products; and c) detecting the presence and / or absence of any of said immunoreaction products, wherein the presence of an immunoreaction product is indicative of an immune response in a patient being due to a natural coronavirus infection, and wherein the absence of an immunoreaction product is indicative of an immune response in said patient being due to a vaccination with spike protein derived antigens.

11. Use of a coronavirus antigen according to any one of claims 1 to 2, a composition according to claim 3, or a coronavirus antigen obtained by the method according to any one of claims 4 to 6, for the manufacture of a kit for detecting anti-coronavirus antibodies in a high-throughput in vitro diagnostic test. ​ ​ ​ ​ ​ ​ ​ ​ 12. Use of the coronantigen according to any one of claims 1 to 2, the composition according to claim 3 or the coronantigen obtained by the method according to any one of claims 4 to 6, for the manufacture of a kit for performing a method for detecting antibodies specific for a coronavirus in an isolated sample.

13. A kit for detecting anti-coronavirus antibodies comprising the coronantigen according to any one of claims 1 to 2, the composition according to claim 3 or the coronantigen obtained by the method according to any one of claims 4 to 6.

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