Corona nucleocapsid antigens for use in antibody-immunoassays

Abstract

The present invention relates to a corona antigen comprising a corona nucleocapsid-specific amino acid sequence, a composition and a reagent kit comprising the same, and a method for producing the same. It also includes a method for detecting anti-corona antibodies in a sample using the corona antigen, and a method for differential diagnosis of a patient's immune response due to natural corona infection or vaccination against corona.

Description

[Technical Field]

[0001] The present invention relates to a corona antigen comprising a corona nucleocapsid-specific amino acid sequence, a composition and a reagent kit comprising the same, and a method for producing the same. It also includes a method for detecting anti-corona antibodies in a sample using the corona antigen, and a method for differential diagnosis of a patient's immune response due to natural corona infection or vaccination against corona. [Background technology]

[0002] background The SARS-CoV-2 virus was discovered by Chinese virologists at the end of 2019 and has since spread relentlessly around the world. SARS-CoV-2, formerly known as nCoV-19 (novel coronavirus 2019), the etiological agent of coronavirus disease 2019 (COVID-19), caused a pandemic in early 2020, resulting in substantial restrictions on public life and severe economic impacts worldwide. Diagnostic tests enabling the detection of acutely infected patients quickly became available. However, the number of available tests could never meet the high demand during the pandemic. Therefore, many patients outside of clinics and hospitals did not undergo testing, as available tests were primarily reserved for patients with very severe symptoms. Statistically, four out of five patients infected with SARS-CoV-2 develop only mild symptoms, such as a mild sore throat, dry cough, or mild fever. As a result, it is currently unknown how many people have been infected or are still infected, and how many have already recovered from the infection.

[0003] To assess the extent of the current pandemic, it would be extremely useful to be able to accurately estimate the infection rate, and therefore the true mortality rate, of SARS-2. Furthermore, patients who have recovered from the disease and are known to have acquired immunity could be excluded from public lockdowns and could help those still in need, for example in clinics and hospitals.

[0004] Therefore, there is a strong need for immunological tests that can detect antibodies against the SARS-CoV-2 virus in patients. Such antibody tests would allow the identification of patients who have previously suffered from an infection (potentially with a mild progression of the disease that they are not even aware of). Therefore, such tests would allow the reliable and first assessment of true infection rates, both within different cohorts and within the entire population. Furthermore, such tests would allow the evaluation of whether vaccines developed against SARS-CoV-2 virus infection are indeed effective in stimulating immune responses in patients and would therefore be crucial in assessing the success of vaccination campaigns.

[0005] However, automated high-throughput assays for detecting anti-SARS CoV-2 antibodies in patients with the necessary sensitivity and specificity are still not available. Currently approved antibody tests can accurately diagnose less than one-third of infected patients, and two-thirds of infected patients are falsely reported. One of the main challenges here is that the tests must be equipped with antigens that can be recognized by anti-SARS CoV-2 antibodies with both high sensitivity and specificity.

[0006] Since the emergence of SARS, first reported in 2002 / 2003, several coronavirus antigens have been known in the art. The spike protein of coronaviruses, especially its receptor-binding domain (RBD), is considered the most promising candidate, as it has previously been shown to be highly immunologically reactive (Wang et al. (Clin Chem (2003) 49 (12), 1989-1996); and He et al. (J. Clin. Microbiol. (2004) 42 (11), 5309-5314), i.e., a strong antibody response is generated against the RBD during the humoral immune response upon infection with SARS CoV. As a result, the receptor-binding domain also serves as the primary antigen in current assay development (Amanat et al., medRxiv, March 2003). 18, 2020). In this very recent manuscript, the authors describe the use of the CoV-2 receptor-binding domain as a capture antigen in an ELISA format. 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 amount of sample analyzed was too small to allow for statistically significant statements regarding sensitivity and specificity. Furthermore, ELISA-format antibody assays often require time-consuming and tedious manual steps, and high-throughput applications are often hindered by limited assay availability.

[0007] Contrary to the prior art preconception that antigens derived from the spike protein are the most promising for the development of coronavirus antibody assays, the present invention relates to an immunological test that uses the nucleocapsid protein of the SARS-CoV-2 virus as an antigen for the reliable detection of anti-SARS-CoV-2 antibodies. Surprisingly, the inventors were able to show that by using the nucleocapsid protein of SARS-CoV-2 as an antigen, both high sensitivity and high specificity of the resulting immunological test can be achieved, allowing the development of an urgently needed and much-needed automated high-throughput coronavirus antibody assay. Summary of the Invention

[0008] Summary of the Invention In a first aspect, the present invention relates to a corona antigen suitable for detecting antibodies against coronavirus in an isolated biological sample, comprising a corona nucleocapsid-specific amino acid sequence, in particular the corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, or a corona nucleocapsid-specific amino acid sequence having 95% sequence identity with the amino acid sequence of SEQ ID NO: 1. In particular, the polypeptide does not comprise any further coronavirus-specific amino acid sequence.

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

[0010] In a third aspect, the present invention provides a method for producing a corona antigen specific for a coronavirus nucleocapsid, the method comprising: a) culturing a host cell, in particular an E. coli cell, transformed with an expression vector to which a recombinant DNA molecule encoding the antigen of the first aspect of the present invention, in particular a recombinant DNA molecule comprising the sequence set forth in SEQ ID NO: 3, has been operably linked; b) expressing the polypeptide; and c) Purification of the polypeptide The present invention relates to a method comprising:

[0011] In a fourth aspect, the present invention relates to a method for detecting coronavirus-specific antibodies in an isolated sample, using 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 as a capture reagent and / or binding partner for said anti-coronavirus antibodies.

[0012] In a fifth aspect, the present invention provides a method for detecting antibodies specific to a coronavirus in an isolated sample, comprising: a) forming an immune reaction mixture by mixing a body fluid sample with 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; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or concentration of any of the immune reaction products The present invention relates to a method comprising:

[0013] In a sixth aspect, the present invention provides a method for identifying whether a patient has been previously exposed to a coronavirus infection, comprising: a) forming an immune reaction mixture by mixing a sample of a patient's body fluid with 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; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or absence of any of the immune reaction products Including, The present invention relates to a method wherein the presence of an immune response product indicates that the patient has been previously exposed to a coronavirus infection.

[0014] In a seventh aspect, the present invention provides a method for differential diagnosis between an immune response in a patient resulting from a natural coronavirus infection and an immune response resulting from vaccination, wherein the vaccination is based on an S protein-derived antigen, an E protein-derived antigen, or an M protein-derived antigen, a) forming an immune reaction mixture by mixing a body fluid sample from a patient with a coronavirus antigen of the first aspect of the invention, a composition comprising the coronavirus antigen of the first aspect of the invention, or a coronavirus antigen obtained by the method of the third aspect of the invention; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or absence of any of the immune reaction products Including, The presence of the immune response product indicates that the immune response in the patient is due to a natural coronavirus infection, and the absence of the immune response product indicates that the immune response in the patient is due to vaccination with a spike protein-derived antigen.

[0015] 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 test for the detection of anti-coronavirus antibodies.

[0016] In a ninth aspect, the present invention relates to a reagent kit for detecting anti-coronavirus antibodies, comprising a coronavirus antigen according to the first aspect of the present invention, a composition according to the second aspect of the present invention, or a coronavirus antigen obtained by the method according to the third aspect of the present invention. [Brief explanation of the drawings]

[0017] [Figure 1]Alignment of nucleocapsid sequences of known coronaviruses with the following UniProt numbers, Gene Bank Acc. numbers and respective SEQ ID NO: 16. Severe acute respiratory syndrome coronavirus 2 N (SARS-CoV-2), β-CoV: UniProt ID P0DTC9; Gene Bank Acc.: MN908947; SEQ ID NO: 16. Severe acute respiratory syndrome coronavirus N (SARS-CoV), β-CoV: UniProt ID P59595; Gene Bank Acc.: AY278741; SEQ ID NO: 17. Middle East respiratory syndrome-related coronavirus N (MERS-CoV), β-CoV: UniProt ID T2BBK0; Gene Bank Acc.: KF600632; SEQ ID NO: 18. Human coronavirus NL63 N (HCoV-NL63), α-CoV: UniProt ID Q6Q1R8; Gene Bank Acc.: AY567487; SEQ ID NO: 19. Human coronavirus 229E N(HCoV-229E), α-CoV:UniProt ID P15130; Gene Bank Acc: Q5MQC6;Gene Bank Acc.:AY597011;SEQ ID NO:22 [Figure 2] Sequence comparison: (A) The degree of sequence identity (%) of the SARS CoV-2 nucleocapsid amino acid sequence to the nucleocapsid sequences of different coronaviruses; (B) The degree of sequence homology (%) of the SARS CoV-2 nucleocapsid amino acid sequence to the nucleocapsid sequences of different coronaviruses. [Figure 3] Graphical representation of EcSlyD-EcSlyD-CoV-2 N(1-419) antigen [Figure 4A] Comparison of immunological reactivity of antigens derived from the S-, E-, and M-proteins of the coronavirus SARS CoV-2 [Figure 4B]Comparison of different antigens derived from the SARS CoV-2 nucleocapsid protein [Figure 5] Comparison of immunological reactivity of full-length nucleocapsids fused to zero, one, or two SlyD-chaperones [Figure 6] Impact of bead pretreatment of ruthenium conjugates (as an additional workflow in the manufacturing process) on assay performance [Figure 7] Sensitivity of the SARS CoV-2 assay; A) Initial results from samples of 129 confirmed SARS CoV-2 patients; and B) Further results including a total of 214 confirmed SARS CoV-2 patients; C) Further results from an additional 292 confirmed SARS CoV-2 patients. [Figure 8] Specificity of the SARS CoV-2 assay: A) results from the first set of measured samples from 5192 patients and 80 potentially cross-reactive samples; B) results from the second set of measured samples from 5261 patients; and C) results from all patients (10453 total). Because cold and coronavirus cross-reactive samples are not routine diagnostics or blood donors, they are excluded from the calculation of overall specificity. [Figure 9] Correlation of assay performance obtained with venous serum samples versus capillary blood samples [Figure 10] Comparison of immunoreactivity of antigens containing SARS CoV-2 nucleocapsid sequences fused to two SlyD- or two SlpA-chaperones [Figure 11A] Reactivity of the N-terminal domain of the nucleocapsid protein from SARS-CoV-2, OC43, NL63, 229E, and HKU1. Measurements were performed in DAGS format on a cobas e411 automated analyzer. The concentrations of biotin conjugate (R1) and ruthenium conjugate (R2) were 100 ng / ml, respectively. Signal readouts (counts) were normalized to the mean of the respective negative values ​​to obtain signal kinetics (s / n). [Figure 11B]Reactivity of the N-terminal domain of the nucleocapsid protein from SARS-CoV-2, OC43, NL63, 229E, and HKU1. Measurements were performed in DAGS format on a cobas e411 automated analyzer. The concentrations of biotin conjugate (R1) and ruthenium conjugate (R2) were 100 ng / ml, respectively. Signal readouts (counts) were normalized to the mean of the respective negative values ​​to obtain signal kinetics (s / n). [Figure 12] Schematic diagram of the four single-point mutation variants of the SARS CoV-2 nucleocapsid antigen [Figure 13] Signal recovery of WT versus 3MUT or 8MUT single point mutation variants of SARS CoV-2 nucleocapsid antigen

[0018] Array List SEQ ID NO: 1: Amino acid sequence of coronavirus SARS CoV-2 nucleocapsid SEQ ID NO: 2: Amino acid sequence of coronavirus SARS CoV-2 nucleocapsid fused to one SlyD chaperone SEQ ID NO: 3: Amino acid sequence of the coronavirus SARS CoV-2 nucleocapsid fused to two SlyD chaperones SEQ ID NO: 4: Nucleotide sequence of coronavirus SARS CoV-2 nucleocapsid SEQ ID NO: 5: Nucleotide sequence of the coronavirus SARS CoV-2 nucleocapsid fused to one SlyD chaperone SEQ ID NO: 6: Nucleotide sequence of the coronavirus SARS CoV-2 nucleocapsid fused to two SlyD chaperones SEQ ID NO: 7: Linker peptide SEQ ID NO: 8: Amino acid sequence of SARS CoV-2-N3 MUT variant SEQ ID NO: 9: EcSlyD-EcSlyD-Amino acid sequence of SARS CoV-2-N3 MUT variant SEQ ID NO: 10: Amino acid sequence of SARS CoV-2-N8 MUT variant SEQ ID NO: 11: Amino acid sequence of EcSlyD-EcSlyD-SARS CoV-2-N 8 MUT variant SEQ ID NO: 12: Amino acid sequence of SARS CoV-2-N12 MUT variant SEQ ID NO: 13: Amino acid sequence of EcSlyD-EcSlyD-SARS CoV-2-N12 MUT variant SEQ ID NO: 14: Amino acid sequence of SARS CoV-2-N15 MUT variant SEQ ID NO: 15: Amino acid sequence of EcSlyD-EcSlyD-SARS CoV-2-N15 MUT variant 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 SEQ ID NO: 17: Amino acid sequence of severe acute respiratory syndrome coronavirus (SARS CoV), β-CoV: UniProt ID P59595; Gene Bank Acc.: AY278741 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 SEQ ID NO: 19: Amino acid sequence of human coronavirus NL63 (HCoV-NL63), α-CoV: UniProt ID Q6Q1R8; Gene Bank Acc: AY567487 SEQ ID NO: 20: Amino acid sequence of human coronavirus 229E (HCoV-229E), α-CoV: UniProt ID P15130; Gene Bank Acc: X51325 SEQ ID NO: 21: Amino acid sequence of human coronavirus OC43 (HCoV-OC43), β-CoV: UniProt ID P33469; Gene Bank Acc.: AY585228 SEQ ID NO: 22: Amino acid sequence of human coronavirus HKU1 (HCoV-HKU1), β-CoV: UniProt ID Q5MQC6; Gene Bank Acc.: AY597011 DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the body of the present specification shall control.

[0021] Each element of the present invention is described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context dictates otherwise.

[0022] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0024] Concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "150 mg to 600 mg" should be interpreted not only to include the explicitly recited value of 150 mg to 600 mg, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, ... 580, 590, 600 mg, etc., and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges reciting only a single numerical value. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics described.

[0025] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0026] A "symptom" of disease is an indication of disease that is noticeable by a tissue, organ, or organism with such disease, and includes, but is not limited to, pain, weakness, tenderness, tension, stiffness, and spasms of a tissue, organ, or individual. A "signal" or "signal" of disease includes, but is not limited to, the presence or absence, increase or elevation, decrease or decline, or other change or alteration, of a particular indicator, such as a biomarker or molecular marker, or the onset, presence, or worsening of a symptom. Symptoms of pain include, but are not limited to, an unpleasant sensation that may be experienced as persistent or variable burning, throbbing, itching, or tingling.

[0027] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition, particularly an abnormal medical condition such as an illness or injury in which a tissue, organ, or individual can no longer perform its function efficiently. Typically, although not necessarily, a disease is associated with specific symptoms or signs that indicate the presence of such a condition. Thus, the presence of such symptoms or signs may indicate a tissue, organ, or individual suffering from a disease. Changes in these symptoms or signs may indicate the progression of such a disease. Disease progression is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a "worsening" or "improvement" of the disease. A "worsening" disease is characterized by a decrease in the ability of a tissue, organ, or organism to perform its function efficiently, whereas an "improvement" of a disease is typically characterized by an increase in the ability of a tissue, organ, or individual to perform its function efficiently. Examples of diseases include, but are not limited to, infectious diseases, inflammatory diseases, skin conditions, endocrine disorders, intestinal disorders, neurological disorders, joint disorders, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0028] The term "coronavirus" refers to a group of related viruses that cause disease in mammals and birds. In humans, coronaviruses cause respiratory tract infections that can range from mild to fatal. Mild illnesses include some cases of the common cold, while more deadly varieties can cause "SARS," "MERS," and "COVID-19." Coronaviruses contain a positive-sense, single-stranded RNA genome.

[0029] The viral envelope is formed by a lipid bilayer to which the membrane (M), envelope (E), and spike (S) structural proteins are anchored. Within the envelope, multiple copies of the nucleocapsid (N) protein form the nucleocapsid, which is bound in a continuous, beads-on-a-string conformation to the positive-sense single-stranded RNA genome. The genome contains Orfs 1a and 1b, which encode the replicase / transcriptase polyprotein, followed by sequences encoding the spike (S)-envelope protein, envelope (E)-protein, membrane (M)-protein, and nucleocapsid (N)-protein. Interspersed among these reading frames are reading frames for accessory proteins, which differ among different virus strains.

[0030] There are several known human coronaviruses, four of which cause fairly mild symptoms in patients: Human coronavirus NL63 (HCoV-NL63), α-CoV Human coronavirus 229E (HCoV-229E), α-CoV Human coronavirus HKU1 (HCoV-HKU1), β-CoV Human coronavirus OC43 (HCoV-OC43), β-CoV

[0031] Three human coronaviruses cause potentially severe illness: Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), β-CoV Severe acute respiratory syndrome coronavirus (SARS-CoV), β-CoV Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), β-CoV

[0032] SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). This strain was first discovered in Wuhan, China, and is therefore sometimes referred to as the Wuhan virus. SARS-CoV-2 is highly contagious to humans, and the World Health Organization (WHO) has designated the ongoing COVID-19 pandemic a Public Health Emergency of International Concern. The earliest known cases are believed to have been discovered on November 17, 2019. The SARS-CoV-2 sequence was first published on January 10, 2020 (Wuhan-Hu-1, GenBank accession number MN908947). After the initial Wuhan outbreak, the virus spread to all provinces in China and over 150 other countries in Asia, Europe, North America, South America, Africa, and the Pacific. Symptoms include high fever, sore throat, dry cough, and exhaustion. In severe cases, pneumonia may develop.

[0033] The term "natural coronavirus" refers to a coronavirus occurring in nature, i.e., any coronavirus disclosed above. Natural coronaviruses are understood to include all proteins and nucleic acid molecules present in naturally occurring viruses. Unlike natural coronaviruses, "virus fragments," "virus-like particles," or corona-specific antigens include only some, but not all, of the proteins and nucleic acid molecules present in naturally occurring viruses. Thus, such "virus fragments," "virus-like particles," or corona-specific antigens are not infectious but can still elicit an immune response in patients. Therefore, vaccination with corona-specific virus fragments, coronavirus-specific virus-like particles, or corona-specific antigens results in the production of antibodies in patients against those virus fragments, virus-like particles, or antigens.

[0034] As used herein, a "patient" refers to any mammal, fish, reptile, or bird that can benefit from the diagnosis, prognosis, or treatment described herein. In particular, a "patient" is selected from the group consisting of a laboratory animal (e.g., a mouse, rat, rabbit, or zebrafish), a farm animal (including, for example, a guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, lizard, or goldfish), or a primate, including a chimpanzee, bonobo, gorilla, and human. It is particularly preferred that the "patient" is a human.

[0035] The terms "sample" or "sample of interest" are used interchangeably herein and refer to a portion or piece of a tissue, organ, or individual, typically smaller than the entire tissue, organ, or individual that is intended to represent such tissue, organ, or individual. Upon analysis, the sample yields information regarding the condition of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. Analysis of a sample can be accomplished visually or chemically. Visual analysis includes, but is not limited to, microscopic imaging or radiological scanning of the tissue, organ, or individual, which allows for morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of specific indicators or changes in their amount, concentration, or level. The sample is an in vitro sample and will be analyzed in vitro and will not be returned to the body.

[0036] The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein and refer to single- or double-stranded oligo- or polymers of deoxyribonucleotides or ribonucleotide bases, or both. A nucleotide monomer is composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed via phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, as well as synthetic forms of nucleic acids containing other linkages (e.g., peptide nucleic acids, as described by Nielsen et al. (Science 254:1497-1500, 1991)). Typically, nucleic acids are single- or double-stranded molecules and are composed of naturally occurring nucleotides. A description of a single strand of a nucleic acid also defines (at least in part) the sequence of the complementary strand. Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. Exemplary double-stranded nucleic acid molecules can have 3' or 5' overhangs, and therefore are not required or expected to be completely double-stranded throughout their entire length. Nucleic acids can be obtained by any method known in the art, including, but not limited to, biological, biochemical, or chemical synthesis methods, or RNA amplification and reverse transcription methods. The term nucleic acid includes chromosomes or chromosomal 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 includes or encodes complementary sequences in addition to any sequence expressly indicated.

[0037] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0038] The term "complementarity" refers to the relationship between two structures that follows the lock-and-key principle. In nature, complementarity is a fundamental principle of DNA replication and transcription, as it is a property shared between two DNA or RNA sequences such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences are complementary.

[0039] The term "sequence comparison" refers to a process in which one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are generally used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity or similarity of the test sequence relative to the reference sequence based on the program parameters. In sequence alignment, the term "comparison window" refers to a stretch of consecutive positions of a sequence that is compared to a reference stretch of consecutive positions of a sequence having the same number of positions. The number of consecutive positions selected may range from 10 to 1000, i.e., 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 about 20 to 800 consecutive positions, about 20 to 600 consecutive positions, about 50 to 400 consecutive positions, about 50 to about 200 consecutive positions, or about 100 to about 150 consecutive positions. Methods for aligning sequences for comparison are well known in the art.Optimal alignment of sequences for comparison can be achieved, for example, by the local algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computer implementations 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 supplement)). Suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in 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 available from the National Center for Biotechnology Information (NCBI).

[0040] The algorithm is published at the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Extension of word hits in each direction is terminated when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989), alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, eg, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993).One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0041] The term "at least 90% sequence identity" is used herein with respect to comparison of amino acid or nucleotide sequences. The term "identical" in the context of two or more nucleic acid or polypeptide amino acid sequences refers to two or more sequences or subsequences that are the same, i.e., contain the same sequence of nucleotides or amino acids. The term "at least 90% sequence identity" 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 identity for the respective amino acid or nucleotide sequences.

[0042] The term "at least 90% sequence identity" is used herein in relation to comparison of amino acid or nucleotide sequences. In addition to identical residues (sequence identity), the percentage of conserved residues with similar physicochemical properties (percent similarity), such as leucine and isoleucine, is also commonly used to "quantify homology." The term "at least 90% sequence identity" 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 identity for the respective amino acid or nucleotide sequences. Optionally, the amino acid sequence in question and the reference amino acid sequence exhibit the indicated sequence identity or sequence homology over a contiguous stretch 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 of interest and the reference nucleic acid sequence exhibit the indicated sequence identity or homology over a contiguous stretch 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.

[0043] The term "recombinant DNA molecule" refers to a molecule produced by the combination of two otherwise separated segments of DNA sequence, accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or chemical synthesis. In doing so, polynucleotide segments of desired functions can be joined together to produce a desired combination of functions. Recombinant DNA techniques for the expression of proteins in prokaryotic or lower eukaryotic or higher eukaryotic host cells are well known in the art. They are described, for example, by Sambrook et al., (1989, Molecular Cloning: A Laboratory Manual).

[0044] The terms "vector" and "plasmid" are used interchangeably herein and refer to a protein or polynucleotide or mixture thereof that can be introduced into a cell, or the proteins and / or nucleic acids contained therein can be introduced into a cell. Examples of plasmids include, but are not limited to, plasmids, cosmids, phages, viruses, or artificial chromosomes.

[0045] The term "amino acid" generally refers to any monomeric unit that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups of any of these groups, or analogs. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other exemplary amino acids include, but are not limited to, amino acids containing photoactivatable crosslinkers, metal-binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, 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, other carbohydrate-modified amino acids, amino acids containing polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids containing one or more toxic moieties. As used herein, the term "amino acid" includes the following 20 naturally occurring or genetically encoded alpha-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).

[0046] The terms "measurement", "measuring", "detect" or "detection" preferably include qualitative, semi-quantitative or quantitative measurements. The term "detecting the presence" refers to a qualitative measurement, indicating presence or absence without a statement about the quantity (e.g., a yes or no statement). The term "detecting the amount" refers to a quantitative measurement where an absolute number is detected (ng). The term "detecting the concentration" refers to a quantitative measurement where the amount is determined for a given volume (e.g., ng / ml).

[0047] The term "immunoglobulin (Ig)," as used herein, refers to immune conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells by their transmembrane regions and include molecules such as, but not limited to, B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, β2-microglobulin (approximately 2M), CD3, CD4, and CDS.

[0048] Typically, the term "antibody," as used herein, refers to a secretory immunoglobulin that lacks a transmembrane region and can therefore be 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, designated by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), and each plays a different role and directs the appropriate immune response to different types of antigens. Different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal, respiratory, and genitourinary tracts, as well as in saliva, tears, and breast milk, where it prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD primarily functions as an antigen receptor for unexposed B cells 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 is involved in allergic reactions through binding to allergens, which triggers histamine release from mast cells and basophils. IgE is also involved in protection against parasites (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 that can cross the placenta and confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four distinct IgG subclasses (IgG1, 2, 3, and 4), named in order of abundance in serum, with IgG1 being the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG4 (approximately 4%).The biological profiles of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in a monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in the early stage of B cell-mediated (humoral) immunity, eliminating pathogens before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are not only found as monomers, but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM of bony fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies are typically made up of four polypeptide chains, including two identical heavy chains and two identical light chains linked via disulfide bonds, resembling a "Y"-shaped macromolecule. Each chain contains several immunoglobulin domains, some of which are constant domains and others are variable domains. Immunoglobulin domains consist of a two-layer sandwich of seven to nine antiparallel chains arranged in two sheets. Typically, an antibody heavy chain contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) domains and one of which is a variable domain (VH). A light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). For example, a human IgG heavy chain is composed of four Ig domains linked from N- to C-terminus in the order VwCH1-CH2-CH3 (also referred to as VwCy1-Cy2-Cy3), while a human IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, and is either kappa or lambda type (VK-CK or VA-CA). By way of example, the constant chain of human IgG contains 447 amino acids.Throughout this specification and claims, the numbering of amino acid positions in immunoglobulins is that of the "EU index" as in Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C. (1991) Sequences of proteins of immunological interest, 5th ed., US Department of Health and Human Services, National Institutes of Health, Bethesda, MD. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Thus, the CH domain in the context of IgG is as follows: "CH1" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0049] The term "binding affinity" generally refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmon resonance-based assays (e.g., BIAcore assays, such as those described in PCT Application Publication WO 2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competitive assays (e.g., RIAs). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0050] The term "antigen (Ag)" refers to a molecule or molecular structure that is bound by an antigen-specific antibody (Ab) or B-cell antigen receptor (BCR). The presence of an antigen in the body typically triggers an immune response. Within the body, each antibody is specifically produced to match the antigen after contact with it by cells of the immune system, allowing for accurate identification or matching of the antigen and the initiation of a personalized response. In most cases, an antibody can only react with and bind to one specific antigen. However, in some instances, antibodies can cross-react and bind to more than one antigen. Antigens are typically proteins, peptides (amino acid chains), and polysaccharides (monosaccharides / single sugar chains), or combinations thereof.

[0051] In diagnostic testing, antigens are often used in serological tests to assess whether a patient has been exposed to a particular pathogen (e.g., a virus or bacteria) and has developed antibodies to such pathogen. Typically, these antigens are recombinantly produced and can be linear peptides or more complex folded molecules intended to represent the native antigen.

[0052] To more closely resemble native antigens and achieve high epitope density, antigens can be generated by polymerizing monomeric antigens through chemical crosslinking. A wide variety of homobifunctional and heterobifunctional crosslinkers are available and well known in the art. However, for use as specific agents in serological assays, chemically induced polymerization of antigens has several significant drawbacks. For example, the insertion of crosslinker moieties into the antigen can impair antigenicity by disrupting the native-like conformation or by masking important epitopes. Furthermore, the introduction of non-native tertiary contacts can interfere with the reversibility of protein folding / unfolding and can cause interference problems in immunoassay mixtures that must be overcome by anti-interference strategies.

[0053] A more recent technique involves fusing an antigen of interest to an oligomeric chaperone, thereby conferring high epitope density to the antigen. The advantages of this technique lie in its high reproducibility and the triple function of the oligomeric chaperone fusion partner: first, the chaperone improves the expression rate of the fusion polypeptide in the host cell (e.g., E. coli); second, the chaperone facilitates the refolding process of the target antigen, improving its overall solubility; and third, the chaperone reproducibly assembles the target antigen into ordered oligomeric structures.

[0054] The term "chaperone" is well known in the art and refers to a protein folding helper that assists in folding and maintaining the structural integrity of other proteins. Examples of folding helpers are described in detail in WO 03 / 000877. By way of example, chaperones of the peptidyl-prolyl isomerase class, such as chaperones of the FKBP family, can be used for fusion to antigen variants. Examples of FKBP chaperones suitable as fusion partners are FkpA (aa 26-270, UniProt ID P45523), SlyD (aa 1-165, UniProt ID P0A9K9), and SlpA (aa 2-149, UniProt ID P0AEM0). Another chaperone suitable as a fusion partner is Skp (aa 21-161, UniProt ID P0AEU7), a trimeric chaperone from the periplasm of Escherichia coli that does not belong to the FKBP family. It is not necessary to use the complete sequence of the chaperone: functional fragments of the chaperone (so-called binding-competent modules) that still have the required capacity and function can also be used (see WO 98 / 13496).

[0055] The antigen may further comprise an "effector group," such as a "tag" or "label." The term "tag" refers to an effector group that provides the antigen with the ability to bind to or be bound to other molecules. Examples of tags include, but are not limited to, a His tag attached to an antigen sequence, for example, to enable purification. A tag may also comprise a bioaffine binding pair partner, which allows the antigen to be bound by the second partner of the binding pair. The term "bioaffine binding pair" refers to two partner molecules (i.e., two partners in a pair) that have a strong affinity for binding to each other. Examples of bioaffine binding pair partners are a) biotin or biotin analog / avidin or streptavidin; b) hapten / anti-hapten antibody or antibody fragment (e.g., digoxin / anti-digoxin antibody); c) saccharide / lectin; d) complementary oligonucleotide sequences (e.g., complementary LNA sequences), and generally e) ligand / receptor.

[0056] The term "label" refers to an effector group that allows for detection of an antigen. 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.

[0057] As used herein, "particle" means a small, localized object to which physical properties such as volume, mass, or average size can be assigned. Thus, particles may be symmetrical, spherical, essentially spherical, or spherical in shape, or may have an irregular, asymmetric shape or form. The size of a particle may vary. The term "microparticle" refers to a particle having a diameter in the nanometer and micrometer range.

[0058] Microparticles, as defined herein above, may comprise or consist of any suitable material known to those skilled in the art, for example, they may comprise, consist of, or consist essentially of inorganic or organic materials. Typically, they may comprise, consist of, or consist essentially of metals or metal alloys, or organic materials, or may comprise, consist of, or consist essentially of carbohydrate elements. Examples of materials contemplated for microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silica, and ferromagnetic metals, alloys, or hybrid materials. In one embodiment, the microparticles are magnetic or ferromagnetic metals, alloys, or hybrids. In further embodiments, the material may have specific properties, such as hydrophobicity or hydrophilicity. Such microparticles are typically dispersed in aqueous solutions, maintaining a small negative surface charge while keeping the microparticles separate and avoiding nonspecific clustering.

[0059] In one embodiment of the present invention, the microparticles are paramagnetic microparticles and their separation in the measurement methods according to the present disclosure is facilitated by magnetic forces: a magnetic force is applied to extract the paramagnetic or magnetic particles from the solution / suspension and optionally retain them, the liquid of the solution / suspension can be removed and the particles can be, for example, washed.

[0060] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent, such as a drug for treating a disorder or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the method of the invention. Typically, the kit may further comprise a carrier means compartmentalized to receive one or more container means, such as vials and tubes, under close custody. In particular, each of the container means contains one of the separate elements used in the method of the first aspect. The kit may further comprise one or more other containers containing additional materials, including, but not limited to, buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. Labeling may be provided on the container to indicate that the composition is to be used in a particular application and may also indicate instructions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly to a computer or data processing device. Additionally, the kit may contain standard amounts of biomarkers, as described elsewhere herein, for calibration purposes.

[0061] "Package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic or drug product, which contain information about the indications, uses, dosage, administration, contraindications of such therapeutic or drug product, other therapeutic products with which the packaged product may be combined, and / or warnings regarding its use.

[0062] Embodiment Currently available ELISA-style immunoassays for detecting anti-SARS CoV-2 virus antibodies in patient samples use spike protein-derived antigens as immunoreactive reagents. However, the inventors have found that these assays lack specificity and produce a significant number of false-positive results. Surprisingly, by restricting the antigens to the coronavirus nucleocapsid, as further described below, the number of false-positive samples can be significantly reduced while maintaining the high sensitivity of the assay.

[0063] Furthermore, all ongoing vaccination strategies are focused on developing spike protein-based vaccines. Using spike protein-derived antigens to detect anti-SARS CoV-2 viral antibodies in samples from vaccinated patients makes it possible to determine whether vaccination was successful and whether the patient developed anti-spike antibodies. However, because the extent to which the long-term effects of vaccination and natural SARS CoV-2 infection interact and affect patients is still unknown, it is important to be able to distinguish whether a patient has been exposed to natural SARS CoV-2 infection or has previously been vaccinated. Therefore, there is an urgent need for an anti-SARS CoV-2 antibody assay that not only detects anti-spike antibodies but also allows for the determination of anti-SARS CoV-2 antibodies against other viral proteins.

[0064] Thus, in a first aspect, the present invention relates to a corona antigen suitable for detecting antibodies against a coronavirus in an isolated biological sample, comprising the corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof. In an embodiment, the corona antigen for detecting antibodies against a coronavirus in an isolated biological sample comprises the corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof.

[0065] In an embodiment, the antigen does not comprise additional coronavirus-specific amino acid sequences.

[0066] In embodiments, the corona antigen is immunoreactive, i.e., antibodies present in a biological sample bind to the antigen. Thus, any peptide derived from a corona nucleocapsid that is not bound by an antibody is not included.

[0067] As shown in Figures 1 and 2, the amino acid sequence of SARS-CoV-2 shows approximately 93% sequence homology and approximately 90% sequence identity to its closest relative, SARS-CoV. The sequence identity and homology with other coronaviruses is still much lower, as shown. Therefore, due to the already limited sequence identity and homology, a corona antigen containing the corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 is specific for detecting SARS-CoV and SARS-CoV-2.

[0068] In an embodiment, the coronavirus is a SARS-CoV or SARS CoV-2 virus, particularly a SARS CoV-2 virus. In a specific embodiment, the coronavirus nucleocapsid is a SARS CoV-2-specific nucleocapsid. In particular, a coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 is specific for detecting SARS CoV-2.

[0069] In embodiments, the corona antigens do not immunologically cross-react, i.e., exhibit significantly reduced or completely absent immunological reactivity, with antibodies or a subset of antibodies raised against corresponding nucleocapsid antigens of other coronaviruses. In particular, the corona antigens do not immunologically cross-react with corresponding nucleocapsid antigens from coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. In particular, the corona antigens do not immunologically cross-react with corresponding nucleocapsid antigens from coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0070] In embodiments, the corona antigens are soluble and therefore suitable for use in in vitro assays aimed at detecting antibodies against the antigens in isolated biological samples.

[0071] Therefore, the corona antigens are suitable for use in in vitro assays aimed at detecting anti-corona antibodies with high sensitivity and specificity. In embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the sensitivity is >99% or >99.5%. In certain embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the specificity is >99% or >99.5%. In certain embodiments, the specificity is >99.8%. In certain embodiments, the sensitivity is 100% and the specificity is 99.8%.

[0072] In embodiments, the corona antigen is suitable for or detects antibodies against coronaviruses in a fluid sample. In certain embodiments, the sample is a human sample, particularly a human body 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.

[0073] In embodiments, the corona antigen is a linear antigen or is in its native state. In certain embodiments, the corona nucleocapsid-specific amino acid sequence contained in the corona antigen is folded in its native state.

[0074] In embodiments, variants of the corona nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 are encompassed. These variants are readily generated by one skilled in the art by conservative or homologous substitutions of the disclosed amino acid sequences (e.g., substitution of cysteine ​​with alanine or serine, etc.). In embodiments, variants exhibit modifications to the amino acid sequence, particularly selected from the group consisting of amino acid exchanges, deletions, or insertions, compared to the amino acid sequence of SEQ ID NO: 1.

[0075] In embodiments, amino acids are deleted at the C-terminus or N-terminus or 1 to 10 amino acids, in one embodiment 1 to 5 amino acids, are inserted at one or both termini. In particular, variants may be isoforms representing the most common protein isoforms. In one embodiment, such substantially similar proteins have at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence identity to SEQ ID NO: 1.

[0076] In embodiments, the coronavirus nucleocapsid variant comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.

[0077] In embodiments, the variant comprises a post-translational modification, in particular selected from the group consisting of glycosylation or phosphorylation.

[0078] It is understood that such variants are classified as corona nucleocapsid variants, i.e., can be detected by binding to anti-corona antibodies present in an isolated sample.

[0079] In embodiments, the overall three-dimensional structure of the corona nucleocapsid remains unchanged, so that epitopes that were previously accessible for binding to antibodies (i.e., wild type) remain accessible in the variant.

[0080] In an embodiment, the corona antigen further comprises at least one chaperone, and thus the corona antigen comprises the corona nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 above or below, and the amino acid sequence of the chaperone.

[0081] In certain embodiments, the corona antigen comprises two chaperones. In certain embodiments, the chaperones are selected from the group consisting of SlyD, SlpA, FkpA, and Skp. In certain embodiments, the chaperone is SlyD, specifically having the amino acid sequence given by UniProt ID P0A9K9.

[0082] In certain embodiments, the corona antigen comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and one SlyD chaperone. In certain embodiments, the corona antigen comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and two SlyD chaperones.

[0083] The fusion of the two chaperones results in higher solubility of the resulting antigen.

[0084] In embodiments, the chaperone is fused to a corona nucleocapsid-specific amino acid sequence at the N-terminus and / or C-terminus of the nucleocapsid, particularly at the N-terminus of the nucleocapsid. Thus, in certain embodiments, a corona antigen comprises one SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence. In certain embodiments, a corona antigen comprises two SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence. In embodiments, a corona antigen comprises one SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence and one SlyD chaperone C-terminal of a corona nucleocapsid-specific amino acid sequence.

[0085] In certain embodiments, the corona antigen further comprises a linker sequence. These sequences are not specific to anti-coronavirus antibodies and are not recognized in in vitro diagnostic immunoassays. In particular, the corona antigen comprises a linker sequence between the corona nucleocapsid sequence and one or more chaperones. In certain embodiments, the linker is a Gly-rich linker. In certain embodiments, the linker has the sequence set forth in SEQ ID NO:7.

[0086] In certain embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO: 2. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0087] In certain embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO: 3. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of SEQ ID NO: 3.

[0088] In embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15.

[0089] It is understood that a corona antigen consisting 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 sequences but may still contain other chemical molecules such as labels and / or tags.

[0090] In certain embodiments, sequence homology to 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 certain embodiments, sequence homology to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is at least 98%.

[0091] In certain embodiments, the sequence homology to 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 certain embodiments, the sequence homology to 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%.

[0092] In embodiments, the corona antigen further comprises a tag or label, and thus the corona antigen comprises the corona 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 set forth above or below, as well as the tag and / or label, and optionally the amino acid sequences of one or more chaperones.

[0093] In certain embodiments, the tag allows for direct or indirect binding of the corona antigen to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, a hapten, or a complementary oligonucleotide sequence (particularly a complementary LNA sequence). In certain embodiments, the tag is biotin.

[0094] In certain embodiments, the label allows for detection of the corona antigen. In certain embodiments, the corona-specific nucleocapsid sequence is labeled. In embodiments in which at least one chaperone is present in the antigen, the corona-specific nucleocapsid sequence is labeled, the at least one chaperone 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 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.

[0095] In a second aspect, the present invention relates to a composition comprising a coronavirus antigen suitable for detecting antibodies to a coronavirus in an isolated biological sample, the coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof. In an embodiment, the coronavirus antigen for detecting antibodies to a coronavirus in an isolated biological sample comprises the coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof.

[0096] In an embodiment, the corona antigen does not comprise additional coronavirus-specific amino acid sequences.

[0097] In embodiments, the corona antigen is immunoreactive, i.e., antibodies present in a biological sample bind to the antigen. Thus, any peptide derived from a corona nucleocapsid that is not bound by an antibody is not included.

[0098] As shown in Figures 1 and 2, the amino acid sequence of SARS-CoV-2 shows approximately 93% sequence homology and approximately 90% sequence identity to its closest relative, SARS-CoV. The sequence identity and homology with other coronaviruses is still much lower, as shown. Therefore, due to the already limited sequence identity and homology, a corona antigen containing the corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 is specific for detecting SARS-CoV and SARS-CoV-2.

[0099] In an embodiment, the coronavirus is a SARS-CoV or SARS CoV-2 virus, particularly a SARS CoV-2 virus. In a specific embodiment, the coronavirus nucleocapsid is a SARS CoV-2-specific nucleocapsid. In particular, a coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 is specific for detecting SARS CoV-2.

[0100] In embodiments, the corona antigens do not immunologically cross-react, i.e., exhibit significantly reduced or completely absent immunological reactivity, with antibodies or a subset of antibodies raised against corresponding nucleocapsid antigens of other coronaviruses. In particular, the corona antigens do not immunologically cross-react with corresponding nucleocapsid antigens from coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. In particular, the corona antigens do not immunologically cross-react with corresponding nucleocapsid antigens from coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0101] In embodiments, the corona antigens are soluble and therefore suitable for use in in vitro assays aimed at detecting antibodies against the antigens in isolated biological samples.

[0102] Therefore, the corona antigens are suitable for use in in vitro assays aimed at detecting anti-corona antibodies with high sensitivity and specificity. In embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the sensitivity is >99% or >99.5%. In certain embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the specificity is >99% or >99.5%. In certain embodiments, the specificity is >99.8%. In certain embodiments, the sensitivity is 100% and the specificity is 99.8%.

[0103] In embodiments, the corona antigen is suitable for or detects antibodies against coronaviruses in a fluid sample. In certain embodiments, the sample is a human sample, particularly a human body 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.

[0104] In embodiments, the corona antigen is a linear antigen or is in its native state. In certain embodiments, the corona nucleocapsid-specific amino acid sequence contained in the corona antigen is folded in its native state.

[0105] In embodiments, variants of the corona nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 are encompassed. These variants can be readily generated by one skilled in the art by conservative or homologous substitutions of the disclosed amino acid sequences (e.g., substitution of cysteine ​​with alanine or serine, etc.). In embodiments, variants exhibit modifications to the amino acid sequence, particularly selected from the group consisting of amino acid exchanges, deletions, or insertions, compared to the amino acid sequence of SEQ ID NO: 1.

[0106] In embodiments, amino acids are deleted at the C-terminus or N-terminus or 1 to 10 amino acids, in one embodiment 1 to 5 amino acids, are inserted at one or both termini. In particular, variants may be isoforms representing the most common protein isoforms. In one embodiment, such substantially similar proteins have at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence identity to SEQ ID NO: 1.

[0107] In embodiments, the coronavirus nucleocapsid variant comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.

[0108] In embodiments, the variant comprises a post-translational modification, in particular selected from the group consisting of glycosylation or phosphorylation.

[0109] It is understood that such variants are classified as corona nucleocapsid variants, i.e., can be detected by binding to anti-corona antibodies present in an isolated sample.

[0110] In embodiments, the overall three-dimensional structure of the corona nucleocapsid remains unchanged, so that epitopes that were previously accessible for binding to antibodies (i.e., wild type) remain accessible in the variant.

[0111] In an embodiment, the corona antigen further comprises at least one chaperone, and thus the corona antigen comprises the corona nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 above or below, and the amino acid sequence of the chaperone.

[0112] In certain embodiments, the corona antigen comprises two chaperones. In certain embodiments, the chaperones are selected from the group consisting of SlyD, SlpA, FkpA, and Skp. In certain embodiments, the chaperone is SlyD, specifically having the amino acid sequence given by UniProt ID P0A9K9.

[0113] In certain embodiments, the corona antigen comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and one SlyD chaperone. In certain embodiments, the corona antigen comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and two SlyD chaperones.

[0114] The fusion of the two chaperones results in higher solubility of the resulting antigen.

[0115] In embodiments, the chaperone is fused to a corona nucleocapsid-specific amino acid sequence at the N-terminus and / or C-terminus of the nucleocapsid, particularly at the N-terminus of the nucleocapsid. Thus, in certain embodiments, a corona antigen comprises one SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence. In certain embodiments, a corona antigen comprises two SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence. In embodiments, a corona antigen comprises one SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence and one SlyD chaperone C-terminal of a corona nucleocapsid-specific amino acid sequence.

[0116] In certain embodiments, the corona antigen further comprises a linker sequence. These sequences are not specific to anti-coronavirus antibodies and are not recognized in in vitro diagnostic immunoassays. In particular, the corona antigen comprises a linker sequence between the corona nucleocapsid sequence and one or more chaperones. In certain embodiments, the linker is a Gly-rich linker. In certain embodiments, the linker has the sequence set forth in SEQ ID NO:7.

[0117] In certain embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO: 2. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0118] In certain embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO: 3. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of SEQ ID NO: 3.

[0119] In embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15.

[0120] It is understood that a corona antigen consisting 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 labels and / or tags.

[0121] In certain embodiments, sequence homology to 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 certain embodiments, sequence homology to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is at least 98%.

[0122] In certain embodiments, the sequence homology to 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 certain embodiments, the sequence homology to 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%.

[0123] In embodiments, the corona antigen further comprises a tag or label. In certain embodiments, the corona-specific nucleocapsid sequence is labeled. In embodiments in which at least one chaperone is present in the antigen, the corona-specific nucleocapsid sequence is labeled, the at least one chaperone is labeled, or both are labeled.

[0124] Thus, the corona antigen comprises the corona 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, as well as a tag and / or label, and optionally the amino acid sequences of one or more chaperones.

[0125] In certain embodiments, the tag allows the antigen to be directly or indirectly bound to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, a hapten, or a complementary oligonucleotide sequence (particularly a complementary LNA sequence). In certain embodiments, the tag is biotin.

[0126] In certain embodiments, the label allows for 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 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.

[0127] In embodiments, the composition comprises one or more additional coronavirus antigens. In certain embodiments, the composition comprises one, two, or three additional antigens. In certain embodiments, the composition comprises one or more additional coronavirus antigens comprising the amino acid sequence of the E protein, M protein, and / or S protein, or portions thereof. In certain embodiments, the composition comprises an additional coronavirus antigen comprising the amino acid sequence of the S protein or portions thereof (e.g., the receptor-binding domain of the S protein).

[0128] In certain embodiments, the additional corona antigen is immunoreactive, i.e., antibodies present in the biological sample bind to the antigen. Thus, any peptides derived from corona that are not bound by anti-corona antibodies are not included.

[0129] In embodiments, the additional coronavirus antigens do not immunologically cross-react with antibodies or antibody subsets raised against corresponding antigens of other coronaviruses, i.e., they exhibit significantly reduced or completely absent immunological reactivity. In particular, the additional coronavirus antigens do not immunologically cross-react with corresponding antigens from coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. In particular, the additional coronavirus antigens do not immunologically cross-react with corresponding antigens from coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0130] In embodiments, the additional coronavirus antigen is soluble, and therefore suitable for use in in vitro assays aimed at detecting antibodies against the antigen in an isolated biological sample.

[0131] In a third aspect, the present invention provides a method for producing a corona antigen specific for a coronavirus nucleocapsid, the method comprising: a) culturing a host cell transformed with an expression vector operably linked with a recombinant DNA molecule encoding a coronavirus antigen as described above for the first aspect of the present invention; b) expressing the polypeptide; and c) Purification of the polypeptide The present invention relates to a method comprising:

[0132] Optionally, as an additional step d), functional solubilization must be performed by refolding techniques known in the art, so that the corona nucleocapsid antigen is in a soluble and immunoreactive conformation.

[0133] In certain embodiments, the host cell is an E. coli cell, a CHO cell, or an HEK cell. In certain embodiments, the host cell is an E. coli cell.

[0134] In embodiments in which the antigen comprises a coronavirus nucleocapsid and one or more chaperones, a recombinant DNA molecule according to the present invention may also include a sequence encoding a linker peptide of 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, the addition of a non-coronavirus-specific linker or peptide fusion amino acid sequence to the coronavirus nucleocapsid is possible because these sequences are not specific for anti-coronavirus antibodies and are not recognized in in vitro diagnostic immunoassays.

[0135] In a specific embodiment, the recombinant DNA molecule comprises the sequence set forth in SEQ ID NO:4.

[0136] In a specific embodiment, the recombinant DNA molecule comprises the sequence set forth in SEQ ID NO:5.

[0137] In a specific embodiment, the recombinant DNA molecule comprises the sequence set forth in SEQ ID NO:6.

[0138] In a fourth aspect, the present invention relates to a method for detecting antibodies specific to coronavirus in an isolated biological sample, using 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 a method according to the third aspect of the invention as a capture reagent and / or binding partner for said anti-coronavirus antibodies.

[0139] In a fifth aspect, the present invention provides a method for detecting antibodies specific to a coronavirus in an isolated biological sample, comprising: a) forming an immunoreaction mixture by mixing an isolated biological sample with a coronavirus antigen or a composition containing a coronavirus antigen; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the isolated biological sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immunoreaction product; and c) detecting the presence, amount and / or concentration of any of the immune reaction products The present invention relates to a method comprising:

[0140] In 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%, or >99.5%. In certain embodiments, the sensitivity is >99% or >99.5%. In certain embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the specificity is >99% or >99.5%. In certain embodiments, the specificity is >99.8%. In certain embodiments, the sensitivity is 100% and the specificity is 99.8%.

[0141] In embodiments, the antibodies detected by the methods of the invention are anti-coronavirus antibodies of the IgG, IgM or IgA subclass, or all three subclasses, in the same immunoassay.

[0142] In embodiments, the antibodies detected are antibodies against the nucleocapsid of a coronavirus, in particular antibodies against the nucleocapsid of the SARS-CoV or SARS CoV-2 virus. In particular embodiments, the antibodies detected are against the nucleocapsid of the SARS CoV-2 virus.

[0143] In embodiments, the isolated biological sample in which coronavirus-specific antibodies are detected is a human sample, particularly a human body 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.

[0144] In an embodiment, the corona antigen mixed with the biological sample isolated in step a) comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof. In an embodiment, the corona antigen does not comprise any additional coronavirus-specific amino acid sequence.

[0145] In embodiments, the corona antigen is immunoreactive, i.e., antibodies present in a biological sample bind to the antigen. Thus, any peptide derived from a corona nucleocapsid that is not bound by an antibody is not included.

[0146] As shown in Figures 1 and 2, the amino acid sequence of SARS-CoV-2 shows approximately 93% sequence homology and approximately 90% sequence identity to its closest relative, SARS-CoV. The sequence identity and homology with other coronaviruses is still much lower, as shown. Therefore, due to the already limited sequence identity and homology, a corona antigen containing the corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 is specific for detecting SARS-CoV and SARS-CoV-2.

[0147] In an embodiment, the coronavirus is a SARS-CoV or SARS CoV-2 virus, particularly a SARS CoV-2 virus. In a specific embodiment, the coronavirus nucleocapsid is a SARS CoV-2-specific nucleocapsid. In particular, a coronavirus antigen comprising the coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 is specific for detecting SARS CoV-2.

[0148] In embodiments, the corona antigens do not immunologically cross-react, i.e., exhibit significantly reduced or completely absent immunological reactivity, with antibodies or a subset of antibodies raised against corresponding nucleocapsid antigens of other coronaviruses. In particular, the corona antigens do not immunologically cross-react with corresponding nucleocapsid antigens from coronavirus strains selected from the group consisting of MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. In particular, the corona antigens do not immunologically cross-react with corresponding nucleocapsid antigens from coronavirus strains selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1.

[0149] In embodiments, the corona antigens are soluble and therefore suitable for use in in vitro assays aimed at detecting antibodies against the antigens in isolated biological samples.

[0150] Therefore, the corona antigens are suitable for use in in vitro assays aimed at detecting anti-corona antibodies with high sensitivity and specificity. In embodiments, the sensitivity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the sensitivity is >99% or >99.5%. In certain embodiments, the sensitivity is 100%. In embodiments, the specificity is >95%, >96%, >97%, >98%, >99%, or >99.5%. In certain embodiments, the specificity is >99% or >99.5%. In certain embodiments, the specificity is >99.8%. In certain embodiments, the sensitivity is 100% and the specificity is 99.8%.

[0151] In embodiments, the corona antigens are soluble, and therefore suitable for use in the present in vitro methods.

[0152] In embodiments, the corona antigen is a linear antigen or is in its native state. In certain embodiments, the corona nucleocapsid-specific amino acid sequence contained in the antigen is folded in its native state.

[0153] In embodiments, variants of the corona nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 are encompassed. These variants can be readily generated by one skilled in the art by conservative or homologous substitutions of the disclosed amino acid sequences (e.g., substitution of cysteine ​​with alanine or serine, etc.). In embodiments, variants exhibit modifications to the amino acid sequence, particularly selected from the group consisting of amino acid exchanges, deletions, or insertions, compared to the amino acid sequence of SEQ ID NO: 1.

[0154] In embodiments, amino acids are deleted at the C-terminus or N-terminus or 1 to 10 amino acids, in one embodiment 1 to 5 amino acids, are inserted at one or both termini. In particular, variants may be isoforms representing the most common protein isoforms. In one embodiment, such substantially similar proteins have at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence identity to SEQ ID NO: 1.

[0155] In embodiments, the coronavirus nucleocapsid variant comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.

[0156] In embodiments, the variant comprises a post-translational modification, in particular selected from the group consisting of glycosylation or phosphorylation.

[0157] It is understood that such variants are classified as corona nucleocapsid variants, i.e., can be detected by binding to anti-corona antibodies present in an isolated sample.

[0158] In embodiments, the overall three-dimensional structure of the corona nucleocapsid remains unchanged, so that epitopes that were previously accessible for binding to antibodies (i.e., wild type) remain accessible in the variant.

[0159] In an embodiment, the corona antigen further comprises at least one chaperone, and thus the corona antigen comprises the corona nucleocapsid-specific amino acid sequence of SEQ ID NO: 1 above or below, and the amino acid sequence of the chaperone.

[0160] In certain embodiments, the corona antigen comprises two chaperones. In certain embodiments, the chaperones are selected from the group consisting of SlyD, SlpA, FkpA, and Skp. In certain embodiments, the chaperone is SlyD, specifically having the amino acid sequence given by UniProt ID P0A9K9.

[0161] In certain embodiments, the corona antigen comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and one SlyD chaperone. In certain embodiments, the corona antigen comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and two SlyD chaperones.

[0162] The fusion of the two chaperones results in higher solubility of the resulting antigen.

[0163] In embodiments, the chaperone is fused to a corona nucleocapsid-specific amino acid sequence at the N-terminus and / or C-terminus of the nucleocapsid, particularly at the N-terminus of the nucleocapsid. Thus, in certain embodiments, a corona antigen comprises one SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence. In certain embodiments, a corona antigen comprises two SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence. In embodiments, a corona antigen comprises one SlyD chaperone N-terminal of a corona nucleocapsid-specific amino acid sequence and one SlyD chaperone C-terminal of a corona nucleocapsid-specific amino acid sequence.

[0164] In certain embodiments, the corona antigen further comprises a linker sequence. These sequences are not specific to anti-coronavirus antibodies and are not recognized in in vitro diagnostic immunoassays. In particular, the corona antigen comprises a linker sequence between the corona nucleocapsid sequence and one or more chaperones. In certain embodiments, the linker is a Gly-rich linker. In certain embodiments, the linker has the sequence set forth in SEQ ID NO:7.

[0165] In certain embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO: 2. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0166] In certain embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO: 3. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of SEQ ID NO: 3.

[0167] In embodiments, the corona antigen comprises the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. In embodiments, the corona antigen does not comprise any additional amino acid sequence. In certain embodiments, the corona antigen consists of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15.

[0168] It is understood that a corona antigen consisting 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 labels and / or tags.

[0169] In certain embodiments, sequence homology to 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 certain embodiments, sequence homology to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 is at least 98%.

[0170] In certain embodiments, the sequence homology to 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 certain embodiments, the sequence homology to 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%.

[0171] In embodiments, the corona antigen further comprises a tag or label, and thus the corona antigen comprises the corona 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 set forth above or below, as well as the tag and / or label, and optionally the amino acid sequences of one or more chaperones.

[0172] In certain embodiments, the tag allows the antigen to be directly or indirectly bound to a solid phase. In certain embodiments, the tag is a partner of a bioaffine binding pair. In certain embodiments, the tag is selected from the group consisting of biotin, digoxin, a hapten, or a complementary oligonucleotide sequence (particularly a complementary LNA sequence). In certain embodiments, the tag is biotin.

[0173] In certain embodiments, the label allows for detection of the corona antigen. In certain embodiments, the corona-specific nucleocapsid sequence is labeled. In embodiments in which at least one chaperone is present in the antigen, the corona-specific nucleocapsid sequence is labeled, at least one chaperone is labeled, or both are labeled.

[0174] 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.

[0175] In embodiments, the method comprises the further step of adding a solid phase to the immune reaction mixture. In embodiments, the solid phase is a solid phase extraction (SPE) cartridge or beads. In certain embodiments, the solid phase comprises or consists 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., the intended capture molecule. Those skilled in the art will be familiar with which coatings are suitable for which analytes. The particles can be made of a variety of different materials. The beads can have different sizes and can include porous or non-porous surfaces.

[0176] In certain embodiments, the particles are microparticles. In embodiments, the microparticles have a diameter of 50 nanometers to 20 micrometers. In embodiments, the microparticles have a diameter of 100 nm to 10 μm. In embodiments, the microparticles have a diameter of 200 nm to 5 μm, particularly 750 nm to 5 μm, and especially 750 nm to 2 μm. In certain embodiments, the microparticles are magnetic or paramagnetic. In particular, the microparticles are paramagnetic.

[0177] In embodiments, the solid phase is added either before adding the sample to the antigen or after the immune reaction mixture is formed. Thus, the addition of the solid phase can occur in step a) of the method, in step b) of the method, or after step b) of the method.

[0178] In an embodiment, the method implemented is an immunoassay for detecting anti-corona antibodies in an isolated biological sample. Immunoassays for detecting antibodies are well known in the art, as are methods for performing such assays and practical applications and procedures. The corona nucleocapsid antigens according to the present invention can be used to improve assays for detecting anti-corona antibodies, regardless of the label used, the detection format (e.g., radioisotope assay, enzyme immunoassay, electrochemiluminescence assay, etc.) or the assay principle (e.g., test strip assay, sandwich assay, indirect test concept, homogeneous assay, etc.).

[0179] In one embodiment, the method implemented is an immunoassay for detecting anti-coronavirus antibodies in a sample isolated according to the so-called double antigen sandwich concept (DAGS). This assay concept is sometimes also called the double antigen bridging concept, since two antigens are bridged by an antibody analyte. Such assays utilize the ability of an antibody to bind to at least two different molecules of a given antigen with its two (IgG, IgE), four (IgA), or ten (IgM) paratopes.

[0180] In an embodiment, an immunoassay for the determination of anti-corona antibodies in a DAGS format is performed by incubating a sample containing anti-corona antibodies with two different coronavirus antigens, namely a first ("capture") coronavirus antigen and a second coronavirus ("detection") antigen, each of the two antigens being specifically bound by an anti-corona antibody.

[0181] In embodiments, the structures of the "capture antigen" and the "detection antigen" are immunologically cross-reactive. A prerequisite for carrying out the method is that the relevant epitope(s) be present on both antigens. Thus, both antigens contain the corona nucleocapsid-specific amino acid sequences described above or below. In embodiments, the two antigens contain the same or different fusion moieties (e.g., SlyD fused to a corona nucleocapsid-specific antigen tagged for binding by a solid phase, and FkpA fused to a corona nucleocapsid-specific antigen labeled for detection), significantly mitigating the problem of nonspecific binding and thus reducing the risk of false-positive results.

[0182] In embodiments, the first antigen can be directly or indirectly bound to a solid phase and typically has an effector group that is part of a bioaffine binding pair. In certain embodiments, the first antigen is conjugated to biotin, and the complementary solid phase is coated with either avidin or streptavidin. In certain embodiments, the second antigen carries a label, alone or complexed with another molecule, that confers specific detectability to this antigen molecule. In certain embodiments, the second antigen carries a ruthenium complex label.

[0183] Thus, in step b) of the method, an immune reaction mixture is formed comprising the first antigen, the sample antibody and the second antigen.

[0184] This ternary complex, consisting of an analyte antibody sandwiched between two antigen molecules, is called an immune complex or immune reaction product.

[0185] In embodiments, the method may comprise the further step of separating the liquid phase from the solid phase.

[0186] Thus, in embodiments, a method for detecting antibodies specific to a coronavirus in an isolated sample comprises: a) adding to the sample a first corona antigen having an effector group that can be directly or indirectly bound to a solid phase and that is part of a bioaffine binding pair, and a second corona antigen having a detectable label, wherein the first and second corona antigens specifically bind to the anti-corona antibody; b) forming an immunoreaction mixture comprising a first antigen, a sample antibody, and a second antigen (a solid phase having a corresponding effector group of the bioaffine binding pair is added before, during, or after the formation of the immunoreaction mixture); c) maintaining the immunoreaction mixture for a sufficient time for anti-corona antibodies against the coronavirus antigens in the body fluid sample to immunoreact with the coronavirus antigens and form an immunoreaction product; d) separating the liquid phase from the solid phase; e) detecting the presence of any of said immune reaction products in the solid phase or liquid phase or both. Includes:

[0187] Finally, the presence of any of the immune reaction products is detected in the solid phase, the liquid phase, or both.

[0188] In an embodiment, the maximum total duration of the method for detecting coronavirus antibodies is less than 1 hour, i.e., less than 60 minutes, in one embodiment less than 30 minutes, in a further embodiment less than 20 minutes, in one embodiment 15-30 minutes, in one embodiment 15-20 minutes. The duration includes pipetting of the sample and reagents necessary to perform the assay, as well as incubation time, optional washing steps, detection steps and final output of results.

[0189] In a sixth aspect, the present invention provides a method for identifying whether a patient has been previously exposed to a coronavirus infection, comprising: a) forming an immune reaction mixture by mixing a sample of a patient's body fluid with 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; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or absence of any of the immune reaction products Including, The presence of an immune response product indicates that the patient has been previously exposed to a coronavirus infection.

[0190] In embodiments, the patient has been exposed to a coronavirus infection prior to the implementation of the method. In particular, the patient has been exposed to a coronavirus infection at least 5 days prior to the implementation of the method. In particular, the patient has been exposed to a coronavirus infection at least 10 days prior to the implementation of the method. In particular, the patient has been exposed to a coronavirus infection at least 14 days prior to the implementation of the method.

[0191] In a seventh aspect, the present invention provides a method for differential diagnosis between an immune response in a patient resulting from a natural coronavirus infection and an immune response resulting from vaccination, wherein the vaccination is based on an S protein-derived antigen, an E protein-derived antigen, or an M protein-derived antigen, a) forming an immune reaction mixture by mixing a body fluid sample from a patient with a coronavirus antigen of the first aspect of the invention, a composition comprising the coronavirus antigen of the first aspect of the invention, or a coronavirus antigen obtained by the method of the third aspect of the invention; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or absence of any of the immune reaction products Including, The presence of an immune response product indicates that the immune response in the patient is due to a natural coronavirus infection, and the absence of an immune response product indicates that the immune response in the patient is due to vaccination with an S-, E-, or M-protein derived antigen.

[0192] In embodiments, the method allows distinguishing between patients who have been naturally infected with a coronavirus and patients who have been vaccinated against a coronavirus, where a patient who has been vaccinated against a coronavirus has been vaccinated with a vaccine using an antigen derived from the coronavirus S, E or M protein.

[0193] In an embodiment, the patient infected with a natural coronavirus is infected with SARS-Cov-1 or SARS-Cov-2, particularly SARS-Cov-2.

[0194] In embodiments, the naturally occurring coronavirus comprises a nucleocapsid protein.

[0195] 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 test for the detection of anti-coronavirus antibodies. In a specific embodiment, the coronavirus antigen according to the first aspect of the invention, the 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 in the method according to the fourth aspect of the invention or the fifth aspect of the invention.

[0196] In a ninth aspect, the present invention relates to a reagent kit for detecting anti-coronavirus antibodies, comprising 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 obtainable by the method according to the third aspect of the invention.

[0197] In certain embodiments, the reagent kit comprises, in separate containers or separate compartments of a single container unit, a coronavirus antigen according to the first aspect of the present invention, a composition according to the second aspect of the present invention, or a coronavirus antigen obtained by the method according to the third aspect of the present invention. In certain embodiments, the coronavirus antigen is covalently bound to biotin.

[0198] In embodiments, the reagent kit further comprises microparticles, in particular avidin or streptavidin coated microparticles, in separate containers or in separate compartments of a single container unit.

[0199] In further embodiments, the present invention relates to the following: 1. A coronavirus antigen suitable for detecting antibodies to coronavirus in an isolated biological sample, comprising a coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, wherein the peptide does not contain any additional coronavirus-specific amino acid sequence.

[0200] 2. The corona antigen of item 1, wherein the coronavirus is a CoV-1 or CoV-2 virus, in particular a CoV-2 virus.

[0201] 3. The corona antigen of item 1 or 2, further comprising at least one chaperone, in particular two chaperones.

[0202] 4. The coronavirus antigen of item 3, wherein the chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

[0203] 5. The corona antigen of items 2 to 4, wherein the chaperone is fused to a corona nucleocapsid-specific amino acid sequence at the N-terminus and / or C-terminus of the nucleocapsid.

[0204] 6. The corona antigen of items 1 to 5, wherein the polypeptide comprises a corona nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 and two SlyD chaperones.

[0205] 7. A coronavirus antigen according to any one of items 1 to 6, which is soluble and immunoreactive.

[0206] 8. A coronavirus antigen described in any one of claims 1 to 7, wherein the SARS CoV-2 coronavirus nucleocapsid variant comprises the amino acid sequence set forth in 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.

[0207] 9. The corona antigen of any of items 1 to 8, further comprising a tag, in particular a tag that allows the antigen (in particular Ru, in particular negatively charged Ru) to be detected, and / or a tag that directly or indirectly binds the antigen to a solid phase (in particular an effector group that is part of a bioaffine binding pair, in particular biotin).

[0208] 10. A composition comprising a coronavirus antigen described in any one of items 1 to 9.

[0209] 11. The composition of item 10, comprising an additional coronavirus antigen, in particular a coronavirus antigen comprising the amino acid sequence of the E protein, M protein, and / or S protein or a portion thereof.

[0210] 12. A method for producing a corona antigen specific to the nucleocapsid of a coronavirus, comprising: a) culturing a host cell, particularly an E. coli cell, transformed with an expression vector to which a recombinant DNA molecule encoding the polypeptide according to any one of items 1 to 9, particularly a recombinant DNA molecule comprising the sequence set forth in SEQ ID NO: 3, has been operably linked; b) expressing the polypeptide; and c) Purification of the polypeptide A method comprising:

[0211] 13. A method for detecting coronavirus-specific antibodies in an isolated sample, using the coronavirus antigen described in any one of items 1 to 9, the composition of items 10 to 11, or the coronavirus antigen obtained by the method described in item 12 as a capture reagent and / or binding partner for anti-coronavirus antibodies.

[0212] 14. A method for detecting antibodies specific to a coronavirus in an isolated sample, comprising: a) forming an immune reaction mixture by mixing a body fluid sample with 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; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or concentration of any of the immune reaction products A method comprising:

[0213] 15. A method for detecting antibodies specific to coronavirus in an isolated sample according to item 14, wherein the immune response is a) adding to the sample a first corona antigen having an effector group that can be directly or indirectly bound to a solid phase and that is part of a bioaffine binding pair, and a second corona antigen having a detectable label, wherein the first and second corona antigens specifically bind to the anti-corona antibody; b) forming an immunoreaction mixture comprising a first antigen, a sample antibody, and a second antigen (a solid phase having a corresponding effector group of the bioaffine binding pair is added before, during, or after the formation of the immunoreaction mixture); c) maintaining the immunoreaction mixture for a sufficient time for anti-corona antibodies against the coronavirus antigens in the body fluid sample to immunoreact with the coronavirus antigens and form an immunoreaction product; d) separating the liquid phase from the solid phase; e) detecting the presence of any of said immune reaction products in the solid phase or liquid phase or both. The method is carried out in a double antigen sandwich format comprising:

[0214] 16. A method for detecting antibodies specific to coronavirus in an isolated sample according to any of items 13 to 15, wherein the antibodies to be detected are IgA, IgG or IgM antibodies, in particular IgG antibodies.

[0215] 17. A method for identifying whether a patient has been previously exposed to a coronavirus infection, comprising: a) forming an immune reaction mixture by mixing a body fluid sample from a patient 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 according to item 12; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or absence of any of the immune reaction products Including, The method, wherein the presence of the immune response product indicates that the patient has been previously exposed to a coronavirus infection.

[0216] 18. A method for differential diagnosis between an immune response resulting from a natural coronavirus infection and an immune response resulting from vaccination, wherein the vaccination is based on an S protein-derived antigen, an E protein-derived antigen, or an M protein-derived antigen; a) forming an immune reaction mixture by mixing a body fluid sample from a patient with a coronavirus antigen of the first aspect of the invention, a composition comprising the coronavirus antigen of the first aspect of the invention, or a coronavirus antigen obtained by the method of the third aspect of the invention; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigen to immunoreact with the coronavirus antigen to form an immune reaction product; and c) detecting the presence and / or absence of any of the immune reaction products Including, The presence of the immune response product indicates that the immune response in the patient is due to a natural coronavirus infection, and the absence of the immune response product indicates that the immune response in the patient is due to vaccination with a spike protein-derived antigen.

[0217] 19. Use of 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 in a high-throughput in vitro diagnostic test for the detection of anti-coronavirus antibodies.

[0218] 20. Use of 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 in the method according to items 13 to 18.

[0219] 21. A reagent kit for detecting anti-coronavirus antibodies, comprising 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.

[0220] 22. A reagent kit according to item 18, comprising at least microparticles coated with avidin or streptavidin and a coronavirus antigen covalently bound to biotin according to any one of items 1 to 9, the composition of items 10 to 11, or microparticles obtained by the method according to item 12, in separate containers or separate compartments of a single container unit.

[0221] 23. A reagent kit according to item 13, comprising at least microparticles coated with avidin or streptavidin and a μ-capture binding partner covalently bound to biotin in separate containers or separate compartments of a single container unit.

[0222] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Example]

[0223] Example 1: Cloning and purification of coronavirus nucleocapsid antigens Cloning of expression cassettes Based on the pET24a expression plasmid from Novagen (Madison, WI, USA), an expression cassette encoding the fusion protein was obtained essentially as described (Scholz, C. et al., J. Mol. Biol. (2005) 345, 1229-1241). The sequence of the nucleocapsid antigen from SARS coronavirus 2 (SARS CoV-2) was retrieved from GenBank under the number MN90847.3. A synthetic gene encoding the nucleocapsid antigen aa 1-419 (i.e., the full-length version of the nucleocapsid or N protein) with a glycine-rich linker region fused in-frame to the N-terminus was purchased from Eurofins (Regensburg, Germany). Because the native amino acid sequence of the Corona N protein does not contain cysteine ​​residues, no amino acid substitutions were necessary to prevent undesired side effects such as oxidation or intermolecular disulfide cross-linking. BamHI and XhoI restriction sites were located at the 5' and 3' ends of the N coding region, respectively. Similarly, an additional synthetic gene encoding one or two EcSlyD units (residues 1–165 of SwissProt accession number P0A9K9) linked via a glycine-rich linker region and containing a portion of the additional linker region at the C-terminus was purchased from Eurofins. NdeI and BamHI restriction sites were located at the 5′ and 3′ ends of this cassette, respectively. The gene and restriction sites were designed to allow in-frame fusion of the chaperone portion, EcSlyD-EcSlyD, with the N antigen portion by simple ligation. To avoid inadvertent recombination processes and increase the genetic stability of the expression cassette in the E. coli host, the nucleotide sequence encoding the EcSlyD unit was modified in the same way as the nucleotide sequence encoding the extended linker region; i.e., different codon combinations were used to encode the same amino acid sequence.

[0224] The pET24a vector was digested with NdeI and XhoI, and a cassette containing tandem-SlyD fused in-frame to the SARS-CoV-2 nucleocapsid (1-419) was inserted. Accordingly, expression cassettes 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 the nucleocapsid fragment from SARS-CoV-2, were constructed. All recombinant fusion polypeptide variants contained a C-terminal hexahistidine tag to facilitate Ni-NTA-assisted purification and refolding. QuikChange (Stratagene, La Jolla, CA, USA) and standard PCR techniques were used to generate point mutations, deletions, insertions, and extension variants or restriction sites in each expression cassette.

[0225] Figure 3 shows a scheme of the nucleocapsid antigen N(1-419) with two SlyD chaperone units fused in-frame to its N-terminus. To indicate the E. coli origin of the SlyD fusion partner, the depicted fusion polypeptide is designated EcSlyD-EcSlyD-CoV-2 N(1-419).

[0226] The insert of the resulting plasmid was sequenced and found to encode the desired fusion protein. The complete amino acid sequences of the antigenic variants CoV-2 N(1-419), EcSlyD-CoV-2 N(1-419), and EcSlyD-EcSlyD-CoV-2 N(1-419) are shown in SEQ ID NOs: 1, 2, and 3, respectively. The amino acid sequence of the linker L is shown in SEQ ID NO: 7.

[0227] Purification of recombinant proteins containing nucleocapsids from SARS coronavirus 2 All nucleocapsid antigen variants were purified using essentially the same protocol. E. coli BLR(DE3) cells harboring the specific pET24a expression plasmid were cultured in LB medium plus kanamycin (30 μg / ml) at 37 °C to an OD of 1.5. 600Cells were grown to 1000 kJ / ml and cytosolic overexpression was induced by adding 1 mM isopropyl-β-D-thiogalactoside. Three hours after induction, cells were harvested by centrifugation (5000 g for 20 min), frozen, and stored at -20°C. For cell lysis, the frozen pellet was resuspended in chilled 50 mM sodium phosphate (pH 8.0), 7.0 M GdmCl, and 5 mM imidazole. 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 containing 5.0 mM TCEP. Subsequent wash steps were adjusted for each 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. The GdmCl solution was then 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-bound protein. To avoid reactivation of co-purifying proteases, a protease inhibitor cocktail (Complete® EDTA-free, Roche) was included in the refolding buffer. A total of 15–20 column volumes of refolding buffer were applied to the overnight reaction. Both TCEP and the Complete® EDTA-free inhibitor cocktail were then removed by washing with 3–5 column volumes of 50 mM potassium phosphate (pH 8.0), 100 mM KCl, and 10 mM imidazole. Subsequently, to remove nonspecifically bound protein contaminants, the imidazole concentration (in 50 mM potassium phosphate (pH 8.0), 100 mM KCl) was increased to 30-50 mM (depending on the respective target protein). Native protein was then eluted with 250 mM imidazole in the same buffer. Protein-containing fractions were assessed for purity by Tricine-SDS-PAGE and pooled.Finally, the protein was subjected to size exclusion chromatography (Superdex HiLoad, Amersham Pharmacia), and protein-containing fractions were pooled and concentrated to 10–20 mg / ml in Amicon cells (YM10).

[0228] After the combined purification and refolding protocol, we were able to obtain protein yields of approximately 10–15 mg from 1 g of wet E. coli cells, depending on the respective target protein (anchaperone N protein approximately 10 mg / g; EcSlyD-N(1-419) approximately 12 mg / g; EcSlyD-EcSlyD-N(1-419) approximately 15 mg / ml).

[0229] Example 2: Spectroscopic measurements Protein concentration was measured using a Uvikon XL double-beam spectrophotometer. The molar extinction coefficient (ε 280 The molar extinction coefficients (ε) used for the different fusion polypeptides were determined using the procedure described in Pace (1995), Protein Sci. 4, 2411-2423. M 280 ) are identified in Table 1. [Table 1]

[0230] The unchaperoned SARS CoV-2 N was cloned in a full-length version (1-419), but the N-terminal methionine is co-translationally cleaved by N-methionyl-aminopeptidase during overproduction in E. coli. Therefore, data for the mature (truncated) SARS CoV-2 nucleocapsid version (2-419) are shown in Table 1. The amino acid sequences of the corona antigen variants are shown in SEQ ID NOs: 1, 2, and 3, respectively.

[0231] Example 3: Coupling of biotin tags and ruthenium complex labels to nucleocapsid antigens The lysine ε-amino groups of the fusion polypeptides were modified with N-hydroxysuccinimide-activated biotin and ruthenium-labeled molecules, respectively, at protein concentrations of 10–30 mg / ml. The label / protein ratio varied from 1:1 to 10:1 (mol:mol) depending on the fusion protein. The reaction buffer was 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 min and terminated by adding buffered L-lysine to a final concentration of 10 mM. To avoid hydrolytic inactivation of the labels, stock solutions were prepared in dry DMSO (seccosolv quality, Merck, Germany). DMSO concentrations of up to 25% in the reaction buffer were well tolerated by all fusion proteins tested. After the coupling reaction, unreacted free label was removed by passing the crude protein conjugate through a gel filtration column (Superdex 200 HiLoad).

[0232] Example 4: Immunological reactivity (i.e., antigenicity) of different nucleocapsid antigen variants in anti-SARS CoV-2 immunoassays The immunological reactivity (i.e., antigenicity) of the polypeptide fusion variants of the coronavirus nucleocapsid antigen was assessed using the automated Elecsys® cobas e 411 analyzer (Roche Diagnostics GmbH). Elecsys® is a registered trademark of the Roche Group. Measurements were performed in a double-antigen sandwich format.

[0233] Signal detection in the Elecsys® and cobas automated analyzers is based on electrochemiluminescence. A biotin-conjugate (i.e., capture antigen) is immobilized on the surface of streptavidin-coated magnetic beads, while the detection antigen carries a complexed ruthenium cation (which switches between redox states 2+ and 3+) as the signaling moiety. In the presence of a specific immunoglobulin analyte, the luminescent ruthenium complex is crosslinked to the solid phase and emits light at 620 nm after excitation at a platinum electrode. The signal output is in arbitrary light units.

[0234] The recombinant corona nucleocapsid antigen was evaluated in a double antigen sandwich (DAGS) immunoassay format. To this end, recombinant corona N antigen was used as a biotin and ruthenium conjugate, respectively, to detect anti-corona nucleocapsid antibodies in human serum.

[0235] The 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 infections. All measurements were performed using either EcSkp-EcSlyD-EcSlyD (EP2893021(B1)) or chemically polymerized, unlabeled EcSlyD-EcSlyD in large excess (5-30 μg / ml) in the reaction buffer as an anti-interfering substance to avoid immunological cross-reactions via the chaperone fusion unit.

[0236] Specifically, this study investigated three nucleocapsid variants derived from SARS-CoV-2: full-length N(1-419) without a fusion partner, full-length N(1-419) fused to one SlyD chaperone, and full-length N(1-419) fused to two SlyD chaperone 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 conjugates in R1 and R2 were approximately 100 ng / ml (unless otherwise indicated). In analytical gel filtration experiments, we found that EcSlyD-EcSlyD-N(1-419) formed soluble and ordered oligomers that exhibited epitope density sufficiently high for binding and detection of M-type immunoglobulins.

[0237] Additionally, EcSlyD fusion polypeptides of putative immunodominant fragments of coronavirus antigens were evaluated using the Elecsys® assay. In particular, fragments of 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) were examined for their antigenicity. All of these chaperone fusion proteins were cloned, purified, biotinylated, and ruthenylated, respectively, essentially as described for the N variants. The fragments were chosen because there was literature suggestion that the corresponding sequences from SARS-CoV-1 were immunologically reactive. Indeed, in the case of SARS-CoV-1, immunodominant epitopes have been described for the corona spike protein (He et al., J. Immunol. (2004); 173: 4050-4057), for the corona M protein (J. Clin. Microbiol. (2005); 43(8): 3718-3726) and for the corona N protein (J. Clin. Microbiol. (2004) 42(2): 5309-5314).

[0238] Unfortunately, human coronavirus seroconversion panels, an essential tool for the development of improved in vitro diagnostic assays, are not yet commercially available. To assess the antigenic characteristics of different nucleocapsid variants during the early stages of SARS-CoV-2 infection, residual sera from clinics and hospitals had to be repeated.

[0239] In the first experiment, all of the candidate coronavirus antigens were evaluated for their immunological reactivity in the previously described DAGS format. To this end, biotinylated and ruthenium-labeled variants of the antigen candidates under investigation were incubated with the samples before the addition of streptavidin-coated beads. Based on the data in Figures 4a and 4b, it is clear that recombinant fusion polypeptides containing fragments of coronavirus proteins do not exhibit immunological reactivity: even at a high concentration of 500 ng / ml, the spike protein fragment 617–649 does not react at all with the five sera from the anti-coronavirus positive panel tested (see Figure 4a). The detected signal is within the system's inherent background of approximately 500 counts, ruling out the presence of the spike (617–649) as an immunodominant epitope. The same is true for another fragment from the spike protein, i.e., fragment 338–516, which encompasses the so-called receptor-binding domain and is considered one of the most immunodominant regions within the coronavirus proteome. Furthermore, the recombinant RBD variant EcSlyD spike (338-516) showed no reactivity, which is in strong contrast to previous reports on the antigenicity of this domain. E protein variants (45-75) and (8-65), both fused to the solubility-enhanced E. coli SlyD protein, showed no reactivity, as did fragments 1-32, 132-163, and 100-222 of the corona M protein. The results for the 100-222 region of the M protein are noteworthy because this is part of the endodomain of the M protein. That is, there was some possibility that this fragment could adopt a native-like conformation and thus present a conformational epitope. However, there was no reactivity at all to the M endodomain. There was also no reactivity with N fragments 151-178 and 374-404 (Figure 4b). In contrast, a weak but significant immunological reactivity is revealed for the full-length nucleocapsid antigen (penultimate column), albeit with a very high background signal.When the SlyD unit is fused to the N-terminus of the nucleocapsid, the solubility of the resulting fusion polypeptide is significantly enhanced, and the background signal is reduced from approximately 490,000 counts to 120,000 counts (Figure 4b, last column). As a result, the signal-to-noise ratio is significantly increased, allowing for excellent discrimination of anti-coronavirus positive sera from negative sera. The background signal is still very high, but can be mitigated by reducing the antigen concentration in the assay.

[0240] Figure 4b shows that fusion of one SlyD unit to the SARS CoV-2 nucleocapsid antigen confers solubility to the target protein, improves its physicochemical properties, and results in an immunoreactive corona antigen well suited for the detection of anti-corona antibodies.

[0241] In the next step, we investigated whether fusion of another SlyD unit would further improve the physicochemical characteristics of the nucleocapsid antigen.

[0242] Figure 5 shows the Elecsys® evaluation of the COVID-2 nucleocapsid antigen in both its unchaperoned form and its fusion to one and two SlyD units. To ensure a fair comparison, identical molar concentrations of each variant were applied. Surprisingly, the addition of one SlyD chaperone unit significantly reduced the background signal, resulting in an improved signal-to-noise ratio. When a second SlyD chaperone unit was added to the corona nucleocapsid antigen, the background signal further improved, further increasing the signal-to-noise ratio. In short, the solubility of the corona N protein strongly benefits from the incorporation of a chaperone such as SlyD. It is also clear from the comparison in Figure 5 that signal recovery is significantly improved when two SlyD units are added to N instead of just one. Long-term stability is a key issue and a prerequisite for any antigen used in immunoassays. When the antigen is incubated under heat stress conditions, such as at 35°C, signal recovery and indeed signal-to-noise recovery should not be severely affected. Table 5 also shows that fusing two SlyD chaperone units to the COVID-19 N antigen improves overall signal recovery, allowing the N to be used in the Elecsys® DAGS format for reliable detection of anti-COVID-19 antibodies. After overnight incubation at 35°C, the signal-to-noise recovery rate is much higher with the EcSlyD-EcSlyD-CoV-2-N conjugate than with the unchaperoned COVID-2 N conjugate. We found the same to be true for the SlpA (SlyD-like protein A)-N fusion protein. E. coli SlpA is closely related to E. coli SlyD and has highly advantageous properties regarding thermostability (see Example 7 below).

[0243] Further optimization of anti-interference additives, buffer composition and antigen concentration in R1 (= Reagent 1; biotin conjugate) and R2 (= Reagent 2; ruthenium conjugate), as well as pre-adsorption of the ruthenium conjugate with beads (Figure 6), ultimately paved the way to an Elecsys®-compatible nucleocapsid antigen with excellent background values ​​(i.e., very low signal in negative sera) and a remarkable signal-to-noise ratio (s / n), facilitating good discrimination between anti-coronavirus positive and negative sera.

[0244] In summary, we conclude that fragments of corona proteins touted as immunodominant epitopes, whether linear (e.g., spike 617-649) or conformational (e.g., the receptor-binding domain RBD contained within the spike protein), do not currently exhibit significant antigenicity. When assessed on the Elecsys® automated analyzer, no antigenicity was observed with promising corona protein fragments, but only with full-length nucleocapsid antigens derived from CoV-2. However, in its native form, the N protein could not be used in the Elecsys® assay due to excessive background signal. Fusing two SlyD chaperone units to the N antigen cured this drawback, making the N antigen suitable for high-throughput applications on the Elecsys® platform.

[0245] Example 5: Sensitivity and specificity of the above anti-SARS CoV-2 immunoassay Initially, 129 patients identified as infected with SARS-CoV-2 by PCR analysis were further tested using our prototype antibody immunoassay based on the nucleocapsid antigen. At different time intervals after a positive PCR test, serum samples were collected and analyzed via the antibody assay described above to determine whether anti-CoV-2 antibodies were present in the samples. Results were classified into three categories: less than 7 days after positive PCR, 7-13 days after the first PCR result, and more than 14 days after.

[0246] Six days after a positive PCR test, 74% of patients could be identified as anti-SARS CoV-2 positive. Between days 7 and 13 after a positive PCR, 95% of patients were already identified as SARS CoV-2 positive. 14 days after a positive PCR, our assay detects 100% of all patients as positive. The results are also shown in Figure 7A.

[0247] In further experiments, a total of 204 samples from 69 symptomatic patients with PCR-confirmed SARS CoV-2 infection were tested with the Elecsys anti-SARS CoV-2 assay as described above. One or more serial specimens from these patients were collected at various time points after PCR confirmation. Results are also shown in Figure 7B.

[0248] In a third experiment, an additional 292 samples from 61 symptomatic patients with PCR-confirmed SARS CoV-2 infection were tested with the Elecsys anti-SARS CoV-2 assay as described above. One or more serial specimens from these patients were collected at various time points after PCR confirmation. Results are also shown in Figure 7C. One sample was nonreactive after 14 days but became reactive after 16 days. Therefore, for this dataset, the sensitivity is 100% after 16 days.

[0249] For specificity testing, the first 1591 diagnostic routine serum and plasma samples collected before December 2019 ("pre-pandemic samples") were analyzed by the antibody assay described above. Due to the date of donation, all samples were classified as SARS CoV-2 antibody negative. Of the 1591 samples, only two were identified as anti-SARS CoV-2 reactive. Therefore, the antibody assay described above has a specificity of 99.87%.

[0250] In further experiments, additional patient samples were analyzed. The first set of samples from 5272 patients analyzed included the original 1591 samples listed above. The following samples were included:

[0251] 3,420 samples from patients in diagnostic routines 1,772 samples from blood donors 40 samples from patients diagnosed with a cold panel, and 40 cross-reactive samples from patients with previous infection with coronaviruses HKU1, NL63, 229E, or OC43 confirmed by PCR.

[0252] All samples were obtained before December 2019 and tested with the Elecsys Anti-SARS CoV-2 assay as described above. Ten false-positive samples were detected. The overall specificity obtained for the first sample set was 99.81%. The 95% lower confidence limit was 99.65%. The results are shown in Figure 8A.

[0253] In the second set, an additional 5261 samples from patients were analyzed. The following samples were included in the specificity test:

[0254] 2376 samples from patients in diagnostic routine 2,885 samples from blood donors

[0255] Additionally, samples from 4696 dialysis patients were analyzed.

[0256] The overall specificity obtained with the second sample set was 99.79%. The 95% lower confidence limit was 99.63%. The results are shown in Figure 8B.

[0257] The combined results of the first and second sets of sample measurements (10453 in total) are shown in Figure 8C.

[0258] Example 6: Capillary blood as a suitable sample type for the above anti-SARS CoV-2 immunoassay To analyze whether capillary blood is suitable for use as a sample type in the anti-SARS CoV-2 immunoassay described above, capillary blood samples were compared with serum samples prepared from venous blood. The effects of three different anticoagulants were also analyzed: Li-heparin plasma, K2-EDTA plasma, and CAT serum. Ten samples were tested for Li-heparin plasma and K2-EDTA plasma, five of which were positive and five of which were negative. Seven samples were tested for CAT serum, five of which were positive and two of which were negative. The results are summarized in Tables 2, 3, and 4 below, and Figures 9A, 9B, and 9C, respectively. [Table 2] [Table 3] [Table 4]

[0259] To address sample volume variability in capillary blood, venous whole blood (collected without clot activator or anticoagulant) was transferred at different volumes to capillary collection tubes containing anticoagulant (300 μl, 400 μl, 600 μl, 800 μl = reference), centrifuged, and tested on a cobas e analyzer with Elecsys Anti-SARS-CoV-2. One negative sample and one spike-positive sample were tested. The results are shown in Table 5 below. [Table 5]

[0260] Example 7: Nucleocapsid antigens fused to alternative chaperones Using the same methods described above in Examples 1-3, the nucleocapsid sequence from SARS coronavirus 2 (SARS CoV-2) was also fused to an alternative chaperone, SlpA. The resulting fusion polypeptides were coupled to either a biotin tag or a ruthenium complex label. Immunoreactivity was tested as described in Example 4 above and compared to the reactivity of the SlyD antigen constructs described above. The results are shown in Figure 10.

[0261] Example 8: Differential diagnosis of SARS CoV-2 against common cold coronaviruses 229E, OC43, NL63, and HKU1 In addition to the nucleocapsid antigen from SARS-CoV-2, it would be valuable to have on hand nucleocapsid homologs from six other well-known human pathogenic coronaviruses: 229E, OC43, SARS-CoV-1, NL63, HKU1, and MERS (listed in order of their appearance in the scientific literature). The so-called common cold coronaviruses 229E, OC43, NL63, and HKU1 still circulate in human populations worldwide and are responsible for cold-like illnesses, especially during the winter months (Human coronavirus circulation in the United States 2014-2017, J. Clin. Virol. 101(2018), 52-56). With the respective antigens on hand, it should be possible to facilitate both anti-interference approaches in anti-SARS-CoV-2 immunoassays and differential diagnosis of suspect sera under investigation. For example, if a false-positive serum in an anti-SARS CoV-2 assay is reactive with the EcSlyD-EcSlyD-N construct derived from rec.229E and NL63 (both alphacoronaviruses), it would be essential to exclude that antibodies raised during relatively harmless alphacoronavirus infections cross-react with the EcSlyD-EcSlyD-CoV-2-N designator used in rec. anti-SARS CoV-2 antibody tests, thereby falsely indicating SARS CoV-2 infection. It should be possible to confirm or exclude a true-positive result by either specific blocking experiments (i.e., addition of unlabeled common cold coronavirus N antigen to the sample under investigation) or differential diagnosis of anti-CoV-2-reactive samples with labeled N variants from common cold coronaviruses, respectively.

[0262] Therefore, we cloned, expressed, and purified (in E. coli Bl21) rec.EcSlyD-EcSly fusion protein versions (of 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, could be obtained in high yields from E. coli and were found to be soluble and stable to date. Protein data and yields are summarized in Table 6. [Table 6]

[0263] Furthermore, we cloned, expressed, and purified the so-called N-terminal domains (NTDs) of the N proteins from SARS-CoV-2, 229E, OC43, NL63, and HKU1 from the E. coli BL21 overproducing strain (following essentially the same purification protocol as described for the full-length N version rec.EcSlyD-EcSlyD-CoV-2-N). In contrast to the full-length N protein, the N-terminal domains do not form dimers or tetramers but are strictly monomeric. Therefore, the NTD is particularly suitable for detecting G-type immunoglobulins. However, M-type immunoglobulins are not recognized by the strictly monomeric NTD when the antigen is used as a capture and detection molecule in a double antigen sandwich (DAGS) format. Physiologically, the NTD binds to and accommodates polyanionic single-stranded viral RNA polymers within coronavirus virions. We were able to demonstrate that the solubility of the NTD is dramatically improved compared to the full-length N protein, and that its heat-induced unfolding is fully reversible, in stark contrast to the full-length N antigen. Melting curves monitored by near-UV CD spectroscopy revealed highly favorable folding behavior of rec.EcSlyD-N_NTD (because the near-UV CD signal was fully restored after thermal 20°C-80°C-20°C unfolding / refolding cycles (data not shown)), indicating high solubility of both the unfolded state and potential folding intermediates. The reversibility of thermally induced unfolding is a highly favorable and welcome feature of the protein, and the lack of aggregation tendency characterizes the NTD as an excellent antigen for use in immunoassays. Protein data and yields of various NTD constructs are shown in Table 7. [Table 7]

[0264] All rec.EcSlyD-N_NTD variants were biotinylated and rutheniumized as described. Each pair of biotin and ruthenium conjugates showed excellent background signals in the Elecsys® assay and was well suited to discriminating between positive and negative sera. Figures 11a+b show the reactivity of NTDs from SARS-CoV-2, OC43, NL63, 229E, and HKU1 with human sera. Because reliable figures for the actual seroprevalence of antibodies to common cold coronaviruses were unavailable, sera were partially precharacterized using the recomLine lateral flow assay SARS CoV-2 IgG [Avidity] RUO (article no. 7374, Mikrogen GmbH, Neuried, Germany). Briefly, we wanted to ensure that at least one of the sera under study was negative for each of the four common cold coronaviruses. Figure 11 shows that a pre-pandemic cold-coronavirus panel from 2019 did not reveal any immunoreactivity against the novel pathogen SARS-CoV-2 (Figure 11a+b, column 1). All CCC sera were anti-SARS-CoV-2 negative, producing electrochemiluminescence signals near the system-specific background (450–600 counts). For the anti-SARS-CoV-2 positive panel (from 2020), the signal from the SARS-CoV-2 NTD was significantly reduced relative to the full-length version of SARS-CoV-2-N. This was expected, since the NTD (46–176) lacks the complete C-terminal portion of the molecule (177–419) and therefore lacks many natural epitopes. Furthermore, due to the strictly monomeric nature of the NTD, many of the anti-coronavirus antibodies in polyclonal patient sera that could target conformationally folded dimers and higher-order oligomeric forms of N are unable to recognize and bind to their target molecule. However, the rather low signal level observed with SARS CoV-2-NTD still appears sufficient to reliably distinguish between positive and negative sera (Fig. 11a+b, column 1).This finding also applies to the OC43- and HKU1-derived common cold coronavirus (CCC) NTDs (Figure 11a+b, columns 2 and 5). As can be inferred from column 2, the incidence of antibodies against OC43 appears to be rather moderate. For this betacoronavirus, we found many sera with background signals close to the system's inherent background in the Elecsys® evaluation. This indicates the excellent solubility of the OC43 antigen in general and the OC43 antigen-ruthenium conjugate in particular. Notably, we also found sera with high signal levels and fairly good signal kinetics, allowing excellent discrimination between positive and negative sera. For NL63 and 229E, initial testing failed to find any truly negative sera with signals within the system's inherent background. All tested sera appeared to be anti-CoV positive for both NL63 and 229E (Figure 11a+b, columns 3 and 4), with very high signal kinetics. The true positive results of the serum were supported by reference measurements in which human serum was replaced with buffer and universal diluent, respectively, for the sample position. In this experimental setup, both NL63 and 229E NTDs were found to exhibit very low background signals ranging from 400 to 650 counts (Figure 11b, bottom three "Buffer" columns). This finding is significant for two reasons: first, it rules out specific or nonspecific association reactions between biotinylated and ruthenylated NL63 and 229E NTD molecules in the assay, which would result in a dramatic increase in signal. Second, it confirms that both NL63 and 229E NTD ruthenium conjugates are highly soluble and do not tend to bind to the surface of streptavidin-coated beads in the Elecsys® assay. Taken together, the data suggest that the high signals for NL63 and 229E measured with human sera are genuine and valid results, highlighting the very high incidence (much higher than for OC43) of antibodies against coronaviruses NL63 and 229E. The data for HKU1 (Fig. 11a+b, column 5) complete the picture.Although the prevalence of this coronavirus strain also seems to be quite high, in contrast to NL63 and 229E, true negative sera for HKU1 were seen and the signal was close to the system's inherent background. According to the rather preliminary data of the inventors, it is attractive to infer the tentative prevalence order of influenza coronavirus OC43 < HKU1 < < NL63, 229E in the analyzed panel based on the small number of sera tested.

[0265] The results of the inventors may be due to chance because of the small number of sera tested, but alpha coronaviruses 229E and NL63 seem to be effectively circulating in the analyzed cohort, especially during the past winter season, while OC43 infections seem to be quite rare.

[0266] Generally, by the expression, purification and modification (i.e., biotinylation and rutheniumation) of the N-terminal domain of the N antigen derived from coronaviruses SARS-CoV-2, OC43, NL63, 229E and HKU1, simple serological discrimination between related influenza coronaviruses could be established. Considering that SARS CoV-2 is spreading continuously worldwide in an unprecedented pandemic, the approach of the inventors could be an attractive option for a simple differential diagnosis that enables differentiating potentially life-threatening SARS CoV-2 infections from harmless influenza induced by one of the four well-known influenza viruses OC43, NL63, 229E and HKU1.

[0267] Example 8: Mutations in the wild-type SARS CoV-2 nucleocapsid antigen Since the number of emerging SARS CoV-2 mutant variants is increasing, the inventors generated four mutant variants containing 3, 8, 12, or 15 single-point mutations (see Figure 12) and expressed each of them fused to two EcSlyD units via the linker of SEQ ID NO: 7 as described above.

[0268] 3 MUT: SEQ ID NO: 8 EcSlyD-EcSlyD-SARS CoV-2-N 3 MUT: SEQ ID NO: 9 8 MUT: SEQ ID NO: 10 EcSlyD-EcSlyD-SARS CoV-2-N 8 MUT: SEQ ID NO: 11 12 MUT: SEQ ID NO: 12 EcSlyD-EcSlyD-SARS CoV-2-N 12 MUT: SEQ ID NO: 13 15 MUT: SEQ ID NO: 14 EcSlyD-EcSlyD-SARS CoV-2-N 15 MUT: SEQ ID NO: 15

[0269] The introduced single point mutations correspond to naturally occurring mutations in SARS CoV-2 mutations currently circulating in the population. The most common circulating mutations are:

[0270] B.1.1.7(UK):D3L,S235F B.1.525 (UK / Nigeria): D3Δ, A12G, T205I COH.20G / 677H(Ohio):P67S,P199L,D377Y B.1.351(South Africa):T205I P.1(B.1.1.28.1):P80R,R203K P.2 (Brazil): A119S P.3 (Philippines): R203K, G204R N.9 (Brazil): I292T EPI_ISL_1360318(India):R203M

[0271] In Figure 12, the single amino acid exchanges contained in the four mutational variants are shown with a patterned bar and the designated amino acid exchange.

[0272] Additional SARS CoV-2 nucleocapsid single point mutations that were less common in the population than those listed above were also introduced and are shown as black bars in Figure 12. These were selected from among the most frequent mutations found in infected individuals worldwide according to the CoV-GLU database published at http: / / cov-glue.cvr.gla.ac.uk / # / home (updated 24 Feb 2021 17:11:05 GMT).

[0273] Two variants containing either three (3 MUT) or eight (8 MUT) single point mutations were evaluated for the impact of selected mutations within the nucleocapsid protein on detection performance. Therefore, we used labeled forms of the nucleocapsid variants in a double antigen sandwich (DAGS) immunoassay format as described above.

[0274] Sera from 50 individuals were tested in parallel with either the wild-type EcSlyD-EcSlyD-nucleocapsid fusion protein or variants of the protein containing either three (3 MUT) or eight (8 MUT) single point mutations. The mean COI recovery of the variants was calculated and compared to wild-type reactivity (see Table 8 and Figure 13). [Table 8]

[0275] Introducing three single point mutations into the nucleocapsid protein sequence resulted in a very small decrease in reactivity (5%) across all samples tested. Because 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 produces valid results when applied to antisera from individuals infected with one of the widespread UK or South African variants. Even exchanges of as many as eight amino acids within the protein sequence resulted in an average COI recovery of 85% and higher signal variability 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 wild-type nucleocapsid sequences, ensuring that the classification of a sample as reactive or non-reactive is not affected by one of the variants. Briefly, despite substitutions of three (D3L, T205I, S235F) and eight (P67S, D103Y, S194L, G204R, A220V, M234I, H300Y, A376T) amino acid residues within the nucleocapsid antigen, respectively, near wild-type reactivity is observed in our N-based Elecsys® antibody assay. We conclude from these observations that positive antisera from individuals infected with one of the previously known SARS CoV-2 variants would be detected as positive anyway.

[0276] Furthermore, we found that the 3-MUT, 8-MUT, and 15-MUT variants adopt a native conformation (i.e., they are natively folded), since their elution behavior in analytical gel filtration (on a Superdex 200 column) is equivalent to that of wild-type nucleocapsid antigen. In the event of partial or total unfolding due to the introduced mutations, apparent widening of the molecule would be expected. Our observation is that the N variants with 3, 8, and 15 mutations, respectively, maintain their overall fold and exhibit elution behavior virtually identical to that of the wild-type N protein.

Claims

1. A coronavirus antigen suitable for detecting antibodies against coronavirus in an isolated biological sample, comprising a coronavirus nucleocapsid-specific amino acid sequence set forth in SEQ ID NO: 1 or a coronavirus nucleocapsid-specific amino acid sequence having 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, The coronavirus antigen further comprises at least one chaperone; the antigen does not contain additional coronavirus-specific amino acid sequences, the coronavirus is the SARS-CoV-2 virus; The chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp. The coronavirus antigen.

2. The coronavirus antigen of claim 1, wherein the SARS CoV-2 coronavirus nucleocapsid comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:

14.

3. The coronavirus antigen of claim 1 or 2, comprising the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO:

15.

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

5. A method for producing a corona antigen specific to a coronavirus nucleocapsid, comprising: a) culturing a host cell transformed with an expression vector to which a recombinant DNA molecule encoding a coronavirus antigen polypeptide according to any one of claims 1 to 3 is operably linked; b) expressing said polypeptide, and c) purifying said polypeptide A method comprising:

6. A method for detecting coronavirus-specific antibodies in an isolated sample, using a coronavirus antigen described in any one of claims 1 to 3, a composition described in claim 4, or a coronavirus antigen obtained by the method described in claim 5 as a capture reagent and / or binding partner for the anti-coronavirus antibodies.

7. 1. A method for detecting antibodies specific to a coronavirus in an isolated sample, comprising: a) forming an immune reaction mixture by mixing a body fluid sample with a coronavirus antigen according to any one of claims 1 to 3, a composition according to claim 4, or a coronavirus antigen obtained by the method according to claim 5; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigens to immunoreact with the coronavirus antigens to form an immune reaction product; and c) detecting the presence and / or concentration of any of said immune reaction products A method comprising:

8. 1. A method for identifying whether a patient has been previously exposed to a coronavirus infection, comprising: a) forming an immune reaction mixture by mixing a body fluid sample of the patient with the coronavirus antigen of any one of claims 1 to 3, the composition of claim 4, or the coronavirus antigen obtained by the method of claim 5; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigens to immunoreact with the coronavirus antigens to form an immune reaction product; and c) detecting the presence and / or absence of any of said immune reaction products Including, The presence of an immune response product indicates that the patient has been previously exposed to a coronavirus infection.

9. 1. A method for differential diagnosis between an immune response resulting from a natural coronavirus infection and an immune response resulting from vaccination, wherein the vaccination is based on an S protein-derived antigen, an E protein-derived antigen, or an M protein-derived antigen; a) forming an immune reaction mixture by mixing a body fluid sample with a coronavirus antigen according to any one of claims 1 to 3, a composition according to claim 4, or a coronavirus antigen obtained by the method according to claim 5; b) maintaining the immunoreaction mixture for a sufficient time for antibodies present in the body fluid sample against the coronavirus antigens to immunoreact with the coronavirus antigens to form an immune reaction product; and c) detecting the presence and / or absence of any of said immune reaction products Including, The presence of an immune response product indicates that the immune response in the patient is due to a natural coronavirus infection, and the absence of an immune response product indicates that the immune response in the patient is due to vaccination with a spike protein-derived antigen.

10. Use of a coronavirus antigen according to any one of claims 1 to 3, a composition according to claim 4, or a coronavirus antigen obtained by the method according to claim 5 in a high-throughput in vitro diagnostic test for the detection of anti-coronavirus antibodies.

11. Use of a coronavirus antigen according to any one of claims 1 to 3, a composition according to claim 4, or a coronavirus antigen obtained by the method according to claim 5 in a method according to any one of claims 6 to 10.

12. A reagent kit for detecting anti-coronavirus antibodies, comprising the coronavirus antigen of any one of claims 1 to 3, the composition of claim 4, or the coronavirus antigen obtained by the method of claim 5.

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