WO2021254868A1

WO2021254868A1 - Multiplex sars-cov-2 immunoassay

Info

Publication number: WO2021254868A1
Application number: PCT/EP2021/065608
Authority: WO
Inventors: Marc Van Den Bulcke; Isabelle Desombere; Maan Zrein; Elodie GRANJON
Original assignee: Sciensano; Infynity Biomarkers
Priority date: 2020-06-19
Filing date: 2021-06-10
Publication date: 2021-12-23

Abstract

The present invention provides methods and tools to determine a serological signature of antibodies against SARS-CoV-2 from a biological sample of a subject. More particularly, a method is provided for the detection of antibodies against SARS-CoV-2 in a biological sample from a subject comprising contacting the sample with a combination of antigens comprising at least one membrane antigen derived from SARS-CoV-2, at least one nucleocapsid antigen derived from SARS-CoV-2, at least one spike antigen derived from SARS-CoV-2, and optionally a T. cruzi antigen, wherein said antigens are immobilized on a solid support spatially separated from one another, and detecting the formed antigen/antibody complexes to determine a serological signature. The invention further provides a solid support and a kit for detecting antibodies against SARS-CoV-2 in a sample according to the invention. The invention further provides a method for the diagnosis and/or prognosis of a SARS-CoV-2 infection in a subject, a method for classifying a subject into a group informative of the clinical severity of a SARS- CoV-2 infection, a method for monitoring the (clinical) progression of a SARS-CoV-2 infection in a subject, a method for predicting the effectiveness of a vaccination in a subject, a method for identifying a subject who has neutralizing antibodies to SARS-CoV-2 and a method for determining a SARS-CoV-2 neutralizing antibody titer, said methods encompassing the determination of a serological signature according to a method of the invention and comparison with an appropriate reference serological signature.

Description

MULTIPLEX SARS-COV-2 IMMUNOASSAY

FIELD OF THE INVENTION

The invention relates to the field of serological profiling. In particular, methods and tools are provided for the detection of antibodies against a plurality of antigens derived from SARS- CoV-2 in a biological sample of a subject, e.g. for the diagnosis and/or prognosis of a SARS- CoV-2 infection in a subject, for distinguishing subjects into groups informative of the clinical severity of a SARS-CoV-2 infection, for monitoring the (clinical) progression of a SARS-CoV-2 infection in a subject, for predicting the effectiveness of a vaccination or a therapy in a subject, and for identifying subjects that have neutralizing antibodies to SARS-CoV-2 and determining a SARS-CoV-2 neutralizing antibody titer in a subject.

BACKGROUND OF THE INVENTION

SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) is a novel beta-coronavirus that has caused the coronavirus disease pandemic of 2019 (COVID-19). In order to identify infected individuals and to contain the spread of the disease, rapid and accurate population wide screening is essential.

Currently, nucleic acid-based tests of nasopharyngeal swabs are the primary method used to detect COVID-19 infected individuals. These tests can only diagnose disease during a narrow window of active infection with an overall clinical sensitivity of 65-72%.

In addition, antibody (Ab) assays are most useful for identifying individuals who have been infected with SARS-CoV-2 and seroconverted. As such, they can be particularly valuable, for example, for identification of subjects who have had an asymptomatic SARS-CoV-2 infection and those who have recovered and would no longer be positive in tests for viral nucleic acids. They would also be particularly useful for serosurveillance, to identify donors for COVID-19 plasma therapy, and to identify individuals who are potentially immune to reinfection. Antibody assays thus fill an essential gap both during and after a SARS-CoV-2 pandemic.

Many serological enzyme-linked immunosorbent assays (ELISA) have been recently developed to detect anti-SARS-CoV-2 antibodies. However, these assays have four important limitations. First, they lack the ability to detect antibodies at early stages of infection. Second, false positive results due to non-specific binding can occur attributable to high levels of pre-existing and cross-reacting antibodies in blood. Third, conventional ELISA assays can only analyze immunoglobulins for one specific target at a time, limiting their ability to profile and understand the underlying immune response. Finally, these assays lack the resolution to quantify variations in the immune response, which may be key in understanding clinical progression and sustainability of a protective immune response.

Hence, there is a need for assays that can inform on the diversity of antibody responses mounted upon SARS-CoV-2 infection and vaccination, and/or that can inform on antibody titer and/or show antibody functionality (e.g., virus neutralization). For pandemics such as the SARS-CoV-2 outbreak, the development of high throughput assays that do not involve handling of infectious material is imperative.

SUMMARY OF THE INVENTION

The present invention solves one or more of the above-mentioned problems in the art. In particular, a multiplex method and tools are provided herein for the detection of antibodies against SARS-CoV-2 in a biological sample from a subject allowing the determination of a serological signature of the subject. More particularly, in the methods and tools of the invention, the sample is contacted with a combination of antigens comprising at least one membrane antigen derived from SARS-CoV-2, at least one nucleocapsid antigen derived from SARS-CoV-2, at least one spike antigen derived from SARS-CoV-2 and optionally a T. cruzi antigen, wherein said antigens are immobilized on a solid support spatially separated from one another, allowing the simultaneous detection of the formed antigen/antibody complexes, yet allowing detection of discrete reactivities on each of the antigens. The methods and tools described herein can be used with blood-derived samples, but advantageously, it was shown that also saliva samples can be used. Saliva samples are particularly attractive given the ease and non-invasiveness of sampling.

It was found that the serological signature as determined by the methods and the tools of the invention allow to distinguish or discriminate subjects into groups, which groups are informative of the clinical severity of the infection in the subject. Accordingly, methods are provided herein for the diagnosis and/or prognosis of a SARS-CoV-2 infection in a subject, for monitoring the (clinical) progression of SARS-CoV-2 infection in a subject and for classifying a SAR-CoV-2 infected patient into a group informative on the clinical severity of the SARS-CoV- 2 infection, which methods are based on the serological signature as determined by a method or tool of the present invention. Furthermore, the methods and tool of the invention can be used to discriminate SARS-CoV-2 vaccine-induced serological signatures from SARS-CoV-2 natural infection-induced serological signatures. Accordingly, methods are provided herein for predicting the effectiveness of a SARS CoV-2 vaccination in a subject and/or selecting an appropriate vaccination method for a subject upon comparing the serological signature of the subject with a vaccine-induced, wherein said serological signatures are determined by a method or tool of the present invention. It was found that the methods and tools described herein also allow to detect neutralizing antibodies to SARS-CoV-2 in a biological sample from a subject and thus provide a simple, fast and safe alternative for in vitro virus seroneutralization assays. Accordingly, methods and tools are also provided herein to identify subjects that have neutralizing antibodies to SARS-CoV-2 such as convalescent patients, which may be potential donors of therapeutic antibodies. The methods and tools provided herein also allow a (semi-quantitative) determination of a SARS-CoV-2 neutralizing antibody titer. The methods and tools are also useful to predict protective immunity against a SARS-CoV-2 (re-)infection in a subject, e.g. a subject who received a vaccine (candidate).

The present invention is in particular captured by any one or any combination of one or more of the below numbered aspects and embodiments (i) to (xxi) wherein:

(i): A method for the diagnosis and/or prognosis of a SARS-CoV-2 infection in a subject comprising:

(i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen to allow the formation of antigen/antibody complexes;

(ii) detecting the antigen/antibody complexes formed in (i) to determine a serological signature; and

(iii) comparing the serological profile with a reference serological signature indicative for the diagnosis and/or prognosis of a SARS-CoV-2 infection; wherein said combination of antigens comprises:

(a) at least one membrane antigen selected from SARS-CoV-2 membrane (M) protein or an antigenic fragment thereof,

(b) at least one nucleocapsid antigen selected from SARS-CoV-2 nucleocapsid (NC) protein or an antigenic fragment thereof,

(c) at least one spike antigen selected from SARS-CoV-2 spike (S) protein, SARS-CoV-2 spike receptor-binding domain (RBD) protein, SARS-CoV-2 SI protein, SARS-CoV-2 S2 protein or an antigenic fragment of any of said proteins, and

(d) optionally a T. cruzi antigen comprising the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7); and wherein said antigens are immobilized on a solid support spatially separated from one another. (ii): A method for categorizing a patient diagnosed with a SARS-CoV-2 infection as asymptomatic, mild symptomatic, severe symptomatic requiring hospitalization or severe symptomatic requiring intensive care treatment, comprising:

(i) contacting a biological sample from the patient with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen to allow the formation of antigen/antibody complexes;

(iii) categorizing the patient as asymptomatic, mild symptomatic, severe symptomatic requiring hospitalization or severe symptomatic requiring intensive care treatment based on the serological signature compared to a reference serological signature indicative for an asymptomatic patient, a mild symptomatic patient, a severe symptomatic patient requiring hospitalization or a severe symptomatic patient requiring intensive care treatment; wherein said combination of antigens comprises:

(d) optionally a T. cruzi antigen comprising the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7); and wherein said antigens are immobilized on a solid support spatially separated from one another.

(iii): A method for monitoring the clinical progression of a SARS-CoV-2 infection in a patient comprising:

(i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen to allow the formation of antigen/antibody complexes; and

(ii) detecting the antigen/antibody complexes formed in (i) to determine a serological signature; wherein said combination of antigens comprises: (a) at least one membrane antigen selected from SARS-CoV-2 membrane (M) protein or an antigenic fragment thereof,

(b) at least one nucleocapsid antigen selected from SARS-CoV-2 nucleocapsid (NC) protein or an antigenic fragment thereof, and

(iv): A method for predicting the effectiveness of a SARS CoV-2 vaccination or a therapy in a subject, said method comprising:

(iii) comparing the serological profile with a reference serological signature indicative for effectiveness of the vaccination or the therapy; wherein said combination of antigens comprises:

(v) A method for predicting protective immunity against a SARS-CoV-2 infection in a subject, said method comprising: (i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen, in particular the CIA antigen, to allow the formation of antigen/antibody complexes;

(iii) comparing the serological profile with a reference serological signature indicative for protective immunity against a SARS-CoV-2 infection; wherein said combination of antigens comprises:

(vi). A method for determining a SARS-CoV-2 neutralizing antibody titer in a subject, said method comprising:

(iii) comparing the serological signature with a reference serological signature indicative for a SARS-CoV-2 neutralizing antibody titer, wherein said combination of antigens comprises:

(b) at least one nucleocapsid antigen selected from SARS-CoV-2 nucleocapsid (NC) protein or an antigenic fragment thereof, (c) at least one spike antigen selected from SARS-CoV-2 spike (S) protein, SARS-CoV-2 spike receptor-binding domain (RBD) protein, SARS-CoV-2 SI protein, SARS-CoV-2 S2 protein or an antigenic fragment of any of said proteins, and

(vii): The method according to any one of (i) to (vi), wherein detecting the antigen/antibody complexes comprises contacting the complexes with a detector molecule capable of binding the antibodies of the formed antigen/antibody complexes, the detector molecule comprising at least one detectably labelled moiety.

(viii): The method according to any one of (i) to (vii), wherein detecting the antigen/antibody complexes comprises determining the presence or absence of the antigen/antibody complexes.

(ix): The method according to any one of (i) to (viii), wherein the detection of said antigen/antibody complexes comprises determining the amount of the antigen/antibody complexes.

(x): The method according to any one of (i) to (ix), wherein the at least one membrane antigen comprises a (synthetic) peptide comprising the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5).

(xi): The method according to any one of (i) to (x), wherein the at least one nucleocapsid antigen comprises a (synthetic) peptide comprising the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6).

(xii): The method according to any one of (i) to (xi), wherein said combination of antigens comprises:

(a) a (synthetic) peptide comprising the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5),

(b) SARS-CoV-2 NC protein and a (synthetic) peptide comprising the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6),

(c) SARS-CoV-2 spike (S) protein, a SARS-CoV-2 spike receptor-binding domain (RBD) protein, a SARS-CoV-2 SI protein and a SARS-CoV-2 S2 protein, and (d) optionally a T. cruzi (synthetic) peptide antigen consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7).

(xiii): The method according to any one of (i) to (xii), wherein the biological sample is a blood sample, a serum sample, a plasma sample or a saliva sample.

(xiv): The method according to any one of (i) to (xiii), wherein said solid support is a microplate, a membrane, or a microarray, preferably a microplate.

(xv): A support for detecting antibodies directed to SARS-CoV-2 in a sample, said support comprising a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen, wherein said antigens are immobilized on said support spatially separated from one another, and wherein said combination of antigens comprises:

(d) optionally a T. cruzi antigen comprising the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7).

(xvi): The support according to (xv), further comprising a positive control, wherein said positive control is capable of detecting human antibodies.

(xvii): The support according to (xvi), wherein said positive control is immobilized in a pattern on said support.

(xviii): The support according to any one of (xv) to (xvii), wherein said solid support is a microplate, a membrane, or a microarray, preferably a microplate.

(xix): A kit for detecting antibodies directed to SARS-CoV-2 in a sample, comprising: a support according to any one of (xv) to (xviii); a wash solution, or components necessary for producing said wash solution; means for detection of the antibody-antigen complexes.

(xx): A SARS-CoV-2 peptide antigen comprising or consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5) or NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6). (xxi): The peptide antigen according to (xx) having a length of less than 40, preferably less than 35, more preferably less than 30 or 25 amino acids.

(xxii): The peptide antigen according to (xx) or (xxi), which is a synthetic peptide.

(xxiii): Use of a SARS-CoV-2 peptide antigen as defined in any one of (xx) to (xxii) for the detection of antibodies directed to SARS-CoV-2.

BRIEF DESCRIPTION OF THE FIGURES

The teaching of the application is illustrated by the following Figures which are to be considered as illustrative only and do not in any way limit the scope of the claims.

Figure 1: Design of a multiplex SARS-CoV-2 immunoassay according to an embodiment of the invention. Synthetic T. cruzi antigen (CIA), synthetic SARS-CoV-2 membrane antigen (MP1), recombinant SARS-CoV-2 nucleocapsid antigen (NCI), synthetic SARS-CoV-2 nucleocapsid antigen (NC2), recombinant SARS-CoV-2 RBD protein antigen (RBD), recombinant SARS-CoV- 2 SI protein antigen (SI), recombinant SARS-CoV-2 Sp2 protein antigen (S2) and recombinant SARS-CoV-2 S protein antigen (S full length) were spotted in duplicate in a single well of a microwell plate, as well as 3 positive controls (PC).

Figure 2: A typical time-scaled sample collection scheme for the infected patients. A patient with some symptoms for COVID is tested for presence of SARS-CoV-2 by RT-PCR, e.g. upon arrival at the hospital. For each infected patient, blood samples were collected at two time intervals (first serology and second serology), allowing to monitor the progression of the immune response.

Figure 3: Boxplot analysis of the multi-SARS-CoV-2 ELISA assay of first and second serology samples from PCR-positive patients. Mean raw values of the CIA antigen (A), the MP1 antigen (B), the NCI antigen (C), the NC2 antigen (D), the RBD antigen (E), the SI antigen (F), the S2 antigen (G) and the S full length antigen (H) obtained on multi-SARS-CoV-2 ELISA assay are given.

Figure 4: Boxplot analysis of IgM (A), IgA (B) and IgG (C) reactivities on the NCI antigen on the multi-SARS-CoV-2 ELISA assay of first and second serology samples from PCR-positive patients.

Figure 5: Schematic illustration of microplate analysis using dedicated reader and image analysis software. (A) Picture capture & matrix detection for analysis. The grid is automatically placed for spot detection. (B) Spot-by-Spot analysis and Pixel intensity Measurement. A Signal is calculated as AveragePixel Intensity minus Background and may range from 0 to 120. (C) Edited report/Data export. An exemplary report for a single well of a microplate is shown. Figure 6: Comparison of the multi-SARS-CoV-2 assay according an embodiment of the invention to detect an immune response against a SARS-CoV-2 infection in a subject compared to assay results of selected commercial ELISAs: Wantai ELISA (RBD), Euroimmun ELISA (SI) and Tecan ELISA (N).

Figure 7: Comparison of the multi-SARS-CoV-2 assay according an embodiment of the invention to detect an immune response against a SARS-CoV-2 infection in a subject compared to assay results of selected commercial ELISAs: DiaSorin ELISA and Abbott ELISA.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/- 5% or less, more preferably +/-!% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed. Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, is clear perse, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to "one embodiment", "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination. The present application relates to methods and tools for detecting antibodies against SARS- CoV-2 in a subject using a multiplex immunoassay.

The term "immunoassay" generally refers to methods known as such for detecting one or more molecules or analytes of interest in a sample, such as e.g., anti-SARS-CoV-2 antibodies, wherein specificity of an immunoassay for the molecule(s) oranalyte(s) of interest is conferred by specific binding between a specific-binding molecule, such as e.g. SARS-CoV-2 antigens, and the molecule(s) or analyte(s) of interest.

By comparison with SARS-CoV and other related coronaviruses, 28 proteins may be encoded by the genome of SARS-CoV-2, including 5 structural proteins (the SI and S2 subunits of the spike protein counting as 2 proteins), 8 accessory proteins and 15 non-structural proteins. Without wishing to be bound by theory, the structural proteins, in particular the membrane protein, the nucleocapsid protein, the full-length spike protein, as well as the SI and S2 subunits and the receptor binding protein RBD, are considered important in eliciting an immune response.

A method is provided herein for detecting antibodies directed against SARS-CoV-2 in a biological sample of a subject comprising:

(i) contacting the sample with a combination of antigens derived from SARS-CoV-2 and optionally a Trypanosoma cruzi antigen, to allow the formation of antigen/antibody complexes, and

(ii) detecting, preferably simultaneously, the antigen/antibody complexes formed in step (i) to determine a serological signature, wherein said combination of antigens comprises:

(a) at least one membrane antigen derived from SARS-CoV-2;

(b) at least one nucleocapsid antigen derived from SARS-CoV-2;

(c) at least one spike antigen derived from SARS-CoV-2; and

(d) optionally a Trypanosoma cruzi antigen, wherein said T. cruzi antigen is a (synthetic) peptide comprising or consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7); and wherein the antigens are immobilized on a solid support spatially separated from one another.

The term "antibody" includes polyclonal and monoclonal antibodies. The term "monoclonal antibody" refers to an antibody composition having a homogeneous population, regardless of the species, the origin and the method for obtaining said antibody. In addition, the term "antibody" includes human antibodies in which at least some of the immunoglobulin domains are present, such as antibody fragments and the so-called VHVL domains (Variable Heavy and Variable Light Chains), and mini-antibodies. The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

Any biological sample that may contain antibodies against SARS-CoV-2 can be used in the methods disclosed herein. Non-limiting examples of suitable biological samples include blood- derived samples, saliva samples, naso-oropharyngeal swab and other fluids (oropharyngeal swab, bronchoalveolar lavage or sputum). Blood-derived samples may include (whole)blood samples, serum samples and plasma samples. In preferred embodiments, the biological sample is a blood sample, a serum sample, a plasma sample or a saliva sample.

In step (i) of the methods discloses herein, the sample is contacted with a combination of antigens to allow the formation of antigen/antibody complexes or immunocomplexes. Said "contacting" is to be understood herein to occur "under conditions sufficient for the antigens and antibodies in the sample to form an antigen/antibody complex", e.g. for sufficient time.

Prior to the detection of the formed antigen/antibody complexes, the methods disclosed herein may contain a washing step to remove any of the sample that has not reacted with the antigens. Standard wash conditions may be used in the methods described herein. For example, a dilute non-ionic detergent medium at an appropriate pH, generally 7-8, can be used as a wash solution. From one to six washes can be employed, with sufficient volume to thoroughly wash non-specifically bound components present in the sample.

In the methods disclosed herein, antibodies against SARS-CoV-2 in the biological sample of the subject are detected through the formed antigen/antibody complexes or immunocomplexes. The detection of the antigen/antibody complexes may be performed by an ELISA (Enzyme-Linked Immunosorbent Assay) technique, known to a person skilled in the art and exemplified in the examples section. Any other technique, however, making it possible to detect and/or measure the antigen/antibody complexes can also be applied in the methods disclosed herein. Non-limiting examples of other suitable techniques include protein arrays and bead- based systems (e.g. the Luminex XMAP bead array system (www.luminexcorp.com)).

In embodiments, the detection of the formed antigen/antibody complexes comprises contacting the complexes with a detector molecule capable of binding to (the antibodies of) the formed antigen/antibody complexes, the detector molecule comprising at least one detectably labelled moiety.

In embodiments, said detector molecule is a labelled anti-human immunoglobulin antibody. Said detector molecule may be able to detect human antibodies in all immunoglobulin classes, or be specific to one or more particular immunoglobulin isotype, e.g. IgG, IgM or IgA. Accordingly, in embodiments, the detector molecule may be, for example, a labelled anti human IgG antibody, a labelled anti-human IgA antibody or a labelled anti-human IgM antibody, or any combination thereof. In embodiments, the formed antigen/antibody complexes are detected by contacting the complexes with a labelled anti-human IgG antibody. In embodiments, the formed antigen/antibody complexes are detected by contacting the complexes with a combination of a labelled anti-human IgG antibody and a labelled anti human IgA antibody. Said anti-human immunoglobulin antibodies may be obtained from any animal, including, without limitation, goat, rabbit, hamster, chicken, rat, guinea pig, sheep, horse, and mouse. These antibodies are commercially available.

By "detectable label" is meant a moiety that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Any type of detectably labelled moiety can be used for labelling the detector molecule. Non-limiting examples of suitable labels include a radiolabel, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a colloidal gold label, a dye moiety, a paramagnetic compound, a detectable enzyme, biotin, avidin, and streptavidin. In embodiments, the detector molecule, such as the anti-human immunoglobulin antibody, is labelled with an enzyme, such as, for example, horseradish peroxidase.

Detection of the antigen/antibody complexes formed may be done according to any methods known in the art, the type of detection of course depending on the type of label used for the detector molecule. For example, in the case where the antigen/antibody complexes emit colorimetric signals, e.g. after addition of an enzyme-conjugated anti-human immunoglobulin antibody (e.g. a horseradish peroxidase (HRP)-conjugated anti-human immunoglobulin antibody) and of a specific substrate chromogen (e.g. TMB chromogen), the colorimetric signal emitted by the antigen/antibody complexes formed can be measured using an absorbance plate reader.

In embodiments, the detection of the antigen/antibody complexes comprises determining the presence or absence of antibodies in the sample for each antigen.

In embodiments, the detection of the antigen/antibody complexes comprises determining or measuring the amount of antibodies in the sample for each antigen.

The terms "amount", "quantity" and "level" are synonymous and as used herein refer to but is not limited to the absolute or relative amount of antibody, and any other value or parameter associated with the latter or which can derive therefrom. Such values or parameters comprise signal intensity values obtained for physical properties of the antibody, obtained by direct or indirect measurement, for example, intensity values in an immunoassay. The readout may be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement. The absolute values obtained for each antigen under identical conditions will display a variability that is inherent in live biological systems and also reflects individual antibody variability as well as the variability inherent between individuals.

As used herein, a "serological signature" or "serological profile" refers to the antigen spectrum of antigens recognized by the antibodies derived from the biological sample of the subject, e.g. as determined by a method for detecting antibodies directed against SARS-CoV-2 as described herein.

In the methods described herein, serological signatures as determined by the methods and tools described herein are compared, e.g. with a refence or control serological signature of sample from another subject, or with a serological signature of a sample collected at another time point. These signatures or profile may be evaluated on a number of points, including, without limitation, the presence/absence of reactive or binding antibodies for one or more, or all, antigens, the total amount of reactive antibodies, the amount of antibodies reactive on one or more particular antigens, any combinations thereof, etc. A statistical test may be utilized to provide a confidence level for a change in the presence and/or amount of antibodies between the test and reference signatures to be considered significant. Non limiting examples of statistical methods that can be utilized include utilize decision tree analysis, classification algorithms, regression analysis, principal components analysis, multivariate analysis, predictive models, supervised and unsupervised machine learning, genetic algorithms, neural networks, and combinations thereof.

The terms "sensitivity" and "specificity" as used herein refer to the accuracy of a test or assay in terms of accurately identifying positive and negative results. The "sensitivity" of a serological assay refers to the ability of the assay to correctly return a positive result for a sample that contains the targeted antibodies. "Specificity" of a serological assay refers to the ability of the assay to correctly return a negative result for a sample that does not contain the targeted antibodies. In general, there is an inverse relationship between sensitivity and specificity: If the threshold required to return a positive result is lowered in order to detect more positive samples, the test might provide positive results for more negative samples ("false positives"). Conversely, if a test is designed to return as few false negative results as possible, it might also miss some positive samples.

The present invention is characterized in that the methods and tools provided herein allow for the detection -simultaneously or sequentially, preferably simultaneously- of a plurality of antibodies against a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen in a single sample (i.e. multiplex methods and tools), yet allowing detection of discrete reactivities on each of the antigens.

The terms "antigen derived from SARS-CoV-2" or "SARS-CoV-2 antigen" as used herein refer to native antigens isolated from SARS-CoV-2, which may have been semi-purified or purified, as well as corresponding recombinant antigens and synthetic antigens.

As used herein, a "recombinant SARS-CoV-2 antigen" is an antigen that is produced using recombinant nucleic acid technology and that is able to specifically detect an antibody selective for SARS-CoV-2. As used herein, an antibody "selective for SARS-CoV-2", also referred to herein as an anti-SARS-CoV-2 antibody, is an antibody that selectively binds to SARS-CoV-2 in that it preferentially binds to SARS-CoV-2 substantially to the exclusion of binding to random or unrelated infectious agents, and optionally substantially to the exclusion of infectious agents that are structurally related. A recombinant antigen is also able to specifically detect the presence of such an antibody in that the recombinant antigen is sufficiently similar to the corresponding native antigen of SARS-CoV-2 to enable such detection. Likewise, a "synthetic SARS-CoV-2 antigen" is an antigen that is (chemically) synthesized and that is able to specifically detect an antibody selective for SARS-CoV-2. The antigens are preferably derived from a structural protein of SARS-CoV-2 or any subunit or domain thereof, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) protein. The whole protein can be used, or any antigenic fragment thereof. An "antigenic fragment" or "immunologically reactive fragment" of a protein or peptide refers to a portion of the protein or peptide that is immunologically reactive with a binding partner, e.g., an antibody, which is immunologically reactive with the protein or peptide itself.

Advantageously, the combination of antigens derived from SARS-CoV-2 that is assayed in the methods and by the tools disclosed herein encompasses antigens derived from different SARS- CoV-2 proteins. Due to sequence homology of SARS-CoV-2(structural) proteins with (structural) proteins of other strains of coronavirus, cross-reactivity may occur, resulting in the detection of non-specific antibodies (i.e. antibodies that bind to multiple strains of coronavirus) and eventually false-positives. It is noted that SARS-CoV-2 nucleoprotein is expected to induce more cross-reactivity than the spike protein. Specificity can be increased, and false-positives decreased, upon using a combination of antigens derived from different SARS-CoV-2 proteins, in particular the well-defined combination of antigens derived from SARS-CoV-2 described herein.

In embodiments, the methods and tools described herein further allow the detection of so- called Trypanosoma-cruzi-Cross Reactive Antibodies (TcCRA).

The terms "T. cruzi antigen", and "Cryptic Inflammatory Antigen (CIA) antigen" are used as synonyms herein and refer to a peptide antigen comprising or consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7). The antigen allows the detection of so- called Trypanosoma-cruzi-Cross Reactive Antibodies (TcCRA). These antibodies were found at a high seroprevalence (40% to 50%) in serum of individuals living in Europe or other non endemic regions, not exposed to T. cruzi (Saba et al. 2013. Anti-Trypanosoma cruzi Cross- Reactive Antibodies Detected at High Rate in Non-Exposed Individuals Living in Non-Endemic Regions: Seroprevalence and Association to Other Viral Serologies. PLoS One. 8(9):e74493).

In particular, the combination of antigens that are detected in the methods and by the tools disclosed herein comprises:

(d) optionally a T. cruzi antigen comprising or consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7).

The SARS-CoV-2 proteins and peptides as described herein encompass native (i.e. naturally occurring), isolated, SARS-CoV-2 proteins and peptides, which may have been semi-purified or purified, as well as variants thereof, and corresponding recombinant proteins or peptides and synthetic proteins or peptides.

The term "protein" as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis corresponding native proteins, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally- occurring protein parts that ensue from processing of such full-length proteins.

The term "polypeptide" as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms "protein" and "polypeptide" may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally-occurring polypeptide parts that ensue from processing of such full- length polypeptides.

The term "peptide" as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids. The term "variants" as used herein encompasses proteins, polypeptides and peptides the amino acid sequence of which is substantially identical (i.e., largely but not wholly identical) to the amino acid sequence of a native protein, polypeptide or peptide, for example at least about 80% identical or at least about 83% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, at least 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical. Preferably, a variant may display such degrees of identity to the (native) protein, polypeptide or peptide when the whole sequence of the variant and the (native) protein, polypeptide or peptide are queried in the sequence alignment (i.e., overall sequence identity).

Sequence identity between proteins, polypeptides and peptides as envisaged herein may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described byTatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89:10915-10919), cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3). Sequence identity as envisaged herein may preferably denote overall sequence identity, i.e., sequence identity calculated from aligning the whole sequences of the compared proteins or peptides. An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (BI2seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters includes: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3. If the two compared sequences share homology, then the output will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the output will not present aligned sequences. Once aligned, the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the query polypeptide, followed by multiplying the resulting value by 100. The percent identity value may, but need not, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It is further noted that the detailed view for each segment of alignment as outputted by BI2seq already conveniently includes the percentage of identities.

The reference to "SARS-CoV-2 membrane (M) protein" denotes the SARS-CoV-2 M protein as commonly known under this designation in the art. The SARS-CoV-2 M protein is a glycoprotein of SARS-CoV2 that is involved in the formation and budding of the viral envelope. It is an integral membrane protein that plays an important role in viral assembly, in particular, it interacts with the nucleocapsid (NC) protein to encapsidate or encapsulate the RNA genome.

The term "SARS-CoV-2 M protein" particularly encompasses the SARS-CoV2 M protein as annotated under GenBank (http://www.ncbi.nlm.nih.gov/) accession number MN908947 comprising or consisting of the amino acid sequence:

MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTL ACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPL HGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQR VAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ (SEQ ID NO:l).

A non-limiting example of an antigenic fragment of SARS-CoV-2 M protein is a peptide, such as a synthetic peptide, comprising, consisting essentially of or consisting of the amino acid sequence: MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5).

The reference to "SARS-CoV-2 nucleocapsid (NC or N) protein" denotes the SARS-CoV-2 NC protein as commonly known under this designation in the art. The "SARS-CoV-2 nucleocapsid (NC) protein" is one of the major structural components involved in many processes of SARS- CoV-2 including viral replication, transcription, and assembly. SARS-CoV-2 nucleocapsid protein is the most abundant and conservative structural protein in coronaviruses. The term "SARS-CoV-2 NC protein" particularly encompasses the SARS-CoV2 NC protein as annotated under GenBank (http://www.ncbi.nlm.nih.gov/) accession number MN908947 comprising or consisting of the amino acid sequence:

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGrPNNTASWFTALTQHGKE DLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYG ANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRS SSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTK KSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPS ASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDK KKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO:2).

A non-limiting example of an antigenic fragment of SARS-CoV-2 NC protein is a peptide, such as a synthetic peptide, comprising, consisting essentially of or consisting of the amino acid sequence: NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6).

The reference to "SARS-CoV-2 spike (S) protein" denotes the SARS-CoV-2 S protein as commonly known under this designation in the art. SARS-CoV-2 spike (S) protein is the main surface glycoprotein of SARS-CoV-2. It is a transmembrane protein with a molecular weight of about 150 kDa. The S-protein mediates host cell entry by binding, with is receptor-binding domain (RBD), to the angiotensin-converting enzyme 2 (ACE2). The S protein forms homotrimers protruding in the viral surface. The S protein is cleaved by the host cell furin-like protease into 2 subunits, namely SI and S2. The SI subunit is responsible for the determination of the host virus range and cellular tropism with the receptor binding domain make-up, while the S2 subunit functions to mediate virus fusion in transmitting host cells.

The term "SARS-CoV-2 S protein" particularly encompasses the SARS-CoV2 S protein as annotated under GenBank (http://www.ncbi.nlm.nih.gov/) accession number YP_009724390.1 comprising or consisting of the amino acid sequence:

MFVFLVLLPLVSSQCW LTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIH

VSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIWNATNW IKVCEFQFCND

PFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKH

TPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFL

LKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT

RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQ

TGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV

EGFNCYFPLQSYGFQPTNGVGYQPYRVW LSFELLHAPATVCGPKKSTNLVKNKCW FNFNGLTGTGVL

TESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDW CTEVP

VAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHWNSYECDIPIGAGICASYQTQTNSPRRARSVASQ SIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSF CTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTL ADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVWQNAQALNTLVKQL SSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLG QSKRVDFCGKGYHLMSFPQSAPHGW FLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFV TQRNFYEPQIITTDNTFVSGNCDW IGIWNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGIN ASVW IQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCS CLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO:3), and the SARS-CoV-2 S protein comprising or consisting of the amino acid sequence of amino acids 16 to 1213 of SEQ ID NO:3.

The term "SARS-CoV-2 SI protein" particularly encompasses the SARS-CoV-2 SI protein comprising the amino acid sequence of amino acids 16 to 685 of SEQ ID NO:3, and the term "SARS-CoV-2 S2 protein" particularly encompasses the SARS-CoV-2 S2 protein comprising the amino acid sequence of amino acids 686 to 1213 of SEQ ID NO:3.

The term "SARS-CoV-2 RBD protein" particularly encompasses the SARS-CoV-2 RBD protein comprising or consisting of the amino acid sequence:

TNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV

IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST

EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVW LSFELLHAPATVCGPENLYFQGHHHHH

H (SEQ ID NO :4).

The combination of antigens used in the methods and tools described herein may comprise one or more than one, such as two, three, four or more antigens in each of a), b) and c). The combination of antigens may comprise the same number or a different number of antigens in each of a), b) and c).

In particular embodiments, the combination of antigens comprises: a) an antigenic fragment of SARS-CoV-2 M protein comprising or consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5); b) SARS-CoV-2 NC protein and an antigenic fragment of SARS-CoV-2 NC protein comprising or consisting of the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6); c) SARS-CoV-2 spike (S) protein, SARS-CoV-2 receptor-binding domain (RBD) protein, SARS- CoV-2 SI protein and SARS-CoV-2 S2 protein; and d) optionally a T. cruzi antigen consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7). In particular embodiments, the combination of antigens comprises: a) an antigenic fragment of SARS-CoV-2 M protein comprising or consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5); b) SARS-CoV-2 NC protein and an antigenic fragment of SARS-CoV-2 NC protein comprising or consisting of the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6); c) SARS-CoV-2 receptor-binding domain (RBD) protein, SARS-CoV-2 SI protein and SARS-CoV- 2 S2 protein; and d) optionally a T. cruzi antigen consisting of the amino acid sequence AAA PA K AA AA P AKT AAA P V (SEQ ID NO:7).

In particular embodiments, the combination of antigens comprises or consists of: a) an antigenic fragment of SARS-CoV-2 M protein comprising or consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5); b) SARS-CoV-2 NC protein and an antigenic fragment of SARS-CoV-2 NC protein comprising or consisting of the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6); c) SARS-CoV-2 spike (S) protein, SARS-CoV-2 receptor-binding domain (RBD) protein and SARS- CoV-2 SI protein; and d) optionally a T. cruzi antigen comprising or consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7).

In further particular embodiments, the combination of antigens comprises:

(a) a (synthetic) peptide consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5),

(b) SARS-CoV-2 NC (recombinant) protein consisting of the amino acid sequence of SEQ ID NO:2 and a (synthetic) peptide consisting of the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6),

(c) SARS-CoV-2 spike (S) (recombinant) protein consisting of the amino acid sequence of amino acids 16 to 1213 of SEQ ID NO:3, SARS-CoV-2 SI (recombinant) protein consisting of the amino acid sequence of amino acids 16 to 685 of SEQ ID NO:3, SARS-CoV-2 S2 (recombinant) protein consisting of the amino acid sequence of amino acids 686 to 1213 of SEQ ID NO:3 and SARS- CoV-2 spike receptor-binding domain (RBD) (recombinant) protein consisting of the amino acid sequence of SEQ ID NO:4, and

(d) optionally a T. cruzi (synthetic) peptide consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7).

In further particular embodiments, the combination of antigens comprises: (a) a (synthetic) peptide consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5),

(c) SARS-CoV-2 SI (recombinant) protein consisting of the amino acid sequence of amino acids 16 to 685 of SEQ ID NO:3, SARS-CoV-2 S2 (recombinant) protein consisting of the amino acid sequence of amino acids 686 to 1213 of SEQ ID NO:3 and SARS-CoV-2 spike receptor-binding domain (RBD) (recombinant) protein consisting of the amino acid sequence of SEQ ID NO:4, and

In further particular embodiments, the combination of antigens comprises:

(c) SARS-CoV-2 spike (S) (recombinant) protein consisting of the amino acid sequence of amino acids 16 to 1213 of SEQ ID NO:3, SARS-CoV-2 SI (recombinant) protein consisting of the amino acid sequence of amino acids 16 to 685 of SEQ ID NO:3 and SARS-CoV-2 spike receptor-binding domain (RBD) (recombinant) protein consisting of the amino acid sequence of SEQ ID NO:4, and

Peptide antigens

A further aspect relates to the newly identified SARS-CoV-2 antigens identified by the present inventors, as well as their use for the detection of antibodies directed to SARS-CoV-2.

Accordingly, a further aspect relates to a SARS-CoV-2 antigen comprising or consisting of a peptide comprising, consisting essentially of or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In embodiments, the peptide comprises, consists essentially of or consists of a fragment of the SARS-CoV-2 M protein, in particular the protein consisting of the amino acid sequence set forth in SEQ ID NO:l, and comprises the sequence set forth in SEQ ID NO:5. In embodiments, the peptide comprises, consists essentially of or consists of a fragment of the SARS-CoV-2 NC protein, in particular the protein consisting of the amino acid sequence set forth in SEQ ID NO:2, and comprises the sequence set forth in SEQ ID NO:6. In embodiments, the peptide has a length of less than 40, preferably less than 35, more preferably less than 30 or less than 25 amino acids. In embodiments, the peptide is a synthetic peptide.

Also disclosed herein is the use of a peptide comprising, consisting essentially of or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 as described herein for the detection of antibodies directed to SARS-CoV-2.

The herein described method can be applied for the diagnosis and/or prognosis of a SARS- CoV-2 infection in a subject. In particular, a method for the diagnosis and/or prognosis of a SARS-CoV-2 infection in a subject is provided herein, said method comprising:

(i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen as described herein, wherein said antigens are immobilized on a solid support spatially separated from one another, to allow the formation of antigen/antibody complexes;

(ii) detecting, preferably simultaneously, the antigen/antibody complexes formed in (i) to determine a serological signature; and

(iii) comparing the serological profile with a reference serological signature indicative for the diagnosis and/or prognosis of a SARS-CoV-2 infection. The method described herein can be used as confirmation test following an initial diagnostic evaluation of a sample from the subject to allow the confirmation/refutation of a SARS-CoV-2 infection in the subject. For example, the subject may initially have been tested positive for a SARS-CoV-2 infection by the detection of viral RNA in a sample from the subject by a nucleic acid-based method (e.g. a PCR-based assay). Accordingly, in embodiments of the method described herein, the subject has been tested positive for a SARS-CoV-2 infection by another detection method such as a nucleic acid-based method (e.g. a PCR-based assay).

The present inventors found that the serological signature as determined by the methods described herein allows to distinguish or discriminate subjects into groups, which groups are informative of the clinical severity of the infection in the patient and/or of the subject's need for therapy. Assessment of this signature of anti-SARS-CoV-2 antibodies in a subject thus allows to provide an appropriate therapy to a subject. In particular, as shown in the experimental section, it was found that the serological signature as determined by the methods described herein allows to distinguish subjects into asymptomatic subjects, mild or moderate symptomatic subjects (not requiring hospitalization), severe symptomatic subjects requiring hospitalization and severe symptomatic subjects requiring intensive care treatment). Advantageously, the use of a multiplex method as described herein, wherein a combination of antigens as described herein is used, can provide a level of discrimination not found with individual antigens.

More particularly, it was found that severe symptomatic subjects requiring intensive care treatment showed relatively high reactivities against an antigenic fragment of SARS-CoV-2 M protein consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5), SARS-CoV-2 NC protein and an antigenic fragment of SARS-CoV-2 NC protein consisting of the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6), SARS-CoV-2 SI protein, and SARS-CoV-2 RBD protein, and relatively low reactivities on the CIA antigen, e.g. as compared to mild symptomatic subjects and/or severe symptomatic subjects requiring hospitalization.

Accordingly, in an aspect, the present invention relates to a method for categorizing a patient diagnosed with a SARS-CoV-2 infection as asymptomatic, mild symptomatic, severe symptomatic requiring hospitalization or severe symptomatic requiring intensive care treatment, said method comprising determining a serological signature of the patient according to the method described herein and comparing said serological signature with a reference or control serological signature. The serological signature of the patient may also be compared with a set of reference signatures. A reference or control serological signature may be a serological signature that is determined from a biological sample of a patient known to be an asymptomatic patient, a mild symptomatic patient, a severe symptomatic patient requiring hospitalization or a severe symptomatic patient requiring intensive care treatment.

In order to identify serological signatures that are indicative of a clinical status, a statistical test may be utilized to provide a confidence level for a change in the presence and/or amount of antibodies between the test and reference signatures to be considered significant as described elsewhere herein.

In embodiments, a serological signature comprising high reactivities on an antigenic fragment of SARS-CoV-2 M protein consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5), SARS-CoV-2 NC protein and an antigenic fragment of SARS-CoV-2 NC protein consisting of the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6), SARS-CoV-2 SI protein, and SARS-CoV-2 RBD protein, and optionally low reactivities on the CIA antigen compared to the reference or control serological signature such as a serological signature determined from a biological sample of a non-infected subject, an asymptomatic patient, a mild symptomatic subject or a severe symptomatic subjects requiring hospitalization indicate that the subject is a severe symptomatic subject requiring intensive care treatment.

Further provided herein is a method for predicting the effectiveness of (or the prognosis of responsiveness to) a therapy in a subject, said method comprising:

(iii) comparing the serological profile with a control or reference serological signature indicative for effectiveness of or responsiveness to the therapy.

In embodiments, the control or reference serological signature is a serological signature as determined by a method disclosed herein from a biological sample of a patient known to be responsive or non-responsive to said therapy. In further embodiments, the serological profile of the subject is compared with a control serological signature, wherein a statistically significant match with a serological signature from a biological sample of a responder to said therapy or a statistically significant difference with a serological signature from a non responder to said therapy is indicative that said subject is responsive to said therapy.

In embodiments, said therapy may comprise the administration of anti-inflammatory drugs (e.g. dexamethasone).

The serological signatures as determined by the methods described herein also allow to compare and/or discriminate SARS-CoV-2 vaccine-induced serological signatures from SARS- CoV-2 natural infection-induced serological signatures. The comparison of these signatures allows to predict the efficacy of a candidate vaccine. It also allows to predict the effectiveness of a vaccine in a subject and/or to provide an appropriate vaccination method to a subject.

Accordingly, yet a further application resides in a method for predicting the effectiveness of a vaccination in a subject, said method comprising determining a serological signature in a biological sample from the subject and comparing said serological signature with a reference or control serological signature indicative for effectiveness of the vaccine such as a SARS-CoV- 2 vaccine-induced serological signature. More particularly, a method for predicting the effectiveness of a SARS CoV-2 vaccination in a subject, said method comprising:

(iii) comparing the serological signature with a reference or control serological signature indicative for effectiveness of the vaccine.

In embodiments, the control serological signature is a serological signature as determined by a method disclosed herein from a biological sample of a vaccinated subject (i.e. a vaccine- induced serological signature).

The herein described methods may also be applied for monitoring the (clinical) progression of a SARS-CoV-2 infection in a subject, and/or eventually for monitoring the efficacy of a therapeutic treatment or a vaccination method. Accordingly, provided herein is a method for monitoring the clinical progression of a SARS-CoV-2 infection in a subject, said method comprising:

(i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen as described herein to allow the formation of antigen/antibody complexes; and

(ii) detecting, preferably simultaneously, the antigen/antibody complexes formed in (i) to determine a serological signature.

In embodiments the method may involve comparing the serological signature with a (set of) reference or control serological signature(s). The control signature may be indicative for a clinical status of a SARS-CoV-2 infection, e.g. a serological signature determined from a biological sample of a subject with a known clinical status. In the case of a method for monitoring the efficacy of a therapy, the control sample can be a biological sample taken from a subject that is known to be responsive to the therapy. In the case of a method for monitoring the efficacy of a vaccination, the control sample can be a biological sample taken from a subject that has been vaccinated.

In embodiments, the method encompasses determining a serological signature according to the methods described herein in biological samples collected from the patient at two or more various time points, e.g. at two or more various time points after a subject has exhibited (clinical) symptoms associated with a SARS-CoV-2 infection, or before and after administration of a therapeutic treatment (e.g. for determining the responsiveness to a therapy) or vaccination (e.g. for determining the responsiveness to a (candidate) vaccine), and comparing said serological signatures.

A further application resides in the use of the methods described herein for identifying subjects that have neutralizing antibodies to SARS-CoV-2 such as convalescent patients, which may be useful as donor for preparation of therapeutic antibodies. Other subjects that may have neutralizing antibodies to SARS-CoV-2 include, for example, recipients of a vaccine (candidate). The method comprises comparing a serological signature determined from a biological sample of a subject according to the methods described herein with a serological signature determined from a biological sample of a subject, e.g. a SARS-CoV-2 infected patient, known to have SARS-CoV-2 neutralizing antibodies. For example, the reference biological sample may be a biological sample that has been tested positive in an in vitro virus seroneutralization assay. In particular, a method is provided for identifying a subject that has neutralizing antibodies to SARS-CoV-2, said method comprising:

(i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen, in particular the CIA antigen, as described herein, wherein said antigens are immobilized on a solid support spatially separated from one another, to allow the formation of antigen/antibody complexes;

(iii) comparing the serological signature with a reference or control serological signature indicative for the presence of neutralizing antibodies to SARS-CoV-2, in particular a serological signature from a biological sample that has been tested positive in an in vitro virus seroneutralization test.

In a further aspect, the methods described herein are used for determining a SARS-CoV-2 neutralizing antibody titer in a subject. In particular, a method is provided herein for determining a SARS-CoV-2 neutralizing antibody titer in a subject, comprising:

(i) contacting a biological sample from the subject with a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen, in particular the CIA antigen, as described herein, wherein said antigens are immobilized on a solid support spatially separated from one another, to allow the formation of antigen/antibody complexes; (ii) detecting, preferably simultaneously, the antigen/antibody complexes formed in (i) to determine a serological signature; and

(iii) comparing the serological signature with a reference serological signature indicative for a SARS-CoV-2 neutralizing antibody titer such as a serological signature from a biological sample wherein a NT₅o neutralization titre has been determined in an in vitro virus seroneutralization test.

Said "determination" may encompass a semi-quantitative determination or estimation or prediction of a neutralizing antibody titer. In particular embodiments, a subject is classified as containing a NT₅o neutralization titre of below 50, between 50 and 500, or more than 500. In embodiments the method may involve comparing the serological signature with a set of reference or control serological signatures, wherein said set comprises a reference or control signature indicative for a NT₅o neutralization titre of below 50, a reference or control signature indicative for a NT₅o neutralization titre of between 50 and 500 and a reference or control signature indicative for a NT₅o neutralization titre of more than 500. These reference or control signatures may be serological signatures determined from a biological sample of a subject with a known SARS-CoV-2 neutralizing antibody titer, for example a biological sample wherein the NT_5O neutralization titre has been determined in an in vitro virus seroneutralization assay.

In contrast to determining in vitro (cell-culture based) virus neutralizing activity of a biological sample, in particular a serum sample, which is a lengthy process (several days) and requires live biological materials and a biocontainment facility level 3, the method disclosed herein, which is based on a serological signature determined from the biological sample, is fast (hours), easy to perform and safe.

The herein described methods and tools may also be used for monitoring a SARS-CoV-2 immune response in a subject, said method comprising determining a serological signature according to the methods described herein in biological samples collected from the subject at two or more various time points, and comparing said serological signatures. In particular embodiments, said method may comprise determining SARS-CoV-2 neutralizing antibody titers according to the methods described herein at two or more various time points, and comparing the determined neutralizing antibody titers.

A related aspect is directed to a method for predicting protective immunity against a SARS- CoV-2 infection in a subject, e.g. a subject who received a vaccine (candidate), said method comprising comparing a serological signature determined from a biological sample of a subject according to the methods described herein with a reference serological signature indicative for protective immunity. As used herein "protective immunity" refers to having an immune response that can prevent future infections of SARS-CoV-2.

In particular, a method is provided for predicting protective immunity against a SARS-CoV-2 infection in a subject, said method comprising:

(iii) comparing the serological signature with a reference or control serological signature indicative for protective immunity against a SARS-CoV-2 infection.

Support

A further aspect relates to a solid support for detecting antibodies directed to SARS-CoV-2 in a sample, said support comprising a combination of antigens as described herein in relation to the methods, wherein said antigens are immobilized on said solid support spatially separated from one another.

The term "solid support" in the current invention refers to any substrate to which a protein or peptide can be immobilized, provided that it retains its immunological reactivity and provided that the background of non-specific binding remains low. Non-limiting examples of suitable solid supports include a microtiter plate or microplate, a membrane (e.g. nitrocellulose, paper, nylon or PVDF), a microsphere (bead) or a microarray (e.g. glass, silicon, plastic). In preferred embodiments, the solid support is a microplate, such as a multi-well, e.g. 96-well, polystyrene microplate.

Techniques for immobilizing proteins and peptides on a solid support are well-known to the skilled person, and include, for example, printing technologies. For example, the protein and peptide antigens can be immobilized on a microplate using a sciFLEXARRAYER printing system (e.g. from SCIENION, Germany) using a non-contact piezoelectric needle. Equivalent operations can be achieved also with a contact printing technology.

Each of the antigens is immobilized, e.g. printed, at a defined concentration. For each antigen a defined concentration to obtain an optimal balance between sensitivity and specificity can be determined using a panel of negative and positive samples. The antigens are immobilized individually (i.e. not as mixtures) and spatially separated from one other on the solid support. Spatial separation of the antigens allows detection of the reactivities on each of the antigens. In preferred embodiments, all antigens (of the combination of antigens described herein) are immobilized on a single solid substrate (e.g. a single well in a microwell plate). This allows the simultaneous detection of the formed antigen/antibody complexes. In other embodiments, each of the antigens is immobilized on a separate solid substrate (e.g. on separate microspheres).

In embodiments, the antigens are immobilized in duplicate on the solid support to enhance readout robustness.

The antigens are preferably immobilized on the solid support in known positions, e.g. defined in X-Y coordinates to allow reliable detection of the specific reacting antibodies. The antigens may be immobilized as dots or spots, lines or other figures.

In addition to the antigens, the solid support may comprise one or more, such as one, two or three, positive controls. For example, the positive control may be an anti-human immunoglobulin antibody, such as anti-human IgG antibody, to detect human antibodies in the samples and the detector molecule.

The positive controls may be immobilized on the solid support in a pattern to allow a precise orientation of the solid support.

Kit

Also disclosed herein is a kit for detecting antibodies directed to SARS-CoV-2 in a sample, said kit comprising: a solid support as described herein; a wash solution, or components necessary for producing said buffer; means for detection of the formed antigen/antibody complexes.

The term "wash solution" means a solution enabling washing of the formed immunocomplexes.

"Means for detection of the formed antigen/antibody complexes" may include detector molecules as described elsewhere herein. In case of enzyme-conjugated detector molecules, means for detection of the formed antigen/antibody complexes may comprise the enzyme- conjugated detector molecule and a specific substrate molecule.

The examples of the present invention presented below are provided only for illustrative purposes and not to limit the scope of the invention. Examples

Example 1: Multiplex serological profiling for SARS-CoV-2 infection

Multiplex assay 8 antigens were printed in each well of a 96-well polystyrene microplate using a sciFLEXARRAYER printing system (SCIENION, Germany). Three spots of positive controls (PC) designed to check for the presence of human samples and enzyme conjugates were printed on the array using a precise orientation pattern. Each antigen was printed at a defined concentration and in duplicate to enhance readout robustness; positioning onto the microplate surface was precisely defined in X-Y coordinates to allow reliable recognition of the specific reacting antibodies. Figure 1 shows a design of the microplate.

Table 1: Antigens used in the multiplex assay.

For each antigen, a panel of negative and positive samples was used to carefully balance the applied concentrations and obtain the optimal conditions with respect to sensitivity and specificity. Each antigen was diluted with carbonate buffer (pH 9.6).

Printing was performed using a non-contact piezoelectric needle under monitored temperature and controlled humidity conditions. Following spotting, plates were incubated overnight in the same conditions, then dried at 37°C for 2h and washed with PBST (phosphate- buffered saline with 0.05% Tween20). A blocking solution was added to the microplate for 2h at room temperature to block nonspecific binding. Following saturation, microplates were washed with PBST. For long term storage, blocked arrayed microplates were stored at 4°C in sealed plastic bag with desiccant. Study Population and Samples

Samples were collected as schematically shown in Figure 2. In brief, subjects with symptoms for COVID were tested for the presence of SARS-CoV-2, e.g. upon arrival at the hospital. Blood samples were collected for each infected patient at two times. Infected patients were classified in three clinical categories: 1) non-hospitalized, mild symptoms, 2) symptoms, hospitalized, but not taken in intensive care unit and 3) hospitalized and taken up in intensive care unit.

Summary of the study subjects:

• 3 different groups -> PCR positive

> Mild Symptoms (non-hospitalized) -> health-care personnel (13 patients; 26 samples)

> Symptoms (hospitalized) (13 patients; 26 samples)

> Severe Symptoms (intensive care) (13 patients; 26 samples)

• 2 different groups -> PCR negative

> Pre-epidemic (N=8)

> Other Coronavirus positive (OC43; 229E; NL63; HKU1) (9 patients; 10 samples)

Table 2: Summary time-metrics for the infected patients.

Multi-SARS-CoV-2 ELISA Assay

The microplates were incubated with the blood donors or patients sera diluted at 1:101 for lh at room temperature and washed three times with PBST. The dilution parameter was optimized during the assay development. Next, horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibodies (Abliance, France), anti-human IgM (Abliance, France) antibodies or anti-human IgA antibodies (Abliance, France) adequately diluted was added to the microplate for lh at room temperature. The microplates were then washed three times before adding a precipitating TMB (3,3',5,5'-tetramethylbenzidine) solution (Seramun, Germany) for 20 min at room temperature in the dark. Then, TMB solution was removed and plates were dried at 37°C for 10 min. Each plate was imaged, then analysed using a microplate reader with digital image analysis capabilities (Scienion, Germany). The software calculates the pixel intensity for each spot. In order to establish the net intensity for each antigen, the mean value of the duplicated spots was used. A schematic of the microplate analysis is shown in Figure 5.

Results

Figure 3 shows the mean raw values for each of the 8 antigens obtained on the multi-SARS- CoV-2 ELISA assay on first and second serology samples from the PCR-positive patients. The CIA antigen showed a remarked decrease in total IgG response in hospitalized patients (Fig. 3A). The immune response to the MP1 antigen was delayed in severely affected patients in early responses but non-discriminatory after 20 days (Fig. 3B). Levels between the three categories were similar after 20 days (Fig. 3B). The immune response to the NCI nucleocapsid antigen was gradually increased over time in all three categories and was consistently higher in hospitalized versus non-hospitalized patients (Fig. 3C). Response to NCI seems to parallel the clinical severity of the SARS-CoV-2 infection and the maturation of the immune response in severely infected patients. The immune response to the NC2 nucleocapsid antigen was solely increased in hospitalized persons with severe symptoms (Fig. 3D). Immune response to this antigen is shown a particular strong indicator of a severe response to SARS-CoV-2 infection. The immune response to the RBD antigen was somewhat increasing over time in all three categories and was consistently higher after 20 days (Fig. 3E). Immune response to RBD antigen seems to parallel the maturation of the immune response. The immune response to SI antigen was somewhat increasing over time in the hospitalized persons and paralleled the severity of infection and the maturation of the immune response in severely infected people (Fig. 3F). The immune response to S2 spike antigen was similar in all three categories, slightly increasing in the persons taken up in intensive care (Fig. 3G).

The three main subclasses of antibodies IgM, IgA and IgG were analysed separately in order to see the variations in immune responses in the patients. The results are shown in Figure 4 for the NCI antigen.

In conclusion, the data shown in Figures 3 and 4 show that the immune response towards SARS-CoV-2 is complex and the immune response differs according to the clinical severity of the SARS-CoV-2 infection. In particular, consistently higher titers were observed in the patients admitted in the intensive care unit for the MP1, NCI, NC2, RBD and SI antigens, and low titers for the CIA antigen. This example demonstrates that the multiplex approach provides specific antibody signatures paralleling the severity of the SARS-CoV-2 infection in a subject (Table 3).

Table 3: Immunogenic response profile (20 days timepoint) as determined by the multi-SARS- CoV-2 ELISA assay.

Example 2: Comparison of the multiplex assay according to the invention with commercial ELISA assays for determining SARS-CoV-2 seropositivity using serum samples

The multi-SARS-CoV-2 ELISA assay as described in Example 1 was compared to commercial ELISAs to detect an immune response to a SARS-CoV-2 infection in a subject. Blood samples

A total of 338 serum samples from 338 individuals in Belgium, in whom the presence of SARS- CoV-2 was confirmed by RT-qPCR, with disease severity varying from asymptomatic to severe symptomatic, were used.

Multi-SARS-CoV-2 ELISA Assay The multi-SARS-CoV-2 ELISA Assay was performed as described in Example 1. An algorithm was computed for SARS-CoV-2 seropositivity in a biological sample (see Example 4). Briefly, a logistic regression formula was designed and validated to identify SARS-CoV-2 infected subjects. The formula returns a probability (p) calculation ranging from 0 to 1. A threshold can be defined to separate seropositive from seronegative populations. In the used test settings, if p is > 0.75, then the biological sample is considered to contain specific antibodies to SARS- CoV-2. ELISA

Three selected ELISAs were performed according to manufacturer's protocol: (1) Wantai SARS-CoV-2 total Ig ELISA from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., China. The ELISA is coated with RBD antigen. (2) Euroimmun Anti-SARS-CoV-2 IgG ELISA assays from EUROIMMUN Medizinische Labordiagnostika AG, Liibeck, Germany. The Euroimmun ELISA is coated with SI antigen. (3) Tecan SARS-CoV-2 IgG ELISA. The Tecan ELISA is coated with NC antigen. The cut-off used for positivity of each assay was as indicated by the manufacturer.

Results

Figure 6 and Table 4 show assay results of the multi-SARS-CoV-2 assay compared to results of commercial ELISAs. These data show that the multi-SARS-CoV-2 assay can detect an immune response against SARS-CoV-2 in a subject with higher sensitivity compared to the commercial ELISAs tested.

Table 4: Comparison of assay results of commercial ELISAs and the multi-SARS-CoV-2 assay

Example 3: Comparison of the multiplex assay according to the invention with commercial ELISA assays for determining SARS-CoV-2 seropositivity using serum samples

In the present example, the multi-SARS-CoV-2 ELISA assay as described in Example 1 was compared to commercial ELISAs to detect an immune response to a SARS-CoV-2 infection in a subject as described in Example 2, but using a different study population and different ELISA's.

Blood samples A total of 238 serum samples from 238 individuals in France, in whom the presence of SARS- CoV-2 was confirmed by RT-qPCR, with disease severity varying from asymptomatic to symptomatic, were used.

ELISA

Two selected ELISAs were performed according to manufacturer's protocol: (1) LIAISON^® SARS-CoV-2 IgG ELISA from DiaSorin. The ELISA is coated with S1/S2 antigen. (2) Architect

SARS-CoV-2 IgG from Abbott. The Abbott ELISA is coated with NC antigen. The cut-off used for positivity of each assay was as indicated by the manufacturer. Results

Assay results of the multi-SARS-CoV-2 assay were compared with those of commercial ELISAs (Fig. 7). Figure 7 shows that the multi-SARS-CoV-2 assay can detect an immune response against SARS-CoV-2 in a subject with higher sensitivity compared to the commercial ELISAs tested.

Example 4: Performance of the multiplex assay according to the invention to determine SARS-CoV-2 seropositivity using serum samples

An algorithm was designed to determine SARS-CoV-2 seropositivity in a biological sample analysed with the multi-SARS-CoV-2 ELISA assay as described in Example 1. To validate the designed algorithm, results obtained with the multi-SARS-CoV-2 ELISA assay were compared with combined RT-qPCR and ELISA data (RBD- and Sl-based).

Study subjects

A group of subjects, participating in Belgian serosurveillance studies, were tested by RT-qPCR at the onset of the COVID-19 epidemic. Since the present validation was performed 3 months after RT-qPCR typing for SARS-CoV-2, antibody levels could have been declined. Therefore, only RT-qPCR positive participants that had antibodies to SARS-CoV-2 according to the ELISA testing (RBD- and Sl-based) were considered and RT-qPCR negative participants without antibodies to SARS-CoV-2 according to the ELISA testing. These stringent selection criteria resulted in a study group of SARS-CoV-2 strictly positive (n=108) and strictly negative (n=89) cases, confirmed by both RT-qPCR and ELISA.

Multi-SARS-CoV-2 ELISA Assay

The multi-SARS-CoV-2 ELISA assay was performed as described in Example 1 using serum samples of the study subjects. An algorithm was computed for SARS-CoV-2 seropositivity in a biological sample. Briefly, a logistic regression formula was designed to identify SARS-CoV-2 infected subjects as determined positive by RT-qPCR. An independent population of true positive and negative cases as described above was used to validate the designed algorithm. The logistic regression formula returns a probability (p) calculation ranging from 0 to 1; if p is > 0.75, then the biological sample is considered to contain antibodies to SARS-CoV-2.

ELISA

Wantai SARS-CoV-2 total Ig ELISA from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.,

China, and Euroimmun Anti-SARS-CoV-2 IgG ELISA assays from EURO I MM UN Medizinische Labordiagnostika AG, Liibeck, Germany were performed according to the manufacturer's protocol. The Wantai ELISA is coated with RBD antigen, and the Euroimmun ELISA is coated with SI antigen. The cut-off used for positivity of each assay was as indicated by the manufacturer.

Results Table 5: Performance of the multi-SARS-CoV-2 ELISA assay and commercial ELISAs (Wantai, Euroimmun) to detect antibodies against SARS-CoV-2 in serum samples from adults. Performance of the commercial ELISA's was determined based on data reported in literature (Lassauniere et al. 2020. Evaluation of nine commercial SARS-CoV-2 immunoassays. medRxiv doi: https://doi.org/10.1101/2020.04.09.20056325; GeurtsvanKessel et al. 2020. An evaluation of COVID-19 serological assays informs future diagnostics and exposure assessment. Nature Communications 11:3436; Bastos et al. 2020. Diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis. BMJ 370:m2516; Elslande et al. 2020. Diagnostic performance of seven rapid IgG/lgM antibody tests and the Euroimmun IgA/lgG ELISA in COVID-19 patients. Clinical Microbiology and Infection 26(8):1082-1087).

Example 5: Multiplex serological profiling for SARS-CoV-2 infection using saliva samples

The multi-SARS-CoV-2 ELISA assay described in Example 1 was optimized for use with saliva samples.

Saliva samples

Saliva samples were obtained from the study population described in Example 4. Multi-SARS-CoV-2 ELISA Assay

The multi-SARS-CoV-2 ELISA assay was performed as described in Example 1 with slight modifications. In particular, the microplates were incubated with larger volumes of sample, in particular 100 pi saliva sample compared to 1 mI serum sample. The same algorithm was used for determining SARS-CoV-2 seropositivity in the saliva samples as for serum samples (see Example 4), the threshold for saliva samples is defined as probability > 0.15 to indicate saliva samples considered to contain antibodies to SARS-CoV-2. ELISA

Euroimmun Anti-SARS-CoV-2 IgG ELISA assay from EURO I MM UN Medizinische Labordiagnostika AG, Liibeck, Germany was performed according to manufacturer's protocol using 100 pi saliva sample. The Euroimmun ELISA is coated with SI antigen. A cut-off value of OD ratio > 0.60 was used for seropositivity of saliva samples.

Results

The Euroimmun ELISA and multi-SARS-CoV-2 assay were converted for antibody detection in saliva as described above. The results are summarized in Table 6.

Table 6: Performance of the multi-SARS-CoV-2 ELISA assay and a commercial ELISA (Euroimmun) to detect antibodies against SARS-CoV-2 in saliva samples.

The multi-SARS-CoV-2 ELISA assay allows to detect antibodies against SARS-CoV-2 in saliva samples at a sensitivity of 91 % and a specificity of 95.50 %.

Example 6: Multiplex serological profiling for SARS-CoV-2 neutralizing antibodies

Performance of the multi-SARS-CoV-2 ELISA Assay as described in Example 1 to assess protective immunity against SARS-CoV-2 was compared with performance of an in vitro virus seroneutralization assay.

Blood samples

A total of 169 serum samples from 169 individuals in Belgium were used. The presence of neutralizing antibodies to SARS-CoV-2 was evaluated with an in vitro virus seroneutralisation assay. Testing these serum samples resulted in a group of 79 serum samples with SARS-CoV- 2 neutralizing antibodies (defined as NT50 >50) and a group of 90 sero-negative samples (defined as NT50 < 50).

In vitro SARS-CoV-2 seroneutralization test

The potential to neutralize SARS-CoV-2 in vitro was evaluated for the serum samples from 169 subjects. Serial dilutions of heat-inactivated serum samples (1/50-1/1600) were incubated for lh (37°C, 7% CO2) with a primary isolate of SARS-CoV-2 (3xTCIDioo_, Wuhan strain). Sample- virus mixtures and controls were added to Vero cells (8 replica/condition). After 5 days of incubation, the cytopathic effect caused by viral growth was scored visually. Results are reported as NT₅o neutralization titre, which is defined as the sample dilution conveying 50% neutralisation in the infected wells. Samples were considered having neutralizing antibodies to SARS-CoV-2 if NT₅o > 50 (reciprocal titre).

Multi-SARS-CoV-2 ELISA Assay The multi-SARS-CoV-2 ELISA Assay was performed as described in Example 1. An interpretation algorithm was computed for neutralizing antibodies to SARS-CoV-2 in the biological sample. Briefly, a logistic regression formula was designed and validated to identify samples defined to contain neutralizing antibodies per virus-culture assay (NT₅o titers > 50). The formula returns a probability (p) calculation ranging from 0 to 1; if p is > 0.5, then the sample is considered to contain neutralizing antibodies.

Results

Table 7: Performance of the multi-SARS-CoV-2 ELISA Assay to assess protective immunity against SARS-CoV-2 in correlation to a virus seroneutralization test.

The multi-SARS-CoV-2 ELISA assay allows to detect neutralizing antibodies against SARS-CoV- 2 in serum samples at a sensitivity of 93.7% and a specificity of 94.4%.

Example 7: Multiplex serological profiling for estimating the titer of SARS-CoV-2 neutralizing antibodies

Performance of the multi-SARS-CoV-2 ELISA Assay as described in Example 1 to estimate titers of SARS-CoV-2 neutralizing antibodies was compared with performance of an in vitro virus seroneutralization assay and with performance of a one-antigen (RBD) model.

Blood samples

A total of 195 serum samples from 195 individuals in Belgium were evaluated in an in vitro virus seroneutralisation assay and NT₅o neutralization titres were measured as described in Example 6. The samples were classified according to the measured NT₅o neutralization titres as negative/low (<50), moderate (50-500) and high (>500) titers of neutralizing antibodies to SARS-CoV-2.

M ul ti-SA RS-CoV-2 ELISA Assay The multi-SARS-CoV-2 ELISA Assay was performed as described in Example 1. An interpretation algorithm was computed to estimate titres of neutralizing antibodies to SARS- CoV-2 in the biological sample. Briefly, linear discriminant analysis was performed to classify the samples as containing negative/low, moderate or high titres of neutralizing antibodies to SARS-CoV-2 defined per virus-culture assay (NT₅o titers of respectively, < 50, 50-500 or >500).

One-antigen model

As a comparative example, linear discriminant analysis was also performed with the RBD antigen of the multi-SARS-CoV-2 ELISA Assay only for classifying the samples as containing negative/low, moderate or high titres of neutralizing antibodies to SARS-CoV-2 defined per virus-culture assay. RBD antigen showed the highest correlation with NT₅o neutralization titres.

Results

Table 8: Performance of the multi-SARS-CoV-2 ELISA Assay to estimate titers of neutralizing antibodies to SARS-CoV-2 in correlation to a virus seroneutralization test.

Table 9: Linear discriminant analysis with RBD antigen only for classifying the samples as containing negative/low, moderate or high titres of neutralizing antibodies in correlation to a virus seroneutralization test.

Table 8 shows that the multi-SARS-CoV-2 ELISA assay allows to classify samples as containing negative/low (<50), moderate (50-500) or high (>500) titers of neutralizing antibodies to SARS- CoV-2 with an accuracy of 76.9%, whereas RBD antigen only is able to classify the samples with an accuracy of 73.3%. In addition to the increase in classification accuracy, the multiplex assay is also more robust compared to the one-antigen (RBD) model in that the probability to misclassify a sample by two classes is much reduced compared to the one-antigen (RBD) model.

Claims

1. A method for the diagnosis and/or prognosis of a SARS-CoV-2 infection in a subject comprising:

2. A method for categorizing a patient diagnosed with a SARS-CoV-2 infection as asymptomatic, mild symptomatic, severe symptomatic requiring hospitalization or severe symptomatic requiring intensive care treatment, comprising:

(a)at least one membrane antigen selected from SARS-CoV-2 membrane (M) protein or an antigenic fragment thereof,

3. A method for monitoring the clinical progression of a SARS-CoV-2 infection in a patient comprising:

(ii) detecting the antigen/antibody complexes formed in (i) to determine a serological signature; wherein said combination of antigens comprises:

4. A method for predicting the effectiveness of a SARS CoV-2 vaccination or a therapy in a subject, said method comprising:

(iii) comparing the serological profile with a reference serological signature indicative for effectiveness of said vaccination or said therapy; wherein said combination of antigens comprises:

5. A method for identifying a subject that has neutralizing antibodies to SARS-CoV-2, said method comprising:

(iii) comparing the serological signature with a reference serological signature indicative for the presence of neutralizing antibodies to SARS-CoV-2, wherein said combination of antigens comprises:

(a) at least one membrane antigen selected from SARS-CoV-2 membrane (M) protein or an antigenic fragment thereof, (b) at least one nucleocapsid antigen selected from SARS-CoV-2 nucleocapsid (NC) protein or an antigenic fragment thereof,

6. A method for determining a SARS-CoV-2 neutralizing antibody titer in a subject, said method comprising:

7. The method according to any one of claims 1 to 6, wherein detecting the antigen/antibody complexes comprises contacting the complexes with a detector molecule capable of binding the formed antigen/antibody complexes, the detector molecule comprising at least one detectably labelled moiety.

8. The method according to any one of claims 1 to 7, wherein detecting the antigen/antibody complexes comprises determining the presence or absence of the antigen/antibody complexes.

9. The method according to any one of claims 1 to 8, wherein the detection of said antigen/antibody complexes comprises determining the amount of the antigen/antibody complexes.

10. The method according to any one of claims 1 to 9, wherein the at least one membrane antigen comprises a peptide comprising the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5).

11. The method according to any one of claims 1 to 10, wherein the at least one nucleocapsid antigen comprises a peptide comprising the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6).

12. The method according to any one of claims 1 to 11, wherein said combination of antigens comprises:

(a) a peptide comprising the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5),

(b) SARS-CoV-2 NC protein and a peptide comprising the amino acid sequence NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6),

(c) SARS-CoV-2 spike (S) protein, SARS-CoV-2 spike receptor-binding domain (RBD) protein, SARS-CoV-2 SI protein and SARS-CoV-2 S2 protein, and

(d) optionally a T. cruzi peptide antigen consisting of the amino acid sequence AAAPAKAAAAPAKTAAAPV (SEQ ID NO:7).

13. The method according to any one of claims 1 to 12, wherein the biological sample is a blood sample, a serum sample, a plasma sample or a saliva sample.

14. The method according to any one of claims 1 to 13, wherein said solid support is a microplate, a membrane, or a microarray, preferably a microplate.

15. A support for detecting antibodies directed to SARS-CoV-2 in a sample, said support comprising a combination of antigens derived from SARS-CoV-2 and optionally a T. cruzi antigen, wherein said antigens are immobilized on said support spatially separated from one another, and wherein said combination of antigens comprises:

(a) at least one membrane antigen selected from SARS-CoV-2 membrane (M) protein or an antigenic fragment thereof, (b) at least one nucleocapsid antigen selected from SARS-CoV-2 nucleocapsid (NC) protein or an antigenic fragment thereof, and

16. The support according claim 15, wherein said solid support is a microplate, a membrane, or a microarray, preferably a microplate.

17. A kit for detecting antibodies directed to SARS-CoV-2 in a sample, comprising: a support according to any one of claims 14 or 15; a wash solution, or components necessary for producing said wash solution; means for detection of the antibody-antigen complexes.

18. A SARS-CoV-2 peptide antigen consisting of the amino acid sequence MADSNGTITVEDLKKLLEQWNLLI (SEQ ID NO:5) or NNNAATVLQLPQGTTLPKGF (SEQ ID NO:6).