CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional patent application 63/088,242 filed October 6, 2020, the entire disclosure of which is herein incorporated by reference.

TECHNICAL FIELD

[0002] This invention relates to the field of epidemiological and population studies of resistance and sensitivity to COVID-19, including a serological testing system and methods which detect SARS-CoV-2 specific antibodies.

BACKGROUND

[0003] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes a respiratory illness known as coronavirus disease 2019 (COVID-19) [1 ],

[0004] SARS-CoV-2 is an enveloped virus; its genome is a single-stranded positive-sense RNA [2], The incubation period of the COVID-19 infection usually ranges from 1 to 14 days. The virus is mainly detected in respiratory secretions, and the general transmission of infection is considered airborne. It has been shown that the virus attaches to pulmonary cells using their ACE-2 receptors, followed by endocytosis [3], An immune response is expected to build starting one week after the infection [4],

[0005] Serological analysis is based on detection of antibodies specific to a particular infection agent in patient’s serum or other bodily fluids. Serological surveys are used in epidemiological studies to determine the prevalence and spread rate of a disease within a population.

[0006] COVID-19 specific IgG antibodies in blood serum of a COVID-19 patient may be detected as early as in one week from the onset of the disease [5] and almost all samples from COVID-19 patients contain COVID-19 specific IgG antibodies after 20-22 days [6], Asymptomatic individuals who test positive for COVID-19 can also develop COVID-19 specific antibodies, but typically in lower concentrations than patients with COVID-19 symptoms [7],

[0007] There is a need in the field for tests that would detect COVID-19 specific antibodies in a biological sample in part because these tests can be used to address which individuals in a population have developed an immune response to COVID-19. [0008] One of many challenges is to develop a test with high sensitivity and specificity [8], A COVID-19 serological testing system must effectively detect SARS- CoV-2 specific antibodies in samples and distinguish them from antibodies specific to other infections. Unfortunately, many COVID-19 serological tests may produce false positive results. For example, The UC Berkley COVID-19 Testing Project demonstrated that out of 14 serological tests analyzed, only three delivered consistent results, and some tests showed specificity of less than 85% [9],

[0009] Thus, there remains a need in the field for a COVID-19 serological testing system which detects SARS-CoV-2 specific antibodies in a biological sample with high specificity and minimizes the level of false positives.

SUMMARY

[0010] This disclosure helps with addressing at least some of these needs. In one embodiment, the disclosure provides a dual-antigen COVID-19 serological testing system for detecting SARS-CoV-2 specific antibodies. In another embodiment, this disclosure provides a dual-antigen test for identifying individuals with an adaptive immune response to SARS-CoV-2 virus.

[0011] In one aspect, this disclosure provides a method for identifying an individual with an adaptive response to SARS-CoV-2 virus, the method comprising: a) contacting a blood serum or blood plasma specimen of the individual with each of two antigens immobilized on a solid support, a first antigen and a second antigen, wherein the first antigen comprises a first SARS-CoV-2 protein or an immunogenic fragment therefore, and the second antigen comprises a second SARS-CoV-2 protein or an immunogenic fragment therefore; b) capturing antibodies specific to the each of the two antigens from a blood serum or blood plasma specimen into complexes with each of the two antigens; c) detecting first complexes comprising the first antigen and captured antibodies and detecting second complexes comprising the second antigen and captured antibodies; and d) reporting the individual as having an adaptive response to SARS-CoV- 2 virus after both the first and the second complexes have been detected.

[0012] In some embodiments of the method, each of the antigens is immobilized in a well of a microplate.

[0013] Some embodiments of the method include those, wherein the detection comprises: e) contacting the first complexes and the second complexes with a tracer antibody conjugated to an enzyme; f) adding a chromogenic substrate for the enzyme and conducting a colorimetric reaction; and g) measuring an optical density of the enzymatic reaction mixture.

[0014] Some embodiments of the method include those, wherein the method also includes contacting in parallel to step a) a first control antibody at a first cut-off concentration with at least one sample of the first antigen and contacting a second control antibody at a second cut-ff concentration with the at least one sample of the second antigen, capturing complexes and detecting the captured complexes, and wherein the optical density of the cut-off reactions as the threshold optical density.

[0015] In some embodiments of the method, the tracer antibody may be coupled to a horseradish peroxidase (HRP) enzyme and the chromogenic substrate contains o-phenylenediamine dihydrochloride (ORD).

[0016] At least in some embodiments, the optical density may be detected at 490 nm.

[0017] In some preferred embodiments of the method, the first antigen may comprise the receptor binding domain (RBD) of the S1 subunit and/or any immunogenic fragment thereof; and/or the second antigen may comprise the nucleocapsid protein N or an immunogenic fragment thereof. [0018] In yet another another aspect, the present disclosure provides a serological testing system for detection of SARS-CoV-2 antibodies in blood plasma or blood serum, the system comprising at least two antigens immobilized on a solid support, a first antigen and a second antigen, wherein the first antigen comprises the nucleocapsid protein N or an immunogenic fragment thereof and/or the second antigen comprises the surface glycoprotein S or an immunogenic fragment thereof. In certain embodiments of the system, each of the antigens may be immobilized in a well of a microplate. In some preferred embodiments of the system, the first antigen may comprise the receptor binding domain (RBD) of the S1 subunit and/or any immunogenic fragment thereof. In some embodiments of the system, the first antigen may contain a peptide with the SEQ ID NO. 1 and/or any immunogenic fragment therefore and/or the second antigen may contain a peptide with the SEQ ID NO. 2 and/or any immunogenic fragment thereof.

[0019] Certain preferred embodiments of the serological testing system include those, wherein the antigens are immobilized in a container comprising wells and wherein the system comprises 3 sets of wells, wherein a first set of wells in the system contains the first antigen immobilized in the first set of wells, a second set of wells contains the second antigen immobilized in the second set of wells; and wherein the system also contains at least one negative control well in which no antigen is immobilized.

[0020] In yet another embodiment, the present disclosure includes a kit comprising the serological testing system according to this disclosure and and one or more of the following: an antibody of known concentration and with specific affinity to at least one of the two antigens in the system, an instruction manual, a tracer antibody, an enzymatic substrate, one or more of dilution buffers, one or more of a enzymatic reaction stopping solutions, a computer software for analyzing optical density reading, and/or a template which maps positions of wells with the first antigen in the container relative to positions of wells with the second antigen in the container.

[0021] In yet another aspect, the present disclosure relates to a method for identifying an individual with the RBD-positive and N-negative serotype, wherein the serotype is indicative of the individual being vaccinated with the surface glycoprotein- based vaccine and/or the individual having a natural resistance to SARS-CoV-2 virus, the method comprising: a) contacting a blood serum or blood plasma specimen of the individual with each of two antigens immobilized on a solid support, a first antigen and a second antigen, wherein the first antigen comprises a first SARS-CoV-2 protein or an immunogenic fragment therefore, and the second antigen comprises a second SARS-CoV-2 protein or an immunogenic fragment therefore; wherein the first antigen comprises the receptor binding domain (RBD) of the S1 subunit and/or any immunogenic fragment thereof; and wherein the second antigen comprises the nucleocapsid protein N or an immunogenic fragment thereof; b) capturing antibodies specific to the each of the two antigens from a blood serum or blood plasma specimen into complexes with each of the two antigens; c) detecting first complexes comprising the first antigen and captured antibodies and detecting second complexes comprising the second antigen and captured antibodies; and d) reporting the individual as having the RBD positive and the N negative serotype after the presence of the first complexes has been detected and the absence of the second complexes has been detected.

[0022] The embodiments of this method include those, wherein each of the antigens is immobilized in a well of a microplate. In some embodiments of this method, the detection comprises: e) contacting the first complexes and the second complexes with a tracer antibody conjugated to an enzyme; f) adding a chromogenic substrate for the enzyme and conducting a colorimetric reaction; and g) measuring an optical density of the enzymatic reaction mixture.

[0023] The embodiments of this method also include those which comprise contacting in parallel to step a) a first control antibody at a first cut-off concentration with at least one sample of the first antigen and contacting a second control antibody at a second cut-ff concentration with the at least one sample of the second antigen, capturing complexes and detecting the captured complexes, and wherein the optical density of the cut-off reactions as the threshold optical density.

[0024] Some preferred embodiments include those, wherein the tracer antibody is coupled to a horseradish peroxidase (HRP) enzyme and the chromogenic substrate contains o-phenylenediamine dihydrochloride (OPD). Some preferred embodiments include those, wherein the optical density is detected at 490 nm. In some preferred embodiments, the first antigen may comprise the receptor binding domain (RBD) of the S1 subunit and/or any immunogenic fragment thereof; and/or the second antigen may comprise the nucleocapsid protein N or an immunogenic fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1A and Fig. 1 B report serum dilution calibration results using RBD antigen (Fig. 1A) and N antigen (Fig. 1 B) for the antibody detection. Red line corresponds to positive antibody control titration results, other lines correspond to COVID-19 positive samples.

[0026] Fig. 2 reports comparison of milk and casein as blocking agents for ELISA with RBD antigen used for antibody detection on a set of 10 COVID-19 negative samples. Blue bars correspond to results with the milk blocking agent and red bars to the casein blocking agent.

[0027] Fig. 3 reports comparison between S1 (blue bars) and RBD (red bars) antigen results. From left to right: eight results are for patients with uncertain status; three PCR+ and five ELISA+ are COVID-19 confirmed cases; five are negative COVID-19 cases; anti-RBD antibodies used as positive control; no serum (noise level). [0028] Fig. 4 reports RBD antigen antibody detection results. First 6 samples are positive COVID-19 samples confirmed by PCR (PCR + 1 -6), next 30 samples are negative. The threshold for identifying false positive results is shown in the blue horizontal line.

[0029] Fig. 5 reports comparison of N-truncated (blue) and N (red) antigen antibody detection results. First 6 samples are positive COVID-19 samples confirmed by PCR (PCR + 1 -6), next 30 samples are negative. Thresholds for identifying false positive results are shown in horizontal lines, blue for N-truncated and red for N. [0030] Fig. 6 reports N-RBD fusion antigen antibody detection results. First 6 samples are positive COVID-19 samples confirmed by PCR (PCR + 1-6), next 30 samples are negative. The threshold for identifying false positive results is shown by the blue horizontal line.

[0031] Fig. 7 reports the SVM linear formulation for separation of positive (red) and negative (blue) samples. Two parameters defining the 24 samples are RBD and nucleocapsid antigen OD values (x and y axes, respectively).

[0032] Fig. 8 reports the SVM linear formulation for separation of positive (red) and negative (blue) samples. Two parameters defining the 120 samples are OD values normalized by positive control for N antigen and RBD antigen (x and y axes, respectively).

[0033] Fig. 9A and Fig. 9B are calibration curves (exemplary) for anti-RBD (Fig. 9A) and anti-N antibodies (Fig. 9B).

[0034] Fig. 10A reports results from dual-antigen ELISA using a threshold cut-off value for distinguishing and eliminating false positive results.

[0035] Fig. 10B reports examples of RBD-positive, N-negative serotype occurrence within one household and in general population tested for COVID-19 antibodies. In Fig. 10B, Negative control is a no serum sample, Positive controls are samples with monoclonal antibodies specific to RBD or N; samples 1301 , 1302, 1303, 1304 are serum samples collected from the members of one household; and samples 200914095, 200826001 are samples from diagnostic test runs.

DETAILED DESCRIPTION

[0036] SARS-CoV-2 is an enveloped single-stranded RNA virus (13). An RNA- based metagenomic next-generation sequencing approach has been applied to characterize its entire genome, which is 29,881 bp in length (GenBank no. MN908947), encoding 9860 amino acids (14). SARS-CoV-2 encodes 4 structural proteins: the surface glycoprotein S which can be also referred to as spike, the envelope protein E, the membrane protein M and the nucleocapsid protein N. (15, 16).

[0037] In one aspect, this disclosure provides a method for identifying an individual with an adaptive response to SARS-CoV-2 virus. The adaptive response may include prior viral infection and/or successful vaccination against SARS-CoV-2 virus.

[0038] In some embodiments, the methods comprise detecting in a biological sample antibodies, preferably IgG and/or IgM antibodies, specific to SARS-CoV-2 proteins, by capturing the antibodies with two antigens, a first antigen and a second antigen, wherein the antigens are preferably attached to a solid support, and wherein the first antigen comprises a first SARS-CoV-2 protein or an immunogenic fragment therefore, and the second antigen comprises a second SARS-CoV-2 protein or an immunogenic fragment therefore. In some preferred embodiments, the first antigen comprises the nucleocapsid protein N or an immunogenic fragment thereof and/or the second antigen comprises the surface glycoprotein S or an immunogenic fragment thereof. Preferred immunogenic fragments of the surface glycoprotein S include, but are not limited to, the S1 subunit and/or any immunogenic fragment thereof. In some preferred embodiments, the S1 immunogenic fragment includes the receptor binding domain (RBD) or any immunogenic fragment thereof. Some preferred biological samples include blood serum, blood plasma, saliva, and/or other bodily fluid.

[0039] One of the technical advantages of the present antibody-capturing and detection methods is that they minimize false positive results. It has been unexpectedly discovered that false positive results can be minimized by performing a dual capture with two different viral antigens. In some embodiments in order to eliminate false positives, the detection is performed by eliminating samples which score below the threshold value.

[0040] Preferred antigens for the surface glycoprotein S may comprise a SARS- CoV-2 surface glycoprotein fragment which comprises at least a portion of the S1 subunit, and preferably at least a portion of a receptor-binding domain (RBD) and at least 20 amino acid residues located C-terminally from the RBD domain, for example at least amino acid residues 542 through 562, 542 through 572, 542 through 572, 542 through 582, 542 through 592, or 542 through 640. In some embodiments, the RBD antigen may contain a least 70%, and preferably at least 80%, and more preferably at least 90% amino acid sequence homology to the following peptide with SEQ ID NO: 1.

[0041] Preferred antigens for the nucleocapsid protein N may contain the full- length nucleocapsid protein. Some preferred N protein antigens may contain a C- terminal fragment of the nucleocapsid protein, including amino acid residues 122-419 or any immunogenic fragment thereof.

[0042] In some preferred embodiments, the N antigen may contain a least 70%, and preferably at least 80%, and more preferably at least 90% amino acid sequence homology to the following peptide with SEQ ID NO: 2.

[0043] In some preferred embodiments, at least one of the antigens may be a fusion protein which contains at least one immunogenic fragment for the RBD antigen and at least one immunogenic fragment for the N protein. The two fragments may be linked together with a linker into one polypeptide.

[0044] In some preferred embodiments, the fusion antigen may contain a least

70%, and preferably at least 80%, and more preferably at least 90% amino acid sequence homology to the following peptide with SEQ ID NO: 3.

[0045] Some preferred methods of this disclosure are conducted with two antigens immobilized to a solid support, wherein the antigens include the peptide with SEQ ID No. 1 or any immunogenic fragment therefore, the peptide with SEQ ID No. 2 or any immunogenic fragment therefore, and/or the peptide with SEQ ID No. 3 or any immunogenic fragment therefore.

[0046] In some preferred embodiments, the antibody-capturing and detection methods of this disclosure include an enzyme-linked immunosorbent assay (ELISA). Other methods which detect an antibody/antigen binding may be also used.

[0047] In some preferred embodiments, the present disclosure relates to a serological antibody method which detects the presence of SARS~CoV~2 specific antibodies in a patient’s blood or blood serum. In some preferred embodiments, the serological antibody method detects antibodies specific to two proteins of SARS-CoV- 2, a surface glycoprotein receptor-binding domain (RBD) and a nucleocapsid phosphoprotein (N).

[0048] In one preferred method embodiment of this disclosure, a COVID-19 antibody test is a dual antigen Enzyme-Linked Immunosorbent Assay (ELISA) for qualitative detection of anti-SARS-CoV-2 antibodies in human blood serum or blood plasma, but other biological samples may be also used. Two SARS-Cov-2 proteins are used for capture and detection of the antibodies: a surface glycoprotein receptorbinding domain (RBD) and a nucleocapsid phosphoprotein (N).

[0049] In some embodiments of the methods, the blood serum or blood plasma samples and control antibodies recognizing the SARS-CoV-2 RBD and N proteins are diluted and incubated with the two SARS-Cov-2 antigens immobilized on a solid support. Preferably, the solid support is a microplate well. The anti-SARS-CoV~2 antibodies form complexes with the immobilized antigens. The test may be conducted in parallel with at least one immobilized antigen sample which is incubated with a threshold concentration of a control antibody. This sample may be referred to as a cut-off sample. The threshold concentration of the control antibody may be determined by calibrating each new antibody batch as shown in Fig. 9 and as described in detail below. In some embodiments, the threshold concentration of the control antibody is the concentration at which the sample produces an optical density value higher than the optical density value of a negative control.

[0050] After washing and removing antibodies that did not bind to the antigens, a secondary (tracer) antibody recognizing human IgG or human IgM is then added to detect the complexes with the immobilized antigens. The secondary antibody may be coupled to a label, e.g. a fluorescent label, radioactive label and/or an enzyme. In some preferred embodiments, the secondary (tracer) antibody is coupled to a horseradish peroxidase (HRP) enzyme, enabling a colorimetric detection of the complex.

[0051] Preferred chromogenic substrates may contain o-phenylenediamine dihydrochloride (OPD). However, other chromogenic substrates for a HRP enzyme may be also used.

[0052] The OPD substrate develops yellow color upon incubation with HRP and hydrogen peroxide. The reaction is stopped, preferably by addition of hydrochloric acid, and the optical density is measured. If the OPD substrate was used, then an optical density can be measured at 490 nm with a plate reader. In some embodiments, measurements can be conducted at another wavelength, e.g. 450 nm, or 460 nm, or 470 nm, etc., depending on the substrate used.

[0053] The presence of anti-SARS-CoV-2 antibodies in a specimen may be determined by comparing the optical density in the specimen to an optical density for a positive control sample wherein the antigen is reacted with a predetermined concentration of an antigen-specific antibody used as a positive control. The optical value of the cut-off sample may be used as a threshold. In these embodiments, only samples which produce an optical density higher than the cuff-off optical density are recorded as positive samples containing an antibody specific to the antigen.

[0054] In another aspect, the present disclosure provides a serological testing system for detection of SARS-CoV-2 antibodies in blood plasma or blood serum.

[0055] In preferred embodiments, the system comprises two antigens immobilized to a solid support. In some preferred embodiments, the first antigen comprises the nucleocapsid protein N or an immunogenic fragment thereof and/or the second antigen comprises the surface glycoprotein S or an immunogenic fragment thereof. Preferred immunogenic fragments of the surface glycoprotein S include, but are not limited to, the S1 subunit and/or any immunogenic fragment thereof. In some preferred embodiments, the S1 immunogenic fragment includes the receptor binding domain (RBD) or any immunogenic fragment thereof. Some preferred bioiogicai samples include blood serum, blood plasma, saliva and/or some other bodily fluid. Suitable antigens include peptides with at least a least 70%, and preferably at least 80%, and more preferably at least 90% amino acid sequence homology at least one of the peptides with SEQ ID Nos. 1 , 2 or 3.

[0056] Some preferred systems of this disclosure include two antigens immobilized to a solid support, wherein the antigens include the peptide with SEQ ID No. 1 or any immunogenic fragment therefore, the peptide with SEQ ID No. 2 or any immunogenic fragment therefore, and/or the peptide with SEQ ID No. 3 or any immunogenic fragment therefore.

[0057] In some preferred embodiments of the system, the antigens are immobilized in a container comprising wells. The system may comprise 3 types of wells which in some embodiments can be positioned in one container. Preferably, a container is a microplate, e.g. a 96-well plate. In some preferred embodiments, a first set of wells in the system contains the first antigen immobilized in the wells. A second set of wells contains the second antigen immobilized in the wells. The system also contains at least one well in which no antigen is immobilized as a negative control.

[0058] In yet another embodiment, the present disclosure provides a kit which comprises one or more systems of this disclosure and one or more of the following: an antibody of known concentration and with specific affinity to at least one of the two antigens in the system, an instruction manual, a tracer antibody, an enzymatic substrate, one or more of dilution buffers, one or more of enzymatic-reaction stopping solutions, a computer software for analyzing optical density values, and/or a template which maps relative positioning of the first antigen and the second antigen in the system.

[0059] The systems, kits and methods according to this disclosure find many practical applications, including epidemiological and population studies of resistance and sensitivity to COVID-19. The present serological methods may help with addressing whether an individual has developed an immune response to infection and/or vaccination and distinguish between individuals who have been vaccinated and also had an exposure to actual COVID-10 virus. This may be helpful in selecting volunteers for clinical studies.

[0060] In developing the system and methods of this disclosure, the inventors analyzed the immune response and a testing potential for a spectrum of candidate antigens derived from the spike and nucleocapsid proteins of SARS-CoV-2. The inventors have developed a dual-antigen testing system in the ELISA format and designed an algorithm for respective data processing, achieving 100% specificity and 93.55% sensitivity of testing at least in some embodiments. Combining the nucleocapsid and the receptor-binding domain of the spike protein for analysis eliminate false positive results, as no samples in studies produced false positives simultaneously for both antigens. This high specificity is critical for serological tests, preventing false assumptions that a person might have antibodies against the COVID- 19 virus.

[0061] The inventors have also tested the system on samples collected from different households, and demonstrated differences of the immune response of COVID-19 patients in comparison to their family members; one asymptomatic case showed high antibody concentration, while others showed no presence of antibodies to any of the studied antigens despite close contacts with the infected individuals.

[0062] In yet another embodiment, the present disclosure is directed to a method in which an individual is identified as having high and efficient resistance to SARS- CoV-2 infection, if antibodies to both antigens, the N antigen and the RBD antigen are detected in the present system and detection methods.

[0063] The dual-antigen serological tests of this disclosure detect SARS-CoV-2 receptor-binding domain of surface glycoprotein (RBD) and nucleocapsid protein (N). RBD is located on the surface of the virus particle and is exposed to the host immunoglobulins as soon as the virus enters the human body. N is bound to viral RNA; it becomes exposed to the host immune system only when the virus penetrates the cells, propagates, and induces the cell lysis, thus releasing the excess of N, which was not packed into the virus particles. Therefore, the presence of antibodies specific to RBD indicates any, even brief, exposure to the virus, which triggered generation of antibodies against the viral antigens exposed on the surface of the virus particle. The presence of antibodies to the N protein, along with antibodies against RBD, indicates an established infection, which resulted in cell infection, lysis, and release of viral proteins.

[0064] The inventors conducted research studies relied on samples collected from 12 donors recovered from the confirmed cases of COVID-2 infection, and 49 COVID-19-negative blood serum samples, collected before December 2019. The research studies establish the thresholds separating the positive and negative samples. The validation studies were performed on 31 samples from donors recovered from the confirmed cases of COVID-2 infection, and 89 COVID-19-negative blood serum samples, collected before December 2019. In the validation run, one out of the PCR-confirmed positive samples tested showed an RBD-positive, N-negative pattern. It was assigned NEGATIVE according to the criteria established in the research studies. One of the 31 PCR-confirmed samples was RBD-positive and N- negative and was assigned NEGATIVE.

[0065] Vaccination with surface glycoprotein-derived antigens should result in appearance of antibodies targeting RBD in the bloodstream of vaccinated people. Appearance of antibodies against N is not expected after vaccination. The dualantigen COVID-19 antibody test and the systems of this disclosure may help in selection of subjects for vaccine clinical studies, identifying individuals with preexisting IgG antibodies targeting RBD of SARS-CoV-2 surface glycoprotein; determining an increase in antibodies targeting RBD post-vaccination; and monitoring the persistence of seroconversion specific to RBD. Accordingly, in yet another embodiment, this disclosure provides a method and systems for detection of postvaccination infection cases can by emerging seroconversion with reaction to the N antigen.

[0066] The inventors analyzed an immune response and a diagnostic potential for several antigens designed based on the SARS-CoV-2 structure under different experimental conditions. The inventors used serum samples obtained from a general asymptomatic population of pre-epidemic individuals (negative samples) and from patients which tested positive with a SARS-CoV-2 RT-PCR assay or alternative ELISAs (positive samples).

[0067] Preferred antigens used for in this analysis comprised the full length nucleocapsid protein (N), nucleocapsid protein truncated from the N-terminus (N- truncated), the first domain of the spike protein (S1 ), the receptor-binding domain of the spike protein (RBD) and the N-RBD fusion construct (N-RBD).

[0068] Through a series of experiments, the inventors have obtained optimal conditions for the ELISA experiments, first determining the necessary serum dilution and blocking reagent.

[0069] Optimal Serum Dilution [0070] 10 positive serum samples confirmed by an alternative ELISA test

(ProteoGenex) and 7 positive serum samples collected by us and confirmed by SARS- CoV-2 RT-PCR assay along with a positive antibody control were used to determine the optimal serum dilution. A series of 2x serum dilutions ranging from 10 to 1240 were prepared and analyzed with ELISA and respective OD signals were obtained on a plate reader at 490 nm. Obtained signals were considered positive if they exceeded noise level (average value from wells with no serum added) at least twice. According to this threshold, all positive samples showed positive signals for dilution up to 80x when RBD antigen was used for the antibody detection, and up to 160x when N antigen was used (Fig 1A and Fig. 1 B). For convenience, we have used the dilution of 50x in our further experiments.

[0071] Optimal Blocking Reagent

[0072] The inventors have tested casein (PBS-T containing 1 % casein) and nonfat dry milk (PBS-T containing 1 % non-fat dry milk) for the ELISAs with 10 positive and 10 negative samples (confirmed positive by ProteoGenex ELISA test). With casein, the RBD-antigen's average signal of negative samples was lowered by 30% (p-value 8.8x10- ¹³ ) (Fig. 2).

[0073] The average signal of positive samples was not affected (the average value raised by 3%, which was comparable with coefficient of variation of 1.3 - 7.6% for different samples). On the graph it can be observed that while low-signal samples are usually not greatly affected by the switch of the blocking agent, casein significantly improves the data for negative samples yielding high signals with milk (see samples 1 , 3, 9), thus lowering the possibility of false positive results occurrence.

[0074] For the N-antigen the average signal of negative samples lowered by 14% (p-value 0.0013). The average positive signal was likewise not affected (it lowered by 2%, while the range of the coefficient of variation for different samples was 0.3% - 9.1 %).

[0075] Since the RBD antigen is expected to have more issues with sensitivity, lowering the overall level of negative sample signals and specifically eliminating high signals from the negative signals was an important step in optimization of assay conditions. Casein was used as a blocking reagent for further experiments.

[0076] Antigen Selection

[0077] For the initial estimation of immunogenicity of studied antigens, we calculated the crude rate of false positive results using the following approach. We identified the signal for a negative sample as a false positive if its optical density (OD) value was above a threshold defined by a commonly used approximation method, where it is set as three standard deviations above the average negative control value

[11].

[0078] To determine the 95% confidence interval (95% Cl) we used exact binomial Bayesian credible interval (Jeffrey's interval) method. It is worth noting that this threshold rule was very strict, identifying some signals as false positives even when they were significantly lower than all positive sample signals, but at this step our goal was to apply a hard filter to the candidate antigens and select optimal ones for further analysis.

[0079] To ensure efficient sensitivity we used 6 samples from the patients with confirmed COVID-19 and indicated the respective result as false negative if the OD value for such sample was less than the highest negative control value, excluding false positives identified at the previous step. No false negative results were obtained for studied antigens at this point.

[0080] The inventors first compared testing capabilities of S1 and RBD antigens. RBD is the part of the S1 domain of the HCoV-2 surface protein. The antibody detection results to those two antigens were expected to be similar at least to some degree. To analyze this correlation, we used 8 positive samples, 8 samples from individuals with an uncertain status (members of households of individuals with confirmed COVID-19 diagnosis) and 5 negative samples. Anti-RBD antibodies were used as an additional positive control, the noise level was assessed in the well where no serum was added. (Fig. 3).

[0081] The S1 antigen demonstrated results highly similar with the RBD antigen results (Pearson's correlation coefficient 0.96), with RBD overall giving higher signals for all positive samples. For both RBD and S1 all the positive samples yielded values greater than negative samples, so efficient separation between positive and negative samples is possible in both cases, which indicates advantages in testing capabilities for either of these antigens. RBD is a part of the surface protein that is responsible for binding to the ACE-2 receptor during infection, so it holds more interest as it should be the primary contributor to the neutralizing capabilities of the respective antibodies. Neutralizing antibodies (nAbs) to the causative agent of the disease represent potential prophylactic and therapeutic options and could help guide vaccine design

[12]. [0082] Out of the two we have thus selected RBD antigen for further analysis. The RBD antigen yielded 1 false positive result on a larger dataset comprising 30 negative samples, reaching the specificity of 96.7% (95% Cl 85.5-99.6%) (Fig 4).

[0083] Next, the inventors analyzed results for nucleocapsid (N) protein and its truncated version (N-truncated) on the same set of samples. The N-truncated antigen yielded 3 false positives, demonstrating overall specificity of 90% (95% Cl 75.7- 97.1 %). The full-length N antigen demonstrated 2 false positive results, corresponding to specificity of 93.3% (95% Cl 80.3-98.6%) (Fig. 5). Overall N-truncated antigen negative samples values were significantly higher, by 87% on average, indicating larger noise level, and the N antigen was thus selected for further experiments.

[0084] Additionally, the inventors analyzed the immunogenicity of an N-RBD fusion construct (Fig. 6). Its noise level was comparable to N-truncated antigen, although it yielded only 1 false positive result, similar to the RBD-antigen. Overall, it demonstrated promising regarding testing capabilities. The interpretation of the respective results remains a moot point, since it is not possible to determine, which viral protein captures the antibodies. Thus, the question about their potential neutralizing capabilities could not be easily answered.

[0085] The false positive results yielded by the RBD and N antigens were not shared. Coming to the conclusion that the combination of N and RBD as two separate antigens might provide maximum specificity of the study, and also allow us to assess the neutralizing capabilities of anti-RBD antibodies in future experiments, the inventors have developed a testing system in the format of ELISA which included both of them (dual-antigen system). Selected antigen concentrations for the plate coating were 3.75 pg/ml for RBD and 4 pg/ml for N.

[0086] The next step was to develop a better threshold algorithm for identifying positive and negative results, since the initial false positive filtering method was too strict for further analysis. In all experiments described above, the lowest positive and highest negative values differed (lowest positive being higher than highest negative). From this, the inventors considered possibly developing a separation procedure to sufficiently distinguish positive and negative samples.

[0087] Other steps included validating that the false positive results between N and RBD antigens were not shared on other data sets and further testing the system sensitivity.

[0088] Efficient Positive and Negative Sample Separation [0089] Using support-vector machines algorithm (SVM) to create linear classifiers with a training set including 24 samples, we have confirmed that the positive and negative samples can be successfully separated using two parameters, N antigen OD value and RBD antigen OD value. The dual-antigen system yielded no false positive and no false negative results (Fig 7).

[0090] We used the 5-fold cross-validation method to estimate the accuracy of the separation results by splitting the data and computing the prediction score five consecutive times with varying training and testing sets. The results were predicted correctly every time. The F1 score (a weighted average of the precision and recall, where an F1 score reaches its best value at 1 and worst score at 0) was 1 every time.

[0091] It is worth noting that the RBD antigen generally provides a much wider range of OD results within different positive samples, thus the threshold for separation of positive and negative samples in case of results for anti-RBD antibodies is ought to be set quite low. Low thresholds, in turn, lead to possible specificity lowering, so using two parameters (and, respectively, two thresholds) simultaneously for identification of positive results is justified.

[0092] The inventors next performed the same procedure on a larger set of samples, comprising 31 positive samples (confirmed by PCR) and 89 negative samples (120 samples total). This experiment included three plates, so the OD values were normalized by positive control of the same concentration on all the plates (anti- N and anti-RBD antibodies).

[0093] Two positive samples had OD values for the N antigen lower than some of the negative sample results (at the same time their RBD values were high). One negative sample, on the other hand, had an RBD value higher than one of the positive samples. While the linear classifier separates most of the samples efficiently, it still yields one false negative result (Fig 8).

[0094] The cross-validation method was again used to assess the robustness of this separation, where the prediction score was computed five consecutive times and the results were predicted correctly with F1 scores of 1 ; 1 ; 1 ; 1 ; 0.91 .

[0095] These observations demonstrated that positive and negative samples could be efficiently separated with high accuracy on large datasets, yielding only a very small number of false negative results with the right classifier selected.

[0096] The inventors concluded based on these findings that while analyzing the ELISA results, false positive results could likewise be avoided by applying a two- parameter threshold, where for a sample to be considered positive, both antigen results should be above a certain value determined for each. A respective threshold selection algorithm was then developed.

[0097] Threshold Algorithm

[0098] To be able to separate positive and negative samples during each following ELISA experiment with new specimens, the inventors have developed a procedure which uses a single positive control for each antigen as a threshold. The use of the positive control allows to identify positive samples without relying solely on an integer number of standard deviations from an average OD value from confirmed negative samples, which requires using training sets with actual negative serum samples for every ELISA and is inconvenient regarding plate space and sample storage requirements.

[0099] For the positive control, the inventors used antibodies against the nucleocapsid protein (anti-N) and the receptor-binding domain of the surface protein (anti-RBD). To determine the correct thresholds for the interpretation of the ELISA results, a training set of negative and positive samples and a calibration curve for a set of anti-N and anti-RBD antibody dilutions were used. The concentrations of both positive controls are determined once per each antibody lot and then used for all future ELISAs.

[00100] The procedure for each threshold selection is as follows. First, the inventors determine the bounds for the window which separates positive samples from negative samples (range between values of the highest negative replica and the lowest positive replica). In the rare case where this range is negative, it usually indicates the existence of false positive results; the inventors remove them from the set, defining result as false positive if its OD value is more than 3 standard deviations over the average negative sample value.

[00101] Second, the inventors obtain values from antibody calibration curve (Fig. 9) which fall within this window and select the lowest possible value which is at least 2-fold higher than average noise level (OD value from the well where no samples or antibodies were added). This is the concentration of the antibody positive control which will be used for further comparison with sample results in all experiments involving same antibody lot. If a new lot is obtained, the calibration curve is calculated again for validation or optimization. [00102] During further experiments, the OD value of each tested sample is compared to the average OD value of at least three replicas of the positive control of each anti-N and anti-RBD antibodies. If the sample value is equal or greater than the control, the result for this antigen is considered positive. The result for the sample is considered overall positive if the thresholds for both antigens are positive. Some of these studies are shown in Fig. 10A.

[00103] Validation of The Dual-Antigen System

[00104] The inventors validated the dual-antigen system performance and the threshold algorithm selection on a validation set including 120 samples (31 positive samples confirmed by SARS-CoV-2 RT-PCR assay and 89 negative samples from the pre-pandemic era). N antigen yielded 2 false positive results and RBD antigen yielded 1 false positive result; they did not intersect. The threshold algorithm requiring both of these antigens to demonstrate positive results successfully eliminated false positive results, bringing specificity to a 100% (95% Cl 97.2 - 100.0%).

[00105] Two false negative results were obtained (both due to N antigen low results), yielding overall sensitivity of the system to 93.6% (95% Cl 80.9 - 98.6%) (See Table A).

Table A. Specificity And Sensitivity Results For N And RBD Antigens Separately And Combined In The Validation Test Including 120 Samples

[00106] The robustness of the dual-antigen system was additionally tested with 32 specimens (11 positive and 21 negative) in 5 repeated experiments done by two different operators over the course of three weeks. Compliant with the threshold algorithm, the sample was indicated as positive if both antigens presented results above respective threshold values obtained using the antibody controls. No false positive results were obtained in either experiment.

[00107] Two positive samples had the signal for the N antigen close to the threshold. The first one appeared as false negative in 3 out of the 5 experiments and the second appeared as false negative in 2 out of the 5 experiments. The ratio to threshold was above 90% in all cases. A result which is this close to the threshold can be considered “borderline.” In actual patient testing settings, this is treated as a suggestion for an individual to retake the test in several days, when the immune system might develop a higher level of antibodies.

[00108] We have tested samples identified as positive and negative by Siemens CLIA testing system (Lenco Diagnostic Laboratory Services). From 96 samples claimed as negative one gave a positive result with our testing system, and from 96 samples claimed as positive one gave a negative result. Overall, 98.4% results matched between two of the tests (Cl 95% 96.7 - 99.8%).

[00109] Case Studies For The Household Members Of COVID-19 Survivors

[00110] Samples collected from several households showed a variety of immune responses of family members of COVID-19 patients, who had or had not reported also having symptoms similar to COVID-19.

[00111] One asymptomatic case demonstrated strong evidence of presence of antibodies for studied antigens. A spouse of a patient with severe symptoms of the SASR-Cov-2 infection claimed to have no symptoms of the disease, but their serum tested positive for antibodies with all of the antigens (N, RBD, S1 , N-truncated< N- RBD fusion) used during the course of our research.

[00112] On the other hand, some members from households of patients with confirmed COVID-19 showed no positive results with any of the antigens. A patient with severe infection symptoms demonstrated strong signals for both N and RBD antigens, but the serum of their live-in partner showed no presence of antibodies to any of the tested antigens.

[00113] Another household included 6 members, all of whom reported mild symptoms of sickness. Three cases were confirmed by an RT-PCR assay to have had SARS-CoV-2 infection and have shown presence of antibodies in our study. Others did not show presence of antibodies for any of the tested antigens.

[00114] The inventors analyzed antibody testing potential for several antigens derived from the surface and nucleocapsid proteins of the SARS-CoV-2 virus. Combining the nucleocapsid and receptor-binding domain of the surface protein for simultaneous analysis eliminates false positive results in our experiments, as no samples yielded them simultaneously for both antigens. The inventors consider this is an important achievement, since incorrectly assuming a person might be safe from the infection due to a false positive serological test result can be particularly dangerous. In general, the inventors developed and validated a dual-antigen testing system in an ELISA format and designed a comprehensive algorithm for respective result processing, consequently achieving 100% specificity and 93.6% sensitivity of testing on the final validation set of 120 samples.

[00115] Testing of family members from same households as confirmed COVID- 19 patients demonstrated that, on one hand, asymptomatic individuals can show strong presence of antibodies to nucleocapsid or surface proteins of the virus, while on the other hand, individuals who have claimed to have COVID-19-like symptoms and lived near confirmed patients may not develop antibodies to either of these antigens.

[00116] Referring to Fig. 10B, it reports results from the dual-antigen ELISA test performed in accordance with this disclosure, wherein blood serum samples were tested for antibodies specific to the RBD antigen or the N antigen. Blood serum samples were collected from all members of one households two weeks after the PCR tests were performed. In Household 1 , no one experienced pronounced symptoms of COVID-19. Subject 1303 from that household, who had a positive PCR test, showed positive signal for RBD-specific IgG. N-specific IgGs were significantly below the threshold.

[00117] 200914095 represents one out of 10 serum samples collected and tested in a diagnostic run; other nine samples from that run were below the threshold for both types of antibodies.

[00118] 200826001 represents one out of 42 serum samples collected and tested in a diagnostic run; two more samples showed the same RBD-positive, N-negative pattern; 4 samples from that run showed above-the-threshold IgG signal for both types of antigens; the remaining 35 samples were double negative.

[00119] In summary, the dual-antigen COVID-19 antibody test reveals the emerging population of individuals with elevated levels of IgGs specific to the surface glycoprotein of SARS-CoV-2, but lacking antibodies recognizing the inner nucleocapsid protein. The RBD positive, N negative serotype may be associated with increased natural resistance to COVID-19 and/or a brief exposure to SARS-CoV-2. The RBD-positive, N negative serotype is expected to appear in individuals vaccinated with the surface glycoprotein-based vaccines.

[00120] Accordingly, yet another technical advantage of the present dual-antigen test according to this disclosure, is this dual-antigen test detects the existence of RBD- positive, N-negative serotype in the population. It is believed that no other currently available test for COVID-19 related antibodies can provide this distinction.

Example 1. Materials and Methods

[00121] Specimens

[00122] The total number of collected samples for this study was 401 . From these, 28 serum samples were collected by us throughout the timeline of the study, of which 11 were from COVID-19 patients confirmed by a SARS-CoV-2 RT-PCR assay and others included their household members. 17 negative samples were collected from before 2019 (pre-epidemic) for different projects. Other specimens were purchased from commercial providers; the complete list is available in Table 1.

Table 1. List of Serum Samples

[00123] Commercial Antigens

[00124] Commercial antigens were purchased from GenScript and comprised of the nucleocapsid protein (Z03488), S1 domain of the surface protein (Z03485), RBD, receptor-binding domain of the surface protein (Z03479, Z03483), nucleocapsid and RBD fusion protein (Z03498) and a C-terminal fragment of the nucleocapsid protein, which includes amino acid residues 122-419.

[00125] Antigen Production And Purification

[00126] The first antigen (RBD) obtained in our laboratory was a fragment of SARS-CoV-2 surface glycoprotein that includes amino acid residues 319-591. It was designed to include the surface glycoprotein receptor-binding domain (amino acid residues 319-541 ) and 50 additional amino acid residues that contain a strong B-cell epitope, according to the BepiPred-2.0 algorithm [10], The humanized sequence encoding the 319-591 fragment was fused to a sequence encoding the surface glycoprotein signal peptide (amino acid residues 1 -14) and to a sequence encoding a C-terminal hexahistidine affinity tag. The resulting construct was expressed from a CMV promoter in transiently transfected HEK293 cells and purified from the conditioned media on Ni-NTA Sepharose and MEP Hypercel resin. The resulting protein was dialyzed to phosphate buffer saline (pH 7.4) containing 20% glycerol. For the ELISA, the microtiter plate wells were coated with 0.1 pg of RBD per well.

[00127] The second antigen (N) was a full-length nucleocapsid protein of SARS- CoV-2. It was cloned into a pET-based vector carrying a C-terminal hexahistidine tag and expressed in a soluble form in a BL21 (DE3) E. co// strain. Purification from the soluble fraction of the bacterial cell lysate was done on Ni-NTA Sepharose. The resulting protein was dialyzed to 20 mM HEPES-Na, pH7.9, 20% glycerol, 100 mM NaCI, 1 mM DTT and 1 mM EDTA. For the ELISA, the microtiter plate wells were coated with 0.05 pg of N per well.

[00128] A comparison of antibody detection capabilities between commercial antigens obtained from GenScript and the same antigens custom-produced in our laboratory was performed; the results were similar overall and demonstrated no difference regarding false positive and false negative sample rates by any of the algorithms used in this study.

[00129] Control Antibodies Against RBD And N Antigens

[00130] SARS-CoV-2 Spike S1 Antibody (HC2001 ), Human Chimeric

(Genscript A02038) monoclonal anti-RBD antibody was used as the positive control for experiments with human anti-RBD antibodies, and SARS-CoV-2 Nucleocapsid Antibody (HC2003), Human Chimeric (Genscript, A02039) monoclonal anti-N antibody was used as the positive control for experiments with human anti-N antibodies.

[00131] Secondary Antibodies

[00132] Monoclonal mouse Anti-Human IgG Fc Antibody (clone

50B4A9, GenScript #01854) was used as the secondary antibody. This antibody is purified on Protein A Sepharose.

Example 2. ELISA Plate Preparation

[00133] In order to prepare a system according to this disclosure, the SARS-CoV- 2 antigens were diluted in 1X PBS and used to coat 96-well ELISA plates (4 HBX, ThermoFisher, 3855). The plates were coated with 100 pl of diluted RBD antigen (0.1 pg) or 50 pl of diluted nucleocapsid (N) antigen (0.05 pg) per well and incubated for 4 hours at 20°C (RT) or at 4°C overnight. Coated ELISA plates were washed 3 times with PBS-T (TWEEN 20 at 0.1 %, Sigma, P1379) and then blocked with PBS-T + 3% milk powder (weight/volume) at 200 pl blocking solution per well at 20°C (RT) for 2 hours. The blocking solution was removed, and the plates were then dried at 20°C (RT) for 2 hours or overnight. The dried plates were then sealed with MicroAmp™ Clear Adhesive Film (ThermoFisher, 4306311 ) and stored at 4°C in a bag with desiccant (Sigma, 1038040001 ) in Silver Metallized Zipper Pouch Bags (ClearBags, ZBGM4S).

Example 3. ELISA Protocol

[00134] This example discloses a method for detecting SARS-Cov-2 specific antibodies in blood serum. For ELISA, wells of the plate prepared according to Example 2, were filled with 100 pl of PBS-T containing 1 % casein (1X Casein in PBS ready to use solution, \ThermoFisher #37528 with 0.1 % TWEEN-20 added). The samples were pre-diluted 1 :5 with 1XPBS in a PCR plate, then 10 pl was added to the wells.

[00135] The plate was incubated for 1 hour at 20°C (RT).

[00136] The plate was then washed 3 times with PBS-T.

[00137] Anti-human IgG HRP conjugated secondary antibody (Mouse Anti-Human IgG Fc Antibody [HRP], mAb, Genscript, A01854) was diluted 1 :3000 in PBS-T containing 1 % casein and 100 pl of the diluted antibody was added to the wells.

[00138] The plate was incubated for 1 hour at 20°C (RT) and then washed 3 times with PBS-T.

[00139] To perform the colorimetric step, SigmaFast OPD (Sigma, P9187) was used with a set of 1 gold and 1 silver tablet dissolved in 20 mL of water following the manufacturer instructions. 100 pl of SigmaFast OPD substrate solution was added to the wells of the plate and after 10-minute incubation the reaction was stopped by addition of 50 pl of 3M HCI.

[00140] The plate was processed on a plate reader at 490 nm.

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