An epitope originating from sars-cov-2 n protein, an antigen containing the epitope, uses thereof and a method for detecting disease caused by coronavirus

Abstract

Epitopes originating from SARS-CoV-2 N protein, vaccine antigens specific for SARS-CoV-2 containing the epitopes useful in the treatment or prevention of disease caused by coronavirus as well as a method for detecting disease caused by coronavirus, in particular COVID-19, especially with an acute course, are disclosed.

Description

[0001] An epitope originating from SARS-CoV-2 N protein, an antigen containing the epitope, uses thereof and a method for detecting disease caused by coronavirus

[0002] The object of the invention is an epitope originating from SARS-CoV-2 N protein, a vaccine antigen specific for SARS-CoV-2 containing the epitope and uses thereof in the treatment or prevention of disease caused by coronavirus as well as a method for detecting disease caused by coronavirus, in particular COVID-19, especially with an acute course.

[0003] Background art

[0004] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 0.5 billion people and caused more than 6 million deaths worldwide.

[0005] SARS-CoV-2 belongs to the genus Betacoronavirus in the family Coronaviridae [1], Two seasonal coronaviruses in the same genus are known: HCoV-HKUl and HCoV-OC43, and two further coronaviruses in the genus Alphacoronavirus: HCoV-229E and HCoV-NL63 [2], Infections due to seasonal coronaviruses are typically common but mild and short-term acquired immunity is reported. Four principal structural proteins are known that form SARS-CoV-2 virions: nucleocapsid (N), spike (S), membrane (M) and envelope (E). N protein is used for viral genome packaging, has a conservative amino acid sequence in the family Coronaviridae, is highly immunogenic and is expressed in high quantities during infection. Patients produce excessive quantities of anti-N antibodies. N protein has been identified as an effective diagnostic tool for detecting SARS-CoV-2 infection and has been suggested to be an interesting vaccine antigen [4].

[0006] Plenty of evidence, albeit frequently contradictory, is available on immunity to SARS-CoV-2 before the pandemic [5], Approx. 20% of individuals have pre-pandemic anti-SARS-CoV-2 antibodies which show cross-reactivity, mainly against the nucleocapsid. High cross-reactivity of anti-SARS-CoV-2 antibodies with other coronaviruses has been found in pre-pandemic samples in Sub-Saharan Africa [6] in which the nucleocapsid was the dominant antigen showing cross-reactivity. The incidence of COVID-19 has been much lower in Sub-Saharan Africa than in the U.S., for example, and has been correlated with higher serological cross-reactivity levels of plasma samples from Tanzania and Zambia compared to those from the U.S. [6], Sagar et al. have shown that patients with a recent and documented history of HCoV common cold had increased survival rates during COVID-19 [7], However, no SARS-CoV-2 nucleocapsid proteins showing cross-reactivity have been found in pre-pandemic Vietnamese plasma samples [8], It is debatable whether these cross-reactive antibodies provide any protection against SARS-CoV-2, because other research has shown that earlier infection with a seasonal coronavirus does not achieve protective immunity against SARS-CoV-2 [9,10]. However, it is important to account for these differences considering the potential immunopathologic action of SARS-CoV-2. A different question is whether past seasonal infections are the only source of cross-reactive SARS-CoV-2 antibodies.

[0007] The objective of the invention is to provide methods for identifying SARS-CoV-2 infections, in particular those with a severe course, tools useful in these methods and means that could be used for the effective treatment or prevention of COVID-19, in particular with an acute course.

[0008] The hereinabove objective has surprisingly been achieved in the present invention. Summary of the invention

[0009] The object of the invention is an epitope originating from SARS-CoV-2 N protein having an amino acid sequence selected from: MSDNGPQNQRNAPRITFGGP (SEQ ID No. 1) and KADETQALPQQRQKKQQTVTL (SEQ. ID No. 2).

[0010] Another object of the invention is a vaccine antigen specific for SARS-CoV-2 containing the epitope originating from SARS-CoV-2 N protein having an amino acid sequence selected from: MSDNGPQNQRNAPRITFGGP (SEQ ID No. 1) and KADETQALPQQRQKKQQTVTL (SEQ ID No. 2).

[0011] Another object of the invention is the aforementioned epitope or the aforementioned antigen for use in the treatment or prevention of disease caused by coronavirus.

[0012] Preferably, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0013] Preferably, the disease is COVID-19, in particular with an acute course.

[0014] Preferably, the aforementioned epitope or antigen is used for vaccination.

[0015] Preferably, the aforementioned epitope or antigen is used for preparing a therapeutic formulation, in particular serum.

[0016] Another object of the invention is a method for identifying coronavirus infection, characterized in that antibodies specific for the aforementioned epitope or the aforementioned antigen are detected in a biological sample collected from a patient, wherein the presence of such antibodies indicates that the patient has coronavirus infection.

[0017] Preferably, the biological sample is blood or saliva.

[0018] Preferably, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0019] Preferably, the result of infection is COVID-19, in particular with an acute course.

[0020] Preferably, current or previous infection is identified.

[0021] Detailed description of the invention

[0022] Humoral and cellular immune response of patients affected by COVID-19 was analyzed. Cross- reactivity of nucleocapsid protein (N protein), a structural protein of SARS-CoV-2, was evaluated. Therefore, sera (pre-pandemic, severe COVID-19 and from convalescents) were used for identifying and mapping nucleocapsid protein epitopes and determining potential cross-reactivity.

[0023] For example, a common KKSAAEASKKPRQKRTATKA epitope was identified, recognized by antibodies from all three groups of sera. Certain motifs in this sequence are found in various coronaviruses or plant or human proteins, which indicates that there may be more causes of cross-reactivity than previous infection with a seasonal coronavirus. Practical usefulness of the epitope is very limited due to low selectivity.

[0024] Surprisingly, two other epitopes, i.e. MSDNGPQNQRNAPRITFGGP (SEQ. ID No. 1) and KADETQALPQQRQKKQQTVTL (SEQ ID No. 2), were identified as specific only for sera from patients in the acute infection phase and convalescents, and they are particularly useful for developing a SARS- CoV-2 vaccine. Based on their selectivity, they are useful also for developing respective diagnostic tools and vaccine antigens. Description of the figures

[0025] Figure 1 presents recognition of the SARS-CoV-2 nucleocapsid by non-immunized sera. (A) shows the result of Western blotting performed in SARS-CoV-2 lysate and sera from pre-pandemic patients. (B) shows the result of ELISA performed in a recombinant SARS-CoV-2 nucleocapsid to measure the level of specific IgG antibodies. (C) shows the result of ELISA performed in a recombinant SARS-CoV-2 nucleocapsid to measure the level of specific IgA antibodies.

[0026] Figure 2 shows immunoreactivity profiles of patients in the acute phase of the disease and convalescents with COVID-19. The sera were collected from each patient on admission to hospital and three weeks after resolution of last infection symptoms. All blots were obtained in identical conditions. The identity of the N protein band was previously confirmed using mass spectrometry of fragments digested with trypsin and their analysis in protein databases.

[0027] Figure 3 shows epitope mapping of SARS-CoV-2 nucleocapsid protein - the pattern of sequences recognized by IgG antibodies. An ELISA test on pins was performed using merged acute phase sera, sera from convalescents with COVID-19 and pre-pandemic sera diluted to 1:1,000. The AP-conjugated human anti-IgG antibody was used in 1:7,500 dilution. The test was performed in five replicates and data show their means with ± SD. The indicated thresholds for each study group (dotted line in the graph) were calculated by computing the mean of all results in the group.

[0028] Figure 4 shows epitope mapping of SARS-CoV-2 nucleocapsid protein - the pattern of sequences recognized by IgA antibodies. An ELISA test on pins was performed using merged acute phase sera, sera from convalescents with COVID-19 and pre-pandemic sera diluted to 1:1,000. The AP-conjugated human anti-lgA antibody was used in 1:30,000 dilution. The test was performed in five replicates and data show their means with ± SD. The indicated thresholds for each study group (dotted line in the graph) were calculated by computing the mean of all results in the group.

[0029] To allow better understanding of the gist of the invention defined above, it has been illustrated by the following examples.

[0030] Example 1. Identification of the epitopes of the invention

[0031] Acquiring human serum samples from different patient groups

[0032] The blood for testing was collected from patients with confirmed SARS-CoV-2 infection admitted to the Infectious Disease Admissions Department of the J. Gromkowski Memorial Hospital in Wroclaw between November 2020 and April 2021. The sera were collected when vaccines were not yet available. The sera were collected on admission to hospital (acute Covid-19, n = 30) and three weeks after resolution of last infection symptoms (Covid-19 convalescents, n = 27). Serum from healthy volunteers (pre-pandemic, n = 16) had been collected before the pandemic and had already been used in our research [11,12], Merged sera were used for experiments, unless specified otherwise.

[0033] Viral protein immunoreactivity testing (immunoblotting)

[0034] The immunoreactivity of viral proteins (viral lysate) separated on polyacrylamide gel was tested using Western blotting. 4-20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, USA) were used for SDS- PAGE electrophoresis and the procedure was identical as before [11,12], 7 pg SARS-CoV-2 lysate samples (cat. NAT41605-500, The Native Antigen Company, United Kingdom) were applied on each lane. 2 pL Precision Plus Protein Dual Color Standards (Bio-Rad, USA) each were used as molecular weight markers. The proteins were transferred to nitrocellulose with 0.45 pm pore size, subsequently blocked with Pierce™ Clear Milk Blocking Buffer (Thermo Fisher Scientific, USA). A sample of merged sera was diluted 1 : 100 times (pre-pandemic sera) or 1 : 350 times (acute and convalescent sera) in Tris buffered normal saline with 0.1% Tween 20 (TBS-T). Anti-human IgA antibodies (SAB3701233, Merck, Germany), anti-human IgG antibodies (cat. SAB3701340, Merck) were diluted 1:2,000 or 1:7,500, respectively, in Pierce™ Clear Milk Blocking in TBS-T. Color reaction was triggered with a 1:1 mixture of nitro blue tetrazolium (NBT, Sigma Aldrich, USA) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma Aldrich), being substrates for alkaline phosphatase. Blots were documented using the Gel Doc System (Bio-Rad).

[0035] Enzyme-linked assay of SARS-CoV-2 nucleocapsid protein

[0036] Immunoreactivity of immunoreactive SARS-CoV-2 nucleocapsid protein was tested using an enzyme- linked assay (ELISA)

[0019] , 96-well MaxiSorp plates were coated overnight with 2 μg / mL SARS-CoV-2 nucleocapsid protein (ab273530, Abeam, United Kingdom) in carbonate buffer. The plates were blocked with SuperBlock™ T20 (cat. 37516, Thermo Fisher Scientific). After rinsing, the plates were incubated with sera diluted to 1:400 (for IgG) or 1:100 (for IgA) in TBS-T for 2 hours. Subsequently, after rinsing, the plates were incubated with secondary anti-human IgG (1 : 7,500) or anti-human IgA (1 : 30,000) antibodies. Color reaction was triggered using Alkaline Phosphatase Yellow (Merck). The plates were read at 405 nm using a plate reader (PowerWave NT, BioTek Instruments, USA).

[0037] In silica prediction of epitope sequences

[0038] The SARS-CoV-2 sequence used for bioinformatic predictions was Uniprot P0DTC9 (NCAP_SARS2). Three servers were used for predicting 20-amino acid B cell epitopes: BepiPred 2.0

[0013] with 0.6 threshold, 0.95116 specificity and 0.09559 sensitivity; BCPred

[0014] with 75% specificity and 0.99 used as the cut-off value; ABCPred

[0015] with 0.6 threshold. NetCTL 1.2

[0016] was used for predicting T cell epitopes of all supertypes at default settings. IFNepitope

[0017] was used for searching for I FNy epitopes in the SARS-CoV-2 sequence using a hybrid Motif and SVM approach. Results of all predictions were compared and overlapping sequences including as many epitopes as possible were selected.

[0039] Peptide synthesis and mapping

[0040] Peptides were synthesized on plastic pins (non-cleavable peptide type, MIMOTOPES, Melbourne, Australia) using the PEPSCAN method

[0018] modified by Jarząb et al.

[0019] , In brief, peptides were synthesized in a 96-well plate by adding one F-moc amino acid derivative to each pin during one coupling reaction until a full-size peptide was obtained. After deprotecting side chains, the pins were dried and stored at -20°C before use.

[0041] The immunoreactivity of pin-bound peptides was tested using ELISA against three groups of merged sera: acute phase COVID-19, COVID-19 convalescents and pre-pandemic sera using a previously published method

[0020] , In brief, plastic pins were blocked with 1% bovine serum albumin (BSA) in TBS- T; incubated with primary antibodies (acute phase COVID-19, COVID-19 convalescents or pre- pandemic sera) diluted to 1 : 1,000 in TBS-T with 0.1% BSA; after rinsing with TBS-T they were incubated with secondary antibodies: anti-human IgG conjugated with alkaline phosphatase (AP) diluted to 1: 7,500 in TBS-T or AP-conjugated anti-human IgA diluted to 1 : 30,000; colorimetric reaction was triggered using Alkaline Phosphatase Yellow Liquid Substrate for ELISA. The plates were read at 405 nm using the PowerWave NT plate reader. After the test, the bound antibodies were removed by sonication in dissociation buffer (1% sodium dodecylsulfate, 0.1% 2-mercaptoethanol and 0.1 M NagPO,}) preheated to 60°C, washed with water, methanol and dried. Each experiment was performed in at least 6 replicates. Data were analyzed in GraphPad 9.3. Epitopes were verified by calculating statistical significance of their immunoreactivity level (absorbance value) with respect to thresholds calculated for each group. The thresholds were calculated according to the previous publication

[0020] by computing the mean of all results in the group. Calculated thresholds: acute phase COVID-19 1.142 for IgG and 0.615 for IgA; COVID-19 convalescents 1.273 for IgG and 0.507 for IgA; group of pre- pandemic sera 0.413 for IgG and 0.348 for IgA.

[0042] Amino acid sequence analysis

[0043] BLAST

[0044] The Basic Local Alignment Search Tool (BLAST) on the UniProt server (https: / / www.uniprot.org / blast / ) was used for searching for similar amino acid sequences in proteins from other organisms

[0021] , UniProtKB settings: reference proteomes plus Swiss-Prot database, E threshold = 10, automatic matrix, no filtering, with gapped sequences.

[0045] IEDB

[0046] The Immune Epitope Database was used for searching for known epitopes having similar sequences

[0022] , Searching was limited to sequences at least 70% identical to the sequence of interest. There were no restrictions in terms of the host, MHC or disease. Duplicated sequences were removed from the list.

[0047] Results

[0048] Polish patients have pre-pandemic immunity to the SARS-CoV-2 nucleocapsid

[0049] Immunoreactivity analysis of five pre-pandemic human sera with SARS-CoV-2 lysate showed that specific anti-nucleocapsid IgA antibodies were contained in one of the sera (Fig. 1A). The same reactivity was not observed when IgG response of the same group of sera was tested (data not shown). To quantitatively determine the immunoreactivity of pre-pandemic sera, ELISA was performed on a recombinant nucleocapsid and results were compared with the response of acute phase COVID-19 and COVID-19 convalescent sera (Fig. IB and 1C). The level of specific anti-nucleocapsid IgG and IgA antibodies was rather low in the pre-pandemic group compared to the acute convalescent groups. However, some readings, in particular in the pre-pandemic IgA group, were relatively high compared to other groups. Based on the unexpected presence of anti-nucleocapsid antibodies in the pre- pandemic sera, it was decided to investigate N protein further.

[0050] Humoral response against the SARS-CoV-2 nucleocapsid is different in convalescents

[0051] The analysis of immunoreactivity of acute phase and convalescent sera showed variable immune response against SARS-CoV-2 N protein (Fig. 2). In general, all patients evaluated had strong anti-N protein IgG and IgA antibody response during the acute phase of infection and three weeks after resolution of last infection symptoms. However, the immunoreactivity profile changed over the three- week period and varied from patient to patient. The immunoreactivity profiles of sera from Patients 2 and 3 had an additional band (37 kDa range) in the convalescent status, both for specific IgG and IgA. The immunoreactivity profiles of serum from Patient 4 seemed to have multiple additional bands in the convalescent status, both for IgG and IgA antibodies.

[0052] Epitope mapping of the SARS-CoV-2 nucleocapsid shows various epitope patterns for serum groups of the acute phase, convalescents and pre-pandemic.

[0053] In silico prediction of SARS-CoV-2 N protein epitopes revealed 20 sequences (Table 1). Most of them were only B cell epitopes. Sequences no. 6 and 19 contain B and T cell epitopes and an I FNy epitope. In the subsequent state, their immunoreactivity with sera from patients of all the groups studied was verified using PEPSCAN and ELISA techniques. Table 1 List of epitopes predicted in silica

[0054] Empirical epitope mapping using sera from patients of the three groups showed that overall reactivity was higher in the SARS-CoV-2 convalescent group compared to other groups (Fig. 3). However, immunoreactivity profiles were rather identical in the study groups, i.e., the same sequences showed the highest immunoreactivity with the test sera. Overall reactivity in the pre-pandemic group was significantly lower than for acute and COVID-19 convalescent groups. The immunoreactivity profile differed compared to the two other groups.

[0055] Thresholds for all the study groups were calculated: acute COVID-19 group 1.142; COVID-19 convalescents 1.273; pre-pandemic group 0.413, and statistical analysis was performed to indicate epitopes (Table 2).

[0056] Table 2 Epitopes identified in the nucleocapsid - with IgG antibodies. Immunoreactivity of each sequence in the group was investigated with respect to a threshold calculated for the whole group using one-way ANOVA and Dunnett's multiple comparison test (a = 0.05): *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001.

[0057] The following three IgG epitopes only were identified in the group of pre-pandemic sera: 14 KKSAAEASKKPRQKRTATKA (****), 9 SDSTGSNQNGERSGARSKQR (**), 3 IGYYRRATRRIRGGDGKMKD (*). Sequences 3 and 9 only were indicated as B cell epitopes in the in silica prediction. Sequence 14 was indicated as the I FNy epitope. It is particularly interesting that most epitopes identified with sera from the acute COVID-19 group were not identified with sera from COVID-19 convalescents. Convalescent sera recognized peptides 6 and 19 (B, T, I FNy epitopes) in a relatively high ratio compared to the group of pre-pandemic sera.

[0058] Overall immunoreactivity profiles for IgA antibodies were more similar than for IgG antibodies. It is a striking fact that there was no significant difference in the immunoreactivity levels of the predicted sequences with the test sera, unlike for IgG antibodies.

[0059] Thresholds for all the study groups were calculated: acute phase COVID-19 group 0.615; COVID-19 convalescents 0.507; pre-pandemic group 0.348, and statistical analysis was performed to identify immunoreactive epitopes (Table 3). Table 3 Epitopes identified in the nucleocapsid - with IgA antibodies. Immunoreactivity of each sequence in the group was investigated with respect to a threshold calculated for the whole group using one-way ANOVA and Dunnett's multiple comparison test (a = 0.05): *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001.

[0060] Again, the same three epitopes were identified in immunoreactivity tests with pre-pandemic sera: 14 KKSAAEASKKPRQKRTATKA (****), 9 SDSTGSNQNGERSGARSKQR (**), 3 IGYYRRATRRIRGGDGKMKD (***). The difference in the recognition of peptides 6 and 19 with convalescent serum with respect to the group of pre-pandemic sera was similar to the pattern of IgG antibodies.

[0061] BLAST analysis of the sequences was performed to show why sequences 3, 9 and 14 were recognized by pre-pandemic sera. SARS-CoV-2 was excluded from the analysis. In addition, a database of known epitopes (IEDB) was searched. The BLAST analysis showed that sequence 14 KKSAAEASKKPRQKRTATKA was widespread in other coronaviruses similar to SARS as well as bat coronaviruses, such as Rhinolophus affinis or BtRs BetaCoV coronavirus. Similar patterns were also found in TCP domain- containing proteins from various plants, such as melon, soya, clementine, sesame, Colorado blue columbine or silver poplar. The TCP domain is highly conservative and is found in plant transcription factors that regulate a number of growth-related processes

[0023] , The SDSTGSNQNGERSGARSKQR sequence is similar for many coronaviruses. The SNQNGXRSGARS sequence has been found in a protein containing the AA_TRNA_LIGASE_II domain in citrus fruit. The IGYYRRATRRIRGGDGKMKD sequence is highly conservative for other coronaviruses. A shorter RRATRRIRG sequence has been found in ants in multiple coagulation factor deficiency protein 2. The TRRIRG pattern has also been found in a protein containing the death domain from Streptomyces sp. CB01883 and glycogen synthase from Planctomycetes bacterium PIA133. Table 4 BLAST analysis of epitopes identified by pre-pandemic sera. Identical amino acids are shown in bold. X denotes variable amino acids.

[0062] Known epitopes deposited in IEDB similar to sequences 14 KKSAAEASKKPRQKRTATKA, 9 SDSTGSNQNGERSGARSKQR and 3 IGYYRRATRRIRGGDGKMKD were searched (Table 5). Again, sequences associated with KKSAAEASKKPRQKRTATKA were the most common. Partial epitopes were found in human proteins, such as nerve injury-induced protein-1 (ninjurin-1) responsible for interactions between immune and endothelial cells and playing a role in nerve regeneration and in programed and necrotic cell death

[0024] ; mediator of DNA damage checkpoint 1 (mdcl), which plays a key role in the response to DNA damage checkpoint

[0025] ; Surfeit locus protein-1 (surf-1), which plays a role in the regulation of cytochrome c oxidase folding

[0026] ; phosphofurin acidic cluster sorting protein 1 (pacsl), a regulator of membrane transport

[0027] ,

[0063] The SDSTGS sequence is common to SARS-CoV-2 protein N and a known epitope from trans-Golgi network integral membrane protein (tgoln2). The protein is located in the Golgi apparatus and may play a role in the formation of exocytic vesicles.

[0064] Two sequences similar to the epitope with sequence GYYRRATRRIRGGDGKMKD, i.e., GYYRRA and YYRRAT, were found in two known epitopes: human serine / threonine-protein phosphatase 5 (ppp5c) and DnaJ homolog subfamily C member 3 (dnajc3), respectively. PppSc dephosphorylates a number of proteins involved in various signaling pathways. Dnajc3 acts as a chaperone protein and binds misfolded proteins

[0028] ,

[0065] Table 2 Results of IEDB analysis

[0066] Conclusions

[0067] There are a number of possible explanations of the pre-pandemic immune response against SARS-CoV- 2. Some authors suggest that antibodies acquired during common cold caused by HCoV infection cross- react with SARS-CoV-2 antigens. Other authors suggest that the pre-existing immunity may be induced by other antigens, such as those that can be found in vaccines

[0029] or commensal bacteria

[0030] , Elucidating and understanding the mechanisms is necessary to design safe and effective therapies and vaccines. The investigation that resulted in this invention involved analysis of cross-reactivity and it was surprisingly discovered that additional sources of pre-pandemic immunity could occur, such as cross-reactivity with own proteins or other proteins found in the environment.

[0068] Anti-SARS-CoV-2 nucleocapsid IgA proteins showing cross-reactivity were found in pre-pandemic sera obtained from the Polish population (Fig. 1A). In addition, there were no specific IgG antibodies, which could indicate pre-existing mucosal immunity based only on IgA antibodies. This discovery is consistent with previously published results. IgA antibodies showing cross-activity were detected in the pre- pandemic milk of African and American mothers

[0031] , Egwang et al. tested human milk using ELISA in terms of anti-SARS-CoV-2 and -HCoV spike protein levels. Mothers in the U.S. and African mothers showed higher frequency of milk IgA antibodies against alphacoronaviruses and betacoronairuses, respectively, which was consistent with the geographic distribution of the viruses. The investigation by the present inventors revealed IgA antibodies showing cross-reactivity against the nucleocapsid without any specific antibodies against spikes. This could result from the adopted methodology. Namely, Western blotting instead of ELISA was used, which targets linear instead of conformational epitopes. Conformational epitopes are typical of S protein and, therefore, the reduced protein in Western blotting is much less immunoreactive than non-reduced protein. For N protein, both variants are immunoreactive at comparable levels

[0032] ,

[0069] To further investigate immunoreactivity, an ELISA test was performed in a larger group of pre- pandemic sera to evaluate antibody response also against non-linear SARS-CoV-2 nucleocapsid epitopes. Overall immunoreactivity of pre-pandemic sera was much lower compared to acute phase COVID-19 and convalescent samples. However, it was shown for IgA antibodies that some individual readings were as high as in the acute phase COVID-19 group (Fig. IB). Again, this may indicate certain pre-existing immunity to SARS-CoV-2 confirmed for the first time in the Polish population. Pre-existing antibodies showing cross-reactivity to SARS-CoV-2 could have had an effect on SARS-CoV-2 vaccination. It was found that S2 protein was the main target of pre-pandemic immunity in healthy humans and SPF mice. Jia et al. showed that production of S2 antibodies showing cross-reactivity was related to commensal intestinal bacteria and had an effect on the titer levels of specific RBD-binding antibodies after SARS-CoV-2 vaccination in humans

[0030] , Furthermore, SARS-CoV-2 S vaccination changed the composition of mouse intestinal microflora.

[0070] Another interesting finding was that antibody response against the nucleocapsid was greater in the convalescent group than in the acute group, in particular for IgG antibodies (Fig. IB). A similar observation was made for S protein by Isho et al.

[0033] , They showed that peak anti-SARS-CoV-2 S IgG levels occurred between days 16-30 after the onset of symptoms, and the level was maintained for 115 days. Anti-S IgA levels were much less durable, because they significantly decreased after the peak between days 16 and 30. Anti-N antibody levels were very similar to those measured for S protein

[0033] , However, when the inventors compared the anti-N antibody response for the acute phase and convalescents using Western blotting, it was clear that the response was greater in the acute group for IgG antibodies (Fig. 2). In addition, the immunoreactivity profile of the response changed over the three-week period after the initial collection of serum samples.

[0071] Epitope mapping using a combined approach: first by in silica epitope identification and subsequently their verification through synthetic peptides and sera from patients of different groups revealed high variability of immunoreactivity profiles (Fig. 3). In particular, immunoreactivity levels of pre-pandemic sera were much lower than in the acute and convalescent groups. However, the levels for IgA antibodies were comparable, and overall profiles were similar (Fig. 4). Seven epitopes recognized by IgG antibodies were identified in acute phase sera, three in convalescents and three in pre-pandemic sera. One epitope (KKSAAEASKKPRQKRTATKA) was recognized by all three groups. The KKSAAEASKKPRQKRTATKA sequence is found in various coronaviruses (Table 4), and its truncated variants are also highly widespread in plant proteins. Some parts of the sequence were also found in known epitopes, such as in human ninjurinl protein. Ninjurinl is a transmembrane protein expressed mainly in endothelial and bone marrow cells and it is induced during inflammation. Ninjurinl plays a role in systemic inflammation by mediating leukocyte migration and modulating TL-4 receptor- depending expression of inflammatory mediators

[0034] , It was already shown in patients with severe Covid-19 that ninjurinl was excessively expressed in macrophages that could increase systemic inflammation

[0035] , It was difficult to determine the cause-and-effect relationship in that case. The SAAEAS motif was already identified in other research as a common motif for SARS-CoV-2 and Surfeit protein 1 locus, a component of the complex necessary for generating the respiratory rhythm in humans

[0036] , The Lucchese and Flöel hypothesis was that the immune targeting of SURF1 and two other proteins could contribute to brainstem-associated respiratory failure in COVID-19 patients. It was found that patients had antibodies against the KKSAAEASKKPRQKRTATKA sequence containing the SAAEAS motif. Phosphofurin acidic cluster sorting protein 1 (PACS-1) was another protein with an epitope sequence similar to KKSAAEASKKPRQKRTATKA. It has been found that PACS-1 is used by viruses to evade immunity, to proliferate and for pathogenesis

[0037] ,

[0072] Other evidence is also available on B cell epitope mapping in protein N. Much research is performed in silica without subsequent empirical verification [38-41], Other authors use peptide synthesis and ELISA to validate epitopes. Amrun et al. used peptide ELISA to identify the NNAAIVLQLPQGTTLPKG sequences as the immunodominant linear B cell epitope which was closely related to disease severity

[0042] , The epitope is found in the terminal RNA-binding N protein domain. Analysis performed by the inventors showed the same sequence, but it was cleaved into two sequences: (4) LNTPKDHIGTRNPANNAAIV and (12) LQLPQGTTLPKGFYAEGSRG, and none of them was identified as epitopes, most likely due to the cleavage. A different approach to epitope mapping involves production of monoclonal proteins. Tian et al. immunized mice with N protein expressed in inset cells and obtained six anti-N monoclonal proteins. They identified DFSKQLQQ as the new conservative B cell epitope

[0043] , The DFSKQLQQ sequence is part of the (18) LDDFSKQLQQSMSSADSTQA peptide tested by the inventors but it was not identified as the epitope using the serum mapping approach. Some research also probes into the cross-reactivity of SARS-CoV-2 N protein. PIWAS analysis of cross-reactivity performed by Haynes et al. showed two epitopes (in sequence 12 LQLPQGTTLPKGFYAEGSRG and sequence 18 LDDFSKQLQQSMSSADSTQA)

[0044] , but none of them was identified as epitopes in the test performed by the present inventors. The differences could result from variability typical of the studied populations, to be taken into consideration when preparing and testing vaccine efficacy.

[0073] The most important result of the research by the present inventors was the identification of epitopes reported for the nucleocapsid protein found in the group of Polish patients who had COVID-19 infection. Sequences MSDNGPQNQRNAPRITFGGP and KADETQALPQRQKKQQTVTL are identified both by serum antibodies from acute infection phase patients and, what is even more important, from convalescents, which indicates that the antibodies are preserved in circulation after infection. In addition, the sequences were not characterized as epitopes in the healthy group, which means that no pre-pandemic immunity against the epitopes occurs and they may be considered adequate vaccine antigens specific for SARS-CoV-2. The use of specific epitopes which have been previously analyzed in terms of potential cross-reactivity is much safer than using a complete protein. This approach reduces the risk of inducing immune response through the mechanism of molecular mimicry

[0045] ,

[0074] To conclude, the presence of antibodies in pre-pandemic sera isolated from the Polish population was found in the research that resulted in the invention, which could indicate pre-existing immunity to SARS-CoV-2. In addition, it was found that the immunoreactive profile of response to N protein was variable during infection. Epitope mapping analysis revealed an epitope recognized by antibodies from sera of all the groups studied. The peptide had common sequences with other proteins, i.e. other coronaviruses of plant or human origin, which showed that the pre-existing immunity to SARS-CoV-2 could have been induced by antibody cross-reactivity against proteins other than the virus. It is the most important that the research by the inventors identified unique peptide sequences recognized by serum antibodies from convalescents, and they are interesting targets as potential vaccine antigens specific for SARS-CoV-2.

[0075] References

[0076] 1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The Species Severe Acute Respiratory Syndrome-Related Coronavirus: Classifying 2019-NCoV and Naming It SARS-CoV-2. Nat. Microbiol. 2020, 5, 536-544, doi:10.1038 / s41564-020-0695-z.

[0077] 2. Killerby, M.E.; Biggs, H.M.; Haynes, A.; Dahl, R.M.; Mustaquim, D.; Gerber, S.I.; Watson, J.T.

[0078] Human Coronavirus Circulation in the United States 2014-2017. J. Clin. Virol. 2018, 101, 52-56, doi:10.1016 / j.jcv.2018.01.019.

[0079] 3. Chen, Y.; Liu, Q..; Guo, D. Emerging Coronaviruses: Genome Structure, Replication, and Pathogenesis. J. Med. Virol. 2020, 92, 418-423, doi:10.1002 / jmv.25681.

[0080] 4. Bai, Z.; Cao, Y.; Liu, W.; Li, J. The SARS-CoV-2 Nucleocapsid Biatko and Its Role in Viral Structure, Biological Functions, and a Potential Target for Drug or Vaccine Mitigation. Viruses 2021, 13, 1115, doi:10.3390 / vl3061115.

[0081] 5. Sealy, R.E.; Hurwitz, J.L. Cross-Reactive Immune Responses toward the Common Cold Human Coronaviruses and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): Mini- Review and a Murine Study. Microorganisms 2021, 9, 1643, doi:10.3390 / microorganisms9081643.

[0082] 6. Tso, F.Y.; Lidenge, S.J.; Pena, P.B.; Clegg, A.A.; Ngowi, J.R.; Mwaiselage, J.; Ngalamika, O.; Julius, P.; West, J.T.; Wood, C. High Prevalence of Pre-Existing Serological Cross-Reactivity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in Sub-Saharan Africa. Int. J. Infect. Dis. 2021, 102, 577-583, doi:10.1016 / j.ijid.2020.10.104.

[0083] 7. Sagar, M.; Reifler, K.; Rossi, M.; Miller, N.S.; Sinha, P.; White, L.F.; Mizgerd, J.P. Recent Endemic Coronavirus Infection Is Associated with Less-Severe COVID-19. J. Clin. Invest. 2021, 131, doi:10.1172 / JCI143380.

[0084] 8. Chau, N.V.V.; Nhan, L.N.T.; Nguyet, L.A.; Tu, N.T.K.; Hong, N.T.T.; Man, D.N.H.; Ty, D.T.B.; Nhu, L.N.T.; Yen, L.M.; Khanh, T.H.; et al. Absence of SARS-CoV-2 Antibodies in Pre-Pandemic Plasma from Children and Adults in Vietnam. Int. J. Infect. Dis. 2021, 111, 127-129, doi:10.1016 / j.ijid.2021.07.072.

[0085] 9. Gombar, S.; Bergquist, T.; Pejaver, V.; Hammarlund, N.E.; Murugesan, K.; Mooney, S.; Shah, N.; Pinsky, B.A.; Banaei, N. SARS-CoV-2 Infection and COVID-19 Severity in Individuals with Prior Seasonal Coronavirus Infection. Diagn. Microbiol. Infect. Dis. 2021, 100, 115338, doi:10.1016 / j.diagmicrobio.2021.115338.

[0086] 10. Anderson, E.M.; Goodwin, E.C.; Verma, A.; Arevalo, C.P.; Bolton, M.J.; Weirick, M.E.; Gouma, S.; McAllister, C.M.; Christensen, S.R.; Weaver, J.; et al. Seasonal Human Coronavirus Antibodies Are Boosted upon SARS-CoV-2 Infection but Not Associated with Protection. Cell 2021, 184, 1858-1864.610, doi:10.1016 / j. cell.2021.02.010.

[0087] 11. Razim, A.; Pacyga, K.; Naporowski, P.; Martynowski, D.; Szuba, A.; Gamian, A.; Gorska, S. Identification of Linear Epitopes on the Flagellar Biatkos of Clostridioides Difficile. Sci. Rep.

[0088] 2021, 11, 9940, doi:10.1038 / s41598-021-89488-7.

[0089] 12. Razim, A.; Pacyga, K.; Aptekorz, M.; Martirosian, G.; Szuba, A.; Pawlak-Adamska, E.; Brzychczy- Wtoch, M.; Myc, A.; Gamian, A.; Gorska, S. Epitopes Identified in GAPDH from Clostridium Difficile Recognized as Common Antigens with Potential Autoimmunizing Properties. Sci. Rep. 2018, 8, 13946, doi:10.1038 / s41598-018-32193-9. 13. Jespersen, M.C.; Peters, B.; Nielsen, M.; Marcatili, P. BepiPred-2.0: Improving Sequence-Based B-Cell Epitope Prediction Using Conformational Epitopes. Nucleic Acids Res. 2017, 45, W24- W29, doi:10.1093 / nar / gkx346.

[0090] 14. EL-Manzalawy, Y.; Dobbs, D.; Honavar, V. Predicting Linear B-Cell Epitopes Using String Kernels. J. Mol. Recognit. JMR 2008, 21, 243-255, doi:10.1002 / jmr.893.

[0091] 15. Saha, S.; Raghava, G.P.S. Prediction of Continuous B-Cell Epitopes in an Antigen Using Recurrent Neural Network. Biatkos 2006, 65, 40-48, doi:10.1002 / prot.21078.

[0092] 16. Larsen, M.V.; Lundegaard, C.; Lamberth, K.; Buus, S.; Lund, O.; Nielsen, M. Large-Scale Validation of Methods for Cytotoxic T-Lymphocyte Epitope Prediction. BMC Bioinformatics 2007, 8, 424, doi:10.1186 / 1471-2105-8-424.

[0093] 17. Dhanda, S.K.; Vir, P.; Raghava, G.P. Designing of Interferon-Gamma Inducing MHC Class-ll Binders. Biol. Direct 2013, 8, 30, doi:10.1186 / 1745-6150-8-30.

[0094] 18. Carter, J.M. Epitope Mapping of a Biatko Using the Geysen (PEPSCAN) Procedure. In Peptide Analysis Protocols; Humana Press: New Jersey, 2014; Vol. 36, pp. 207-224 ISBN 0-89603-274-4.

[0095] 19. Jarząb, A.; Witkowska, D.; Ziomek, E.; Dąbrowska, A.; Szewczuk, Z.; Gamian, A. Shigella Flexneri 3a Outer Membrane Biatko C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity. PLoS ONE 2013, 8, e70539, doi:10.1371 / journal. pone.0070539.

[0096] 20. Pacyga, K.; Razim, A.; Martirosian, G.; Aptekorz, M.; Szuba, A.; Gamian, A.; Myc, A.; Gorska, S. The Bioinformatic and In Vitro Studies of Clostridioides Difficile Aminopeptidase M24 Revealed the Immunoreactive KKGIK Peptide. Cells 2020, 9, 1146, doi:10.3390 / cells9051146.

[0097] 21. Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, DJ. Gapped BLAST and PSI-BLAST: A New Generation of Biatko Database Search Programs. Nucleic Acids Res. 1997, 25, 3389-3402, doi:10.1093 / nar / 25.17.3389.

[0098] 22. Vita, R.; Mahajan, S.; Overton, J.A.; Dhanda, S.K.; Martini, S.; Cantrell, J.R.; Wheeler, D.K.; Sette, A.; Peters, B. The Immune Epitope Database (IEDB): 2018 Update. Nucleic Acids Res. 2019, 47, D339-D343, doi:10.1093 / nar / gkyl006.

[0099] 23. Manassero, N.G.U.; Viola, IL.; Welchen, E.; Gonzalez, D.H. TCP Czynnik transkrypcyjnys: Architectures of Plant Form. Biomol. Concepts 2013, 4, 111-127, doi:10.1515 / bmc-2012-0051.

[0100] 24. Weerasinghe-Mudiyanselage, P.D.E.; Kim, J.; Choi, Y.; Moon, C.; Shin, T.; Ahn, M. Ninjurin-1: A Biomarker for Reflecting the Process of Neuroinflammation after Spinal Cord Injury. Neural Regen. Res. 2020, 16, 1331-1335, doi:10.4103 / 1673-5374.301033.

[0101] 25. Stewart, G.S.; Wang, B.; Bignell, C.R.; Taylor, A.M.R.; Elledge, SJ. MDC1 Is a Mediator of the Mammalian DNA Damage Checkpoint. Nature 2003, 421, 961-966, doi:10.1038 / nature01446.

[0102] 26. Khan, T.; Kayani, S. Natural History Study of SURF-1 (5137). Neurology 2021, 96.

[0103] 'Ll. Youker, R.T.; Shinde, U.; Day, R.; Thomas, G. At the Crossroads of Homoeostasis and Disease: Roles of the PACS Biatkos in Membrane Traffic and Apoptosis. Biochem. J. 2009, 421, 1-15, doi:10.1042 / BJ20081016.

[0104] 28. Regulated Association of Misfolded Endoplasmic Reticulum Lumenal Biatkos with P58 / DNAJc3. EMBOJ. 2008, 27, 2862-2872, doi:10.1038 / emboj.2008.199.

[0105] 29. Reche, P.A. Potential Cross-Reactive Immunity to SARS-CoV-2 From Common Human Pathogens and Vaccines. Front. Immunol. 2020, 11.

[0106] 30. Jia, L.; Weng, S.; Wu, J.; Tian, X.; Zhang, Y.; Wang, X.; Wang, J.; Yan, D.; Wang, W.; Fang, F.; et al. Pre-Existing Antibodies Targeting a Linear Epitope on SARS-CoV-2 S2 Cross-Reacted with Commensal Gut Bacteria and Shaped Vaccine Induced Immunity 2022, 2021.07.13.21260404.

[0107] 31. Egwang, T.G.; Owalla, T.J.; Okurut, E.; Apungia, G.; Fox, A.; De Carlo, C.; Powell, R.L. Differential Pre-Pandemic Breast Milk IgA Reactivity against SARS-CoV-2 and Circulating Human Coronaviruses in Ugandan and American Mothers. Int. J. Infect. Dis. 2021, 112, 165-172, doi:10.1016 / j.ijid.2021.09.039.

[0108] 32. Maghsood, F.; Shokri, M.-R.; Jeddi-Tehrani, M.; Torabi Rahvar, M.; Ghaderi, A.; Salimi, V.;

[0109] Kardar, G.A.; Zarnani, A.-H.; Amiri, M.M.; Shokri, F. Identification of Immunodominant Epitopes on Nucleocapsid and Spike Biatkos of the SARS-CoV-2 in Iranian COVID-19 Patients. Pathog. Dis. 2022, 80, ftacOOl, doi:10.1093 / femspd / ftac001.

[0110] 33. Isho, B.; Abe, K.T.; Zuo, M.; Jamal, A.J.; Rathod, B.; Wang, J.H.; Li, Z.; Chao, G.; Rojas, O.L.; Bang, Y.M.; et al. Persistence of Serum and Saliva Antibody Responses to SARS-CoV-2 Spike Antigens in COVID-19 Patients. Sci. Immunol. 2020, 5, eabe5511, doi:10.1126 / sciimmunol.abe5511.

[0111] 34. Jennewein, C.; Sowa, R.; Faber, A.C.; Dildey, M.; von Knethen, A.; Meybohm, P.; Scheller, B.; Drose, S.; Zacharowski, K. Contribution of Ninjurinl to Toll-Like Receptor 4 Signaling and Systemic Inflammation. Am. J. Respir. Cell Mol. Biol. 2015, 53, 656-663, doi:10.1165 / rcmb.2014-0354QC.

[0112] 35. Xu, Q.; Yang, Y.; Zhang, X.; Cai, JJ. Association of Pyroptosis and Severeness of COVID-19 as Revealed by Integrated Single-Cell Transcriptome Data Analysis. ImmunoInformatics 2022, 6, 100013, doi:10.1016 / j. immuno.2022.100013.

[0113] 36. Lucchese, G.; Flöel, A. Molecular Mimicry between SARS-CoV-2 and Respiratory Pacemaker Neurons. Autoimmun. Rev. 2020, 19, 102556, doi:10.1016 / j.autrev.2020.102556.

[0114] 37. Thomas, G.; Aslan, J.E.; Thomas, L.; Shinde, P.; Shinde, U.; Simmen, T. Caught in the Act - Biatko Adaptation and the Expanding Roles of the PACS Biatkos in Tissue Homeostasis and Disease. J. Cell Sci. 2017, 130, 1865-1876, doi:10.1242 / jcs,199463.

[0115] 38. Kumar, J.; Qureshi, R.; Sagurthi, S.R.; Qureshi, LA. Designing of Nucleocapsid Biatko Based Novel Multi-Epitope Vaccine Against SARS-COV-2 Using Immunoinformatics Approach. Int. J. Pept.

[0116] Res. Then 2021, 27, 941-956, doi:10.1007 / sl0989-020-10140-5.

[0117] 39. Rakib, A.; Sami, S.A.; Islam, M.A.; Ahmed, S.; Faiz, F.B.; Khanam, B.H.; Marma, K.K.S.; Rahman, M.; Uddin, M.M.N.; Nainu, F.; et al. Epitope-Based Immunoinformatics Approach on Nucleocapsid Biatko of Severe Acute Respiratory Syndrome-Coronavirus-2. Molecules 2020, 25, 5088, doi:10.3390 / molecules25215088.

[0118] 40. Dai, Y.; Chen, H.; Zhuang, S.; Feng, X.; Fang, Y.; Tang, H.; Dai, R.; Tang, L.; Liu, J.; Ma, T.; et al. Immunodominant Regions Prediction of Nucleocapsid Biatko for SARS-CoV-2 Early Diagnosis: A Bioinformatics and Immunoinformatics Study. Pathog. Glob. Health 2020, 0, 1-8, doi:10.1080 / 20477724.2020.1838190.

[0119] 41. Tilocca, B.; Soggiu, A.; Sanguinetti, M.; Musella, V.; Britti, D.; Bonizzi, L.; Urbani, A.; Roncada, P. Comparative Computational Analysis of SARS-CoV-2 Nucleocapsid Biatko Epitopes in Taxonomically Related Coronaviruses. Microbes Infect. 2020, 22, 188-194, doi:10.1016 / j.micinf.2020.04.002.

[0120] 42. Amrun, S.N.; Lee, C.Y.-P.; Lee, B.; Fong, S.-W.; Young, B.E.; Chee, R.S.-L.; Yeo, N.K.-W.; Torres- Ruesta, A.; Carissimo, G.; Poh, C.M.; et al. Linear B-Cell Epitopes in the Spike and Nucleocapsid Biatkos as Markers of SARS-CoV-2 Exposure and Disease Severity. EBioMedicine 2020, 58, 102911, doi:10.1016 / j.ebiom.2020.102911.

[0121] 43. Tian, Y.; Zhang, G.; Liu, H.; Ding, P.; Jia, R.; Zhou, J.; Chen, Y.; Qi, Y.; Du, J.; Liang, C.; et al. Screening and Identification of B Cell Epitope of the Nucleocapsid Biatko in SARS-CoV-2 Using the Monoclonal Antibodies. Appl. Microbiol. Biotechnol. 2022, 106, 1151-1164, doi:10.1007 / s00253-022-11769-6.

[0122] 44. Haynes, W.A.; Kamath, K.; Bozekowski, J.; Baum-Jones, E.; Campbell, M.; Casanovas-Massana, A.; Daugherty, P.S.; Cruz, C.S.D.; Dhal, A.; Farhadian, S.F.; et al. High-Resolution Mapping and Characterization of Epitopes in COVID-19 Patients 2020, 2020.11.23.20235002.

[0123] 45. Segal, Y.; Shoenfeld, Y. Vaccine-Induced Autoimmunity: The Role of Molecular Mimicry and Immune Crossreaction. Cell. Mol. Immunol. 2018, 15, 586-594, doi:10.1038 / cmi.2017.151.

Claims

Claims1. An epitope originating from SARS-CoV-2 N protein having an amino acid sequence selected from: MSDNGPQNQRNAPRITFGGP (SEQ ID No. 1) and KADETQALPQQRQKKQQTVTL (SEQ ID No. 2).

2. A vaccine antigen specific for SARS-CoV-2 containing the epitope originating from SARS-CoV-2 N protein having an amino acid sequence selected from: MSDNGPQNQRNAPRITFGGP (SEQ. ID No. 1) and KADETQALPQQRQKKQQTVTL (SEQ ID No. 2).

3. The epitope of Claim 1 or the antigen Claim 2 for use in the treatment or prevention of disease caused by coronavirus.

4. The epitope or the antigen for use of Claim 3, characterized in that the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

5. The epitope or the antigen for use of Claim 3, characterized in that the disease is COVID-19, in particular with an acute course.

6. The epitope or the antigen for use of Claim 3, characterized in that it is used for vaccination.

7. The epitope or the antigen for use of Claim 3, characterized in that it is used for preparing a therapeutic formulation, in particular serum.

8. A method for identifying coronavirus infection, characterized in that antibodies specific for the epitope of Claim 1 or the antigen of Claim 2 are detected in a biological sample collected from a patient, wherein the presence of such antibodies indicates that the patient has coronavirus infection.

9. The method of Claim 8, characterized in that the biological sample is blood or saliva.

10. The method of Claim 8, characterized in that the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

11. The method of Claim 8, characterized in that the disease is COVID-19, in particular with an acute course.

12. The method of Claim 8, characterized in that current or previous infection is identified.

Images