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  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • G—PHYSICS
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• • G—PHYSICS
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  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• 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.
• 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.
• 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].
• 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.
• 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).
• 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).
• 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.
• the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• the disease is COVID-19, in particular with an acute course.
• the aforementioned epitope or antigen is used for vaccination.
• the aforementioned epitope or antigen is used for preparing a therapeutic formulation, in particular serum.
• 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.
• the biological sample is blood or saliva.
• the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• the result of infection is COVID-19, in particular with an acute course.
• current or previous infection is identified.
• N protein nucleocapsid protein
• SARS-CoV-2 a structural protein of SARS-CoV-2
• 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.
• MSDNGPQNQRNAPRITFGGP SEQ. ID No. 1
• KADETQALPQQRQKKQQTVTL SEQ ID No. 2
• 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.
• 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.
• 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.
• 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.
• 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 catalog. SAB3701340, Merck
• TBS-T Tris buffered normal saline with 0.1% Tween 20
• Immunoreactivity of immunoreactive SARS-CoV-2 nucleocapsid protein was tested using an enzyme- linked assay (ELISA) [19], 96-well MaxiSorp plates were coated overnight with 2 ⁇ g/mL SARS-CoV-2 nucleocapsid protein (ab273530, Abeam, United Kingdom) in carbonate buffer. The plates were blocked with SuperBlockTM 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.
• ELISA enzyme- linked assay
• 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).
• 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 [13] with 0.6 threshold, 0.95116 specificity and 0.09559 sensitivity; BCPred [14] with 75% specificity and 0.99 used as the cut-off value; ABCPred [15] with 0.6 threshold.
• NetCTL 1.2 [16] was used for predicting T cell epitopes of all supertypes at default settings.
• IFNepitope [17] 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.
• Peptides were synthesized on plastic pins (non-cleavable peptide type, MIMOTOPES, Melbourne, Australia) using the PEPSCAN method [18] modified by Jarz ⁇ b et al. [19], 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.
• pin-bound peptides were 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 [20], 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.
• BSA bovine serum albumin
• the Immune Epitope Database was used for searching for known epitopes having similar sequences [22], 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.
• 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.
• Epitope mapping of the SARS-CoV-2 nucleocapsid shows various epitope patterns for serum groups of the acute phase, convalescents and pre-pandemic.
• 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).
• 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.
• 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).
• 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).
• 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.
• IEDB epitopes
• 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 [23],
• 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.
• 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 [24]; mediator of DNA damage checkpoint 1 (mdcl), which plays a key role in the response to DNA damage checkpoint [25]; Surfeit locus protein-1 (surf-1), which plays a role in the regulation of cytochrome c oxidase folding [26]; phosphofurin acidic cluster sorting protein 1 (pacsl), a regulator of membrane transport [27],
• 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.
• GYYRRATRRIRGGDGKMKD 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 [28],
• 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 [31], 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.
• 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 [30], Furthermore, SARS-CoV-2 S vaccination changed the composition of mouse intestinal microflora.
• 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 [34], It was already shown in patients with severe Covid-19 that ninjurinl was excessively expressed in macrophages that could increase systemic inflammation [35], 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 [36], 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.
• PACS-1 Phosphofurin acidic cluster sorting protein 1
• LNTPKDHIGTRNPANNAAIV LNTPKDHIGTRNPANNAAIV
• (12) LQLPQGTTLPKGFYAEGSRG LQLPQGTTLPKGFYAEGSRG
• DFSKQLQQ as the new conservative B cell epitope [43]
• 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) [44], 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.
• 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.
• 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 [45],
• 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.

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