An isolated polypeptide or antigenic fragment thereof and its uses for diagnosing or predicting a disease such as long covid or post-VAC-syndrome

Abstract

The present invention inter alia relates to an use of an isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11, 16-20 and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids for diagnosing Long COVID or Post-Vac-Syndrome, or for predicting a risk of developing Long COVID or Post-Vac-Syndrome. The present invention also relates to the isolated polypeptide or antigenic fragment thereof for use in preventing, treating or ameliorating Long COVID or Post-Vac-Syndrome., as well as to a method of detecting a presence of antibodies against SARS-CoV-2 in a subject, comprising the step of contacting a sample of said subject with at least one of the polypeptide or antigenic fragment thereof.

Description

AN ISOLATED POLYPEPTIDE OR ANTIGENIC FRAGMENT THEREOF AND ITS USES FOR DIAGNOSING OR PREDICTING A DISEASE SUCH AS LONG COVID OR POST-VAC-SYNDROMEFIELD OF THE INVENTION

[0001] The present invention relates to an use of an isolated polypeptide or an antigenic fragment thereof for diagnosing Long COVID or Post-Vac-Syndrome, and / or for predicting a risk of developing Long COVID or Post-Vac-Syndrome. The present invention also relates to a method of detecting a presence of antibodies against SARS- CoV-2 in a subject, comprising the steps of contacting a sample of the subject with at least one of the polypeptides or antigenic fragments thereof; and detecting in said sample the presence of antibodies that specifically bind said at least one polypeptide or antigenic fragment thereof. Furthermore, the invention relates to an isolated polypeptide or an antigenic fragment thereof for use in treating Long COVID or Post-Vac-Syndrome, and / or for use in predicting a risk of developing Long COVID or Post-Vac-Syndrome.BACKGROUND OF THE INVENTION

[0002] The consequences of the COVID-19 pandemic go far beyond the socioeconomic impact or the number of deaths following SARS-CoV-2 infection. To this day, people suffer from symptoms that persist for more than three months after an acute SARS-CoV-2 infection (e.g., anosmia, fatigue, dyspnea, cough) and substantially impair the quality of life (Davis et al., 2023; Nalbandian et al., 2021). This Long COVID, recently renamed post-acute sequelae of COVID-19 (PASC) by the WHO, can occur after mild and more severe courses of the disease (Stallmach et al., 2022) and is distinct from the sequelae of serious COVID-19 infection associated with hospitalization (Huang et al., 2021).

[0003] According to the German expert guideline for Long COVID, about 2% of patients are affected to an extent that normal daily life is no longer possible (S1 guideline Long COVID, 2021; AWMF-register No. 020 / 027). The risk of developing Long COVID after infection with variant-of-concern (VOC) Delta appears to be twice as high as after infection with Omicron (Antonelli et al., 2022).

[0004] Over the past few months, the list of possible causes of Long COVID has grown. Persistence of SARS-CoV-2 RNA and antigen fragments in the gastrointestinal tract as well as tissue damage are thought to be responsible for persistent inflammation and immune stimulation (Gaebler et al., 2021; Gupta et al., 2020). Rheology appears tobe affected by altered red blood cell morphology (Renoux et al., 2021; Thomas et al., 2020), fibrin amyloid microclots causing platelet aggregation (Pretorius et al., 2022), hypercoagulability due to elevated fibrinogen levels and consumption of factor XII (Wygrecka et al., 2022), and autoantibodies targeting phospholipids and phospholipidbinding proteins (Zuo et al., 2020).

[0005] However, other causes of Long COVID have also recently been discussed. For example, in acute SARS-CoV-2 infection, a dysregulation of the immune system has been observed, e.g., suppressive plasma factor-induced T-cell hyporeactivity (Renner et al., 2021), linked to increased glycolysis and impaired ketogenesis (Karagiannis et al., 2022). In patients suffering from Long COVID, a strong activation of the innate immune system with increased expression of type I and type III interferons persists beyond the acute infection (Phetsouphanh et al., 2022). This dysregulation appears to promote autoantibody formation (Su et al., 2022), particularly against G-protein coupled receptors (Wallukat et al., 2021).

[0006] Despite the recent progress made in understanding causes and symptoms of Long COVID, accurate diagnosis and specific therapies are still lacking.

[0007] Accordingly, it is an object of the present invention to address these issues and to provide improved care for patients suffering from Long COVID.SUMMARY OF THE INVENTION

[0008] This object is inter alia accomplished by the polypeptides, the uses, the kits, the compositions the methods having the features of the respective independent claims. In a first aspect, the invention provides an use of an isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11 , 16-20 and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids, for (in vitro / Ex-vivo) diagnosing Long COVID or Post-Vac-Syndrome or for (in vitro / Ex-vivo)predicting a risk of developing Long COVID or Post-Vac-Syndrome.

[0009] In a second aspect, the invention provides an isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11 , 16-20 and 23-28; wherein the isolated polypeptide ishaving a length of no more than 200 contiguous amino acids for use in preventing, treating or ameliorating Long COVID or Post-Vac-Syndrome.

[0010] The inventors have found that surprisingly, the isolated polypeptides or antigenic fragments thereof as herein provided may be used for diagnosis of e.g. Long COVID and / or Post-Vac Syndrome and thus represent a valuable biomarker. For example, the isolated polypeptides or antigenic fragments thereof as herein provided may be used as an indication - e.g. together with other parameters - in a method for diagnosing e.g. Long COVID and / or Post-Vac Syndrome in order to help the medical practitioner to make a diagnosis. The isolated polypeptides or antigenic fragments thereof may further advantageously be used in e.g. treating, preventing and / or ameliorating a disease such as Long COVID and / or Post-Vac-Syndrome.

[0011] For example, the disease may be Long COVID and / or Post-Vac-Syndrome.

[0012] In a third aspect, the invention provides a pharmaceutical composition comprising the isolated polypeptide or antigenic fragment thereof according to the present invention.

[0013] In a fourth aspect, the invention provides a method of detecting a presence of antibodies against SARS-CoV-2 in a subject, comprising(a) contacting a sample of said subject with at least one of the polypeptide or antigenic fragment thereof as herein provided;(b) detecting in the sample the presence of antibodies that specifically bind said polypeptide or antigenic fragment thereof.

[0014] In a fifth aspect, the invention provides a kit for diagnosing a disease such a Long COVID or Post-Vac-Syndrome in a subject, comprising the isolated polypeptide or antigenic fragment thereof as herein provided.In a sixth aspect, the invention provides a method for treating or preventing a disease such as Long COVID or Post-Vac-Syndrome, comprising administering the pharmaceutical composition comprising the isolated polypeptide or antigenic fragment thereof as herein provided into a patient being diagnosed with Long COVID or Post-Vac-Syndrome.

[0015] All aspects of the invention provide the above described advantages and improvements related to the potential diagnosis of e.g. Long COVID and / or Post-Vac Syndrome and / or the advantageous use of the polypeptides in e.g. treating, preventing and / or ameliorating a disease such as Long COVID and / or Post-Vac-Syndrome.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:

[0017] Fig. 1 shows a schematical representation of the applied method of epitope mapping for determining peptide sequences specifically recognized by anitibodies present in patients suffering from Long COVID and / or Post-Vac-Syndrome

[0018] Fig. 2 shows a graphical representation of results from a micropeptide assay relating to a specific part of printed 15-amino acid-long peptides.

[0019] Fig. 3 shows a graphical representation of results from a micropeptide assay relating to a specific part of printed 15-amino acid-long peptides.

[0020] Fig. 4 shows a different graphical representation of results from the micropeptide assay of Fig. 2-3, wherein results of specifically chosen polypeptides are shown in detail.

[0021] Fig. 5 shows shows a different graphical representation of results from the micropeptide assay of Fig. 2-3, wherein results of specifically chosen polypeptides are shown in detail.

[0022] Fig. 6 shows shows a different graphical representation of results from the micropeptide assay of Fig. 2-3, wherein results of specifically chosen polypeptides are shown in detail.

[0023] Fig. 7 shows a graphical representation of results of flag-controls for the ELISA assays as shown in Fig 8.

[0024] Fig. 8 shows a graphical representation of results of ELISA assays for the respective polypeptides

[0025] Fig. 9 shows a graphical representation of the validation of the epitoperecognition using four different cohorts.DETAILED DESCRIPTION OF THE INVENTIONAs explained above, in a first aspect the invention is directed to an use of an isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11 , 16-20and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids for diagnosing Long COVID or Post-Vac-Syndrome.

[0026] The term "sequence identity" or "identity" as used in the present invention means the percentage of pair-wise identical residues, following homology alignment of a sequence of a polypeptide and or nucleic acid of the present invention with a sequence in question, with respect to the number of residues in the longer of these two sequences.

[0027] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). The percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSLIM 62; gap costs: 11.1; cutoff value set to 10-3) including the respective sequences. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0028] The term “isolated polypeptide” as referred to herein relates to polypeptides separated or outside from the natural environment as e.g. cells, and comprises e.g. synthetically generated polypeptides or polypeptides generated by recombinant processes. Specifically, all commonly known standard techniques that may be used for the generation of the polypeptides as herein presented are envisaged.

[0029] It is noted in this context that it has been surprisingly found here that the isolated polypeptides or antigenic fragments thereof as herein provided may be used for (in vitro / Ex-vivo) diagnosis of e.g. Long COVID and / or Post-Vac Syndrome and thus represent valuable biomarkers. The isolated polypeptides or antigenic fragments thereof may further advantageously be used in e.g. diagnosing, treating, preventing and / or ameliorating a disease such as Long COVID and / or Post-Vac-Syndrome.

[0030] For example, the polypeptide region of the isolated polypeptide or antigenic fragment thereof may have at least 60%, or at least 61%, or at least 62 %, or at least 63%, or at least 64%, or at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%, or at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at leat 97%, or at least 98%, or at least 99%, or 100% sequenceidentity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11, 16-20 and 23-28.

[0031] According to an embodiment, the polypeptide region of the isolated polypeptide or antigenic fragment thereof may have one or more conservative amino acid substitutions relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of 1-11 , 16-20 and 23-28. For example, the polypeptide region of the isolated fragment thereof may have 1 , 2, 3, 4, 5, 6, 7, 8, or 9 conservative amino acid substitutions relative relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of 1-11, 16-20 and 23-28.

[0032] The expression antigenic fragment as used herein relates to the region of the polypeptide having an amino acid sequence which is sufficient and necessary for being recognized by an antibody of a patient suffering from Long COVID and / or Post- Vac-Syndrome. For example, the antigenic fragment may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, or at least 14 contiguous amino acids.

[0033] According to an embodiment, the polypeptide region may have at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 12-15, 21-22 and 29-31.

[0034] For example, the polypeptide region of the isolated polypeptide or antigenic fragment thereof may have at least 60%, or at least 61%, or at least 62 %, or at least 63%, or at least 64%, or at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%, or at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at leat 97%, or at least 98%, or at least 99%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 12-15, 21-22 and 29-31.

[0035] According to an embodiment, the polypeptide region of the isolated polypeptide or antigenic fragment thereof may have one or more conservative amino acid substitutions relative to an amino acid sequence as set forth in any one of the SEQID No’s selected from the group consisting of 12-15, 21-22 and 29-31. For example, the polypeptide region of the isolated fragment thereof may have 1 , 2, 3, 4, 5, 6, 7, 8, or 9 conservative amino acid substitutions relative relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No.: 12-15, 21-22 and 29-31.

[0036] For example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 12 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.7, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.8, and the entire polypeptide region having at least60% Sequence identity with SEQ ID No.9. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQID No. 12 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0037] According to another embodiment, the polypeptide region having at least60% sequence identity with SEQ ID No. 14 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.1 , the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.2, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.3, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.4, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.5, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.6, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.7, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.8, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.9, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.10, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.11. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 14 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0038] According to another embodiment, the polypeptide region having at least 60% sequence identity with SEQ ID No. 15 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.2, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.3, the entire polypeptideregion having at least 60% Sequence identity with SEQ ID No.4, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.5, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.6, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.7, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.8, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.9, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.10, This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least60% sequence identity with SEQ ID No. 15 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0039] According to another embodiment, the polypeptide region having at least 60% sequence identity with SEQ ID No. 21 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.15, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.16, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.17, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.18, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.19, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.20. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 21 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0040] According to another embodiment, the polypeptide region having at least 60% sequence identity with SEQ ID No. 22 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No 17, entire polypeptide region having at least 60% Sequence identity with SEQ ID No.18, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.19. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 22 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0041] According to another embodiment, the polypeptide region having at least 60% sequence identity with SEQ ID No. 29 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No 23, entire polypeptide regionhaving at least 60% Sequence identity with SEQ ID No.24, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.25, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No 26, entire polypeptide region having at least 60% Sequence identity with SEQ ID No.27, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.28. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 29 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0042] According to another embodiment, the polypeptide region having at least 60% sequence identity with SEQ ID No. 30 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No 23, entire polypeptide region having at least 60% Sequence identity with SEQ ID No.24, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.25, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No 26, and the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.27. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 30 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0043] According to another embodiment, the polypeptide region having at least 60% sequence identity with SEQ ID No. 31 may comprise the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.24, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No.25, the entire polypeptide region having at least 60% Sequence identity with SEQ ID No 26, entire polypeptide region having at least 60% Sequence identity with SEQ ID No.27. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID. It is envisaged that, for example, the polypeptide region having at least 60% sequence identity with SEQ ID No. 31 may comprise more potential epitopes / antigenic fragments that may be specific for Long COVID.

[0044] According to an embodiment, the length of the polypeptide may be in a range between 4 and 200 contiguous amino acids, or between 4 and 150 contiguous amino acids, or between 4 and 100 contiguous amino acids, or between 4 and 50 contiguous amino acids, or between 4 and 20 contiguous amino acids, or between 5 and 200 contiguous amino acids, or between 5 and 150 contiguous amino acids, or between5 and 100 contiguous amino acids, or between 5 and 50 contiguous amino acids, or between 5 and 20 contiguous amino acids, or between 6 and 200 contiguous amino acids, or between 6 and 150 contiguous amino acids, or between 6 and 100 contiguous amino acids, or between 6 and 50 contiguous amino acids, or between 6 and 20 contiguous amino acids, or between 7 and 200 contiguous amino acids, or between 7 and 150 contiguous amino acids, or between 7 and 100 contiguous amino acids, or between 7 and 50 contiguous amino acids, or between 7 and 20 contiguous amino acids, or between 8 and 200 contiguous amino acids, or between 8 and 150 contiguous amino acids, or between 8 and 100 contiguous amino acids, or between 8 and 50 contiguous amino acids, or between 8 and 20 contiguous amino acids, or between 9 and 200 contiguous amino acids, or between 9 and 150 contiguous amino acids, or between 9 and 100 contiguous amino acids, or between 9 and 50 contiguous amino acids, or between 9 and 20 contiguous amino acids, or between 10 and 200 contiguous amino acids, or between 10 and 150 contiguous amino acids, or between 10 and 100 contiguous amino acids, or between 10 and 50 contiguous amino acids, or between 10 and 20 contiguous amino acids.

[0045] In some embodiments, the polypeptide may consist of any one of the polypeptide sequences selected from the group consisting of of SEQ ID No.: 1 - 31;

[0046] For example, the polypeptides as herein presented may be linear or cyclic.

[0047] In some embodiments, one or more amino acid residues of the polypeptides of the invention may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications, for example phosphorylation, acetylation and deamidation (Engelhard et al., 2006). Chemical modifications may not correspond to those that may be present in vivo. For example, the N or C terminal ends of the polypeptide may be modified improve the stability, bioavailability and or affinity of the polypeptides (see for example, Brinckerhoff et al., 1999). Further examples of nonnatural modifications may include incorporation of photoreactive cross-linking amino acids, N-methylated amino acids, backbone reduction, retroinversion by using d-amino acids, N-terminal methylation and C-terminal amidation and PEGylation.

[0048] For example, the antigenic fragment of a polypeptide as herein presented may consist of at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11 , or at least 12 amino acids in length. It is envisaged that a polypeptide as herein presented may comprise more than one antigenic fragment.

[0049] In accordance with an embodiment, the polypeptide region may have at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least90%, or at least 95%, or at least 97%, or 100% sequence identity with the polypeptide sequence as set forth in SEQ ID No. 13, wherein the polypeptide is cyclic.

[0050] According to a further aspect, the polypeptide or antigenic fragment thereof as herein presented may be used for preventing, treating or ameliorating of a disease, such as Long COVID or Post-Vac-Syndrome.

[0051] For example, the use may further comprise using the polypeptide or antigenic fragment thereof in adsorbing extracorporeally autoantibodies of a patient being diagnosed with the disease from blood or blood plasma of the patient; preferably wherein the disease is Long COVID.

[0052] According to an embodiment, the use may comprise in vivo depleting an anti-SARS-CoV-2 antibody in the patient that binds to the isolated polypeptide or antigenic fragment thereof.

[0053] Alternatively, the use may further comprise using the polypeptide or antigenic fragment thereof in adsorbing extracorporeally autoantibodies of a patient being diagnosed with the disease from blood or blood plasma of the patient wherein the disease is Post-Vac Syndrome and wherein the polypeptide region is having at least 60% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No. 6, 14, 15, 24, 29, 30 and 31.

[0054] According to some embodiments, it is herewith provided a use of the isolated polypeptide or antigenic fragment thereof as herein described for diagnosing a disease, such as Long COVID or Post-Vac-Syndrome; and / or for predicting a risk of developing a disease, such as Long COVID or Post-Vac-Syndrome.

[0055] The expression “Long COVID” as referred to herein relates to (1) ongoing symptomatic COVID-19 for people who still have symptoms between 4 and 12 weeks after the start of acute symptoms; and (2) post-COVID-19 syndrome for people who still have symptoms for more than 12 weeks after the start of acute symptoms. Long COVID as used herein thus relates to patients presenting with new or ongoing symptoms 4 weeks or later after acute COVID infection.

[0056] The expression “Post-Vac-Syndrome” as herein understood relates a persistent health impairment resulting from and temporally associated with COVID-19 vaccination.

[0057] Preferably, the use further comprises the use of the isolated polypeptide or antigenic fragment thereof in a method for determining a presence of an antibody againstSARS-CoV-2 in a sample of a subject, wherein the antibody against SARS-CoV-2 is capable of binding to the the isolated polypeptide or antigenic fragment thereof, preferably further wherein the antibody is capable of binding to the Spike protein of SARS-CoV-2.

[0058] For example, the disease may be Long COVID.

[0059] Alternatively, the disease may be Post-Vac Syndrome, and the isolated polypeptide or antigenic fragment thereof may comprise a polypetide region having at least 60% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No. 6, 14, 15, 24, 29, 30 and 31.

[0060] Furthermore, it is herein provided a pharmaceutical composition comprising the isolated polypeptide or antigenic fragment as herein provided.

[0061] In an embodiment, the pharmaceutical composition may further comprise an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof. In some embodiments, one or more amino acid residues of the polypeptides included in the pharmaceutical composition may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications, for example phosphorylation, acetylation and deamidation (Engelhard et al., 2006). Chemical modifications may not correspond to those that may be present in vivo. For example, the N or C terminal ends of the polypeptide may be modified improve the stability, bioavailability and or affinity of the polypeptides (see for example, Brinckerhoff et al., 1999). Further examples of non-natural modifications may include incorporation of photoreactive cross-linking amino acids, N-methylated amino acids, backbone reduction, retroinversion by using d-amino acids, N-terminal methylation and C-terminal amidation and PEGylation.

[0062] In another aspect, the invention also provides a method of detecting a presence of antibodies against SARS-CoV-2 in a subject, comprising(a) contacting a sample of said subject with at least one of the polypeptide or antigenic fragment thereof as herein described;(b) detecting in said sample the presence of antibodies that specifically bind said polypeptide or antigenic fragment thereof.

[0063] In some embodiments, the method may further comprise a step of comparing a detected amount or concentration of the antibody with a reference;preferably wherein the reference may be a reference for a healthy condition and / or for Long COVID or Post-Vac-Syndrome.

[0064] According to an embodiment, the increased amount of detected antibody in the sample of the subject compared to the reference may be indicative for Long COVID or Post-Vac-Syndrome;

[0065] According to some embodiments, for example, the sample may comprise serum, plasma, whole blood, saliva, cerebrospinal fluid, interstitial fluid or urine.

[0066] According to an embodiument, the sample may be contacted with a plurality of the polypeptide or antigenic fragments thereof as herein presented.

[0067] For example, the sample may be contacted with at least one of the isolated polypeptides or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 1-15; and the sample may further be contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID No. 16 - 31. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID.

[0068] Alternatively, the sample may be contacted with the isolated polypeptide or antigenic fragment thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 16-22, and the sample may further be contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 23 - 31. This may allow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID.

[0069] Alternatively, the sample may be contacted with at least one of the isolated polypeptides or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 1-15; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 16-22; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragment thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 23 - 31. This mayallow for detection of e.g. more Long COVID specific antibodies in patients suffering from Long COVID.

[0070] According to a further aspect, there is provided a kit for treating or diagnosing a disease such a Long COVID or Post-Vac-Syndrome in a subject, comprising the isolated polypeptide or antigenic fragment as herein described.

[0071] For example, the kit may further comprise a suitable buffer solution which may be used in a method for diagnosing a disease such as Long COVID or Post-Vac- Syndrome in a subject and / or in a method for treating a disease such as Long COVID or Post-Vac-Syndrome.

[0072] It is also provided a method for treating or preventing a disease such as Long COVID or Post-Vac-Syndrome, comprising administering the pharmaceutical composition comprising the isolated polypeptide or antigenic fragment thereof as herein provided into a patient being diagnosed with Long COVID or Post-Vac-Syndrome.

[0073] In an embodiment, the method may further comprise the step of in vivo depleting an anti-Covid 19 antibody in the patient that binds to the isolated polypeptide or antigenic fragment thereof.

[0074] The invention will be further illustrated by the following non-limiting Experimental Examples.

[0075] Sequences as used herein are depicted in below Table 1.

[0076] Table 1. Sequences as used herein.Experimental Examples

[0077] Example 1. Peptide Microarray for Identification of Peptides Suitable for Binding of Antibodies to SARS-CoV-2 spike that are specifically associated with Long COVID.Long COVID is characterized by persistent symptoms after acute SARS-CoV-2 infection that significantly affect quality of life. To date, no well-characterized biomarker is available. To decipher the humoral immune response to this virus, Long COVID patients (n=64) and convalescent controls (n=64) matched for sex, age, and time since acute infection were recruited. Long COVID was associated with significantly elevated IgG (not IgA and IgM) antibodies to SARS-CoV-2 spike and spike subdomains but not to nucleocapsid protein. These antibodies were functionally active and neutralized wild-type SARS-CoV-2 significantly better than sera from convalescent controls. In contrast, both cohorts showed similar reactivity to seasonal coronaviruses. A peptide microarray showed pronounced reactivity of Long COVID sera against different epitopes in the S1and S2 domains of SARS-CoV-2, which was confirmed by a Luminex-based bead array. This pronounced reactivity against different specific epitopes in the S1 and S2 domain of SARS-CoV-2 detected in sera of Long COVID and / or Post-Vac-syndrome patients was not visible in convalescent controls, naive controls, or vaccinated controls, respectively. Results of this assay demonstrates an enhanced and spike-specific immune response that e.g. may form the basis of a diagnostic tool for e.g. Long COVID or Post-Vac syndrome. A schematical overview of the assay used to identify polypeptides suitable as a biomarker is shown in Fig. 1.

[0078] 1.1 Materials and Methods

[0079] Recruitment of study cohortsA total of 64 Long COVID patients infected during the first and second SARS-CoV-2 wave in Germany were recruited at the Long COVID center of Donaustauf Hospital, which specializes in the treatment of patients with respiratory and psychosomatic diseases. An equally sized control cohort of convalescent subjects were recruited at the Kreisklinik Trostberg, Trostberg, Germany. The collection of blood, respiratory and fecal samples from Long COVID patients and respective control cohorts was approved by the Ethical Commission of the Faculty for Medicine, University of Regensburg, as part of the COVUR (Ref. No. 20-1785- 101) and Trostberg study (Ref. No. 20-1896-101), respectively. Long COVID patients and subjects from the respective control cohorts (convalescent control, naive control and vaccinated control, respectively) provided written informed consent. For the ELISA assays as herein provided, furthermore, independent control cohorts have been used for validation of the results. With respect to these independent cohorts, sampling of blood from PCS patients in Homburg and Dresden was approved by the Medical Association of Saarland and the ethical commission of the Technical University Dresden (Ref. nos. 62 / 20 and BO-EK- 208052023). All participants provided written informed consent. The dominant SARS- CoV-2 variant was classified at the time of acute infection as the causative variant.

[0080] Peptide MicroarrayRecognition of SARS CoV-2 N- and S-protein epitopes was determined using a customized PEPperCHIP® microarray (PEPperPRINT GmbH, Heidelberg, Germany) covering the fulllength N- and S-protein of SARS-CoV-2 Wuhan-1 (UniProt ID: P0DTC9, 419 aa and UniProt ID: P0DTC2, 1273 aa, respectively) with 15-amino acid-longpeptides, designed with an overlap of 13 amino acids and elongated with neutral C- and N-terminal GSGSGSG (SEQ ID No. 204) linkers. A total of 847 individual peptides were printed in duplicates on each microarray. Influenza hemagglutinin (HA) and polio control peptides (YPYDVPDYAG (SEQ ID No 205) and KEVPALTAVETGAT (SEQ ID No. 206) respectively) were included with 48 spots per control peptide for each sub-array. Peptide microarrays were incubated for 15 min at room temperature (RT) with orbital shaking at 140 rpm in PBS supplemented with 0.05% Tween 20, and then blocked for 30 min at RT using Rockland Blocking Buffer (Rockland Immunochemicals, Pottstown, USA). Prior to serum incubation, microarrays were stained with goat anti-Human IgG Fc Cross- Adsorbed Secondary Antibody DyLight 680 (Invitrogen, Carlsbad, USA) at a final concentration of 5pg / ml, and then shaken at RT and 140 rpm for 45 min to determine nonspecific background. After three washing steps with PBS / 0.05% Tween 20, microarrays were carefully immersed in 1 mM TRIS pH 7.4, dried in a pressurized air stream to remove antibody residues, and scanned using the LI-COR Odyssey Classic Imaging System (scanning offset 0.65 mm, resolution 21 pm, intensity 7 for channel 700 / 800) (Bad Homburg, Germany). After imaging, microarrays were equilibrated in PBS / 0.05% Tween 20 and 10% Rockland Blocking Buffer at RT for 15 min with orbital shaking at 140 rpm. The buffer was discarded and peptide microarrays were incubated with patient sera diluted 1:1000 in PBS / 0.05% Tween 20 and 10% Rockland Blocking Buffer overnight at 4°C with orbital shaking at 140 rpm. Microarrays were washed three times with PBS / 0.05% Tween 20 and then stained with goat anti-Human IgG Fc Cross- Adsorbed Secondary Antibody DyLight 680 (see above) at a final concentration of 0.5 pg / ml for 45 min at RT and orbital shaking at 140 rpm to detect peptide binding. Microarrays were washed, air dried and scanned as described above, and then equilibrated in PBS / 0.05% Tween 20 and 10% Rockland Blocking Buffer for 15 min with orbital shaking at 140 rpm to prepare for HA and polio control peptide staining. After removal of the buffer, a mouse monoclonal anti-HA control antibody (12CA5)-DyLight800 (PEPperPRINT) was added at a final concentration of 0.5pg / ml. After incubation at RT for 45 min with orbital shaking at 140 rpm, microarrays were washed, air dried and scanned. The three 16-bit-grayscale TIFF files were analyzed using MAPIX Analyzer Software (Innopsys, Carbonne, France). Mean background intensity, reflecting nonspecific binding, was determined for each peptide spot using the first scan before serum incubation. This nonspecific background was subtracted for each microarray analysis and peptide spot. The fluorescence intensity of each peptide was calculated as mean of duplicates at the referenced wavelength.

[0081] Data analysis and statisticsNon-parametric data of two groups and three or more groups were compared using Mann-Whitney-U test or Kruskal-Wallis test, respectively. Two-sided p-values below 0.05 were considered significant. Graphs were generated and statistical analyses performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA).

[0082] ELISA AssayNunc MaxiSorp plates were coated overnight at 4°C with 50 pl neutravidin at a concentration of 1 pg / ml (Thermo Fisher Scientific). Plates were washed three times (HydroFlex Microplate Washer, Tecan, Mannedorf, Switzerland) with 200 pl PBS containing 0.1% Tween 20 (PBS-T), coated with 50 pl peptides (0.5 pM; Intavis Peptide Services GmbH, Tubingen, Germany) at 37°C for 30 minutes, and then blocked for 1h at RT with 200 pl of 5% fat-free milk powder (Hugli Nahrungsmittel GmbH, Radolfzell, Germany) in PBS-T. After washing three times with 200 pl PBS-T, 50 pl serum diluted 1 :100 with 1% fat-free milk powder and 2.5 pg / ml neutravidin in PBS-T was added and incubated for 1h at RT. The plates were then washed ten times with 200 pl PBS-T and filled with 50 pl of the secondary polyclonal rabbit anti-human IgG / HRP conjugate (Dako, Glostrup, Denmark), diluted 1 :5,000 in PBS-T. After 1h of incubation at RT, plates were washed ten times with 200 pl PBS-T. After adding 50 pl of the substrate solution (recomWell Substrate TMB fast2, Mikrogen GmbH, Neuried, Germany) to each well, plates were incubated for 8 min at RT and stopped by adding 50 pl of 1.0 N sulfuric acid (Roth). Optical density was determined using the Microplate Reader Model 680 (BioRad, Hercules, CA, USA) in three technical replicates. For background correction, OD630 values were subtracted from OD450 values.

[0083] 1.2. Peptide Microarray assay analysisThe results of Micropeptide assay analysis are shown in Fig. 2, Fig.3, Fig. 4, Fig. 5, and Fig. 6.

[0084] Fig. 2 shows the results of the Micropeptide assay analysis of a subset of reactive SARS-CoV-2 S1 domain overlapping polypeptides that have been analysed in the assay. Specifically, the respectively tested polypeptide is indicated at the left-hand side of the graphical representation of the results of the micropeptide assay (the firstpage [2 / 16 of drawing pages] of Fig. 2 indicates at the left hand site from top to bottom Seq ID No. 32-34, 208, 35, 209, 36-40, 210, 41-55, 211 , 56-57, 212, and 58-62; the second page [3 / 16 of drawing pages] of Fig. 2. indicates at the left-hand site from top to bottom Seq ID No. 63 - 99; the third page [4 / 16 of the drawing pages] of Fig. 2. indicates at the left-hand site from top to bottom Seq ID No.100 - 104, 1 - 10, and 105 - 125). “Post-COVID” (Samples 1-14) relate to samples of patients suffering from Long COVID, “Convalescent” (Samples 15-28) relate to samples of individuals recovered from COVID- 19 disease, “Vaccinated” (Samples 29-36) relate to samples from individuals that have received a vaccination against COVID-19 disease, “Post-Vac” (Sample 37) relate to a sample of a patient suffering from Post-Vac-Syndrome, “Naive” (Samples 38 - 42) relate to samples from naive control subjects. The color intensity is indicative of the Fluorescence displayed on a logarithmic (Iog10) scale (right hand side). The designation “A-K” relate to polypeptides which are further specifically depicted in Fig. 4.

[0085] Fig. 3 shows the results of the Micro peptide assay analysis of a subset of polypeptides overlapping the SARS-CoV-2 S2 domain that have been analysed in the assay. Specifically, the respectively tested polypeptide is indicated at the left-hand side of the graphical representation of the results of the micropeptide assay (the first page [5 / 16 of drawing pages] of Fig. 3 indicates at the left hand site from top to bottom Seq ID No. 126-155; the second page [6 / 16 of drawing pages] of Fig. 3. indicates at the lefthand site from top to bottom Seq ID No. 156 - 178, 213, and 179 - 184; the third page [7 / 16 of the drawing pages] of Fig. 3. indicates at the left-hand site from top to bottom Seq ID No.185, 16 - 20, 186-202, 23 - 28, and 203). “Post-COVID” (Samples 1-14) relate to samples of patients suffering from Long COVID, “Convalescent” (Samples 15- 28) relate to samples of individuals recovered from COVID-19 disease, “Vaccinated” (Samples 29-36) relate to samples from individuals that have received a vaccination against COVID-19 disease, “Post-Vac” (Sample 37) relate to a sample of a patient suffering from Post-Vac-Syndrome, “Naive” (Samples 38 - 42) relate to samples from naive control subjects. The color intensity is indicative of the Fluorescence displayed on a logarithmic (Iog10) scale (right-hand side). The designations “L-P” and “Q-W”, respectively, relate to polypeptides which are further specifically depicted in Fig. 5 and 6, respectively.

[0086] Fig. 4 shows the degree of reactivity against specifically depicted polypeptides KFLPFQQFGRDIADT (SEQ ID No. 1), LPFQQFGRDIADTTD (SEQ ID No. 2), FQQFGRDIADTTDAV (SEQ ID No. 3), QFGRDIADTTDAVRD (SEQ ID No. 4), GRDIADTTDAVRDPQ (SEQ ID No. 5), DIADTTDAVRDPQTL (SEQ ID No. 6),ADTTDAVRDPQTLEI (SEQ ID No. 7), TTDAVRDPQTLEILD (SEQ ID No. 8), DAVRDPQTLEILDIT (SEQ ID No. 9), VRDPQTLEILDITPC (SEQ ID No. 10), DPQTLEILDITPCSF (SEQ ID No. 11), shown as violin plots relating to Long COVID patients (“Post-COVID”) (n=14), a patient suffering from Post-Vac-Syndrome (n=1), control (n=14), vaccinated (n=8) and naive subjects (n=5). Statistics were performed using one-way ANOVA. *p<0.05, ***p<0.001. The complete region of the SARS-CoV-2 protein covered by the overlapping polypeptides according to SEQ ID 1-11 is having the amino acid sequence KFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF (SEQ ID No. 14) (Core sequence in Bold letters and underlined).

[0087] Fig. 5 shows the degree of reactivity against specifically depicted polypeptides PLQPELDSFKEELDK (SEQ ID No.16), QPELDSFKEELDKYF (SEQ ID No. 17), ELDSFKEELDKYFKN (SEQ ID No. 18), DSFKEELDKYFKNHT (SEQ ID No. 19), FKEELDKYFKNHTSP (SEQ ID No. 20), shown as violin plots relating to Long COVID patients (“Post-COVID”) (n=14), a patient suffering from Post-Vac-Syndrome (n=1), control (n=14), vaccinated (n=8) and naive subjects (n=5). Statistics were performed using one-way ANOVA. *p<0.05, ***p<0.001. The complete region of the SARS-CoV-2 protein covered by the overlapping polypeptides according to SEQ ID 16- 20 is having the amino acid sequence PLQPELDSFKEELDKYFKNHTSP (SEQ ID No. 21) (Core sequence in Bold letters and underlined).

[0088] Fig. 6 shows the degree of reactivity against specifically depicted polypeptides CSCGSCCKFDEDDSE (SEQ ID No. 23), CGSCCKFDEDDSEPV (SEQ ID No. 24), SCCKFDEDDSEPVLK (SEQ ID No. 25), CKFDEDDSEPVLKGV (SEQ ID No. 26), FDEDDSEPVLKGVKL (SEQ ID No. 27), EDDSEPVLKGVKLHY (SEQ ID No. 28), and GCCSCGSCCKFDEDD (SEQ ID No. 202) shown as violin plots relating to Long COVID patients (“Post-COVID”) (n=14), a patient suffering from Post-Vac-Syndrome (n=1), control (n=14), vaccinated (n=8) and naive subjects (n=5). Statistics were performed using one-way ANOVA. *p<0.05, ***p<0.001. The complete region of the respective SARS-CoV-2 protein covered by the overlapping polypeptides according to SEQ ID 23-28, is having the amino acid sequence CSCGSCCKFKDEDDSEPVLKGVKLHY (SEQ ID No. 29) (Core sequence in Bold letters and underlined).

[0089] 1.3 ELISA assay analysis. The results of Micropeptide assay analysis are shown in Fig. 7-9.

[0090] Fig. 7 shows standard curves of 30 ELISA titrations of a monoclonal antibody against the immobilized flag peptide to demonstrate the inter-assay reproducibility.

[0091] Fig. 8 shows the results of ELISA assays using specifically the polypeptides DAVRDPQTLEILDIT (SEQ ID No. 9), ADTTDAVRDPQTLEILDIT (SEQ ID No. 12), GGGDAVRDPQTLEILDITGGG (SEQ ID No. 13), QPELDSFKEELDKYF (SEQ ID No. 17), and CSCGSCCKFDEDDSEPVLKGVKL (SEQ ID No. 30). “PCS+” relate to samples of patients suffering from Long COVID, seropositive, “PCS-“ relate to samples of patients not suffering from Long COVID, seronegative, “Convalescent” relate to samples of individuals recovered from COVID-19 disease, “Vaccinated” relate to samples from individuals that have received a vaccination against COVID-19 disease, “Naive” relates to samples from naive control subjects, and “Independent PCT+” relates to samples from patients suffering from Long-COVID which were recruited independently from the original cohort.

[0092] Fig. 9 shows the results of the validation of the epitope-recognition with respect to the polypeptide QPELDSFKEELDKYF (SEQ ID No. 17) using four different cohorts.“Naive” relates to samples from naive control subjects, “Convalescent” relates to individuals recovered from COVID-19 disease, and “Post-Covid” relates to individuals suffering from Long-COVID, comprising the original PCS cohorts as well as validation cohorts I (Donaustauf, black), II (Donaustauf, dark grey), III (Homburg, grey) and IV (Dresden, light grey).

[0093] 1.3 ResultsRecruitment of Long COVID and convalescent control cohort. A total of 64 patients suffering from Long COVID were recruited at the outpatient center at Donaustauf Hospital and compared with an equally sized convalescent control cohort (Fig. 1). All subjects were infected with European wildtype (D614G) or variant-of-concern (VOC) Alpha in the first and second wave of SARS-CoV-2 in Germany, respectively. A total of53 (82.8%) Long COVID patients and all convalescent controls had mild to moderate disease. Long COVID patients, but not controls, continued to have symptoms beyond 12 weeks after acute infection. A total of 10 Long COVID patients had received one dose of vaccine prior to sampling. Both cohorts were matched for gender, age, and time since acute infection. Half of the Long COVID cohort reported 4-6 concurrent symptoms, of which fatigue, dyspnea, and cognitive / memory impairment occurred in more than 70% of patients. Sleep disturbances, tachycardia / palpitations, anosmia / ageusia, depression, and myalgia / arthralgia occurred less frequently in 25.4-34.9% of patients. The three most common comorbidities were respiratory / otolaryngologic, metabolic, and / or cardiovascular disease in 34.4-40.6% of patients. Overall, 14.1%, 17.2%, and 18.8% of patients reported regular use of cardiovascular medications, psychotropic drugs, and antiasthmatics, respectively. For 6-minute walk distance, Long COVID patients achieved only 82.8% of the desired value, and 41.3% of patients were below 80% of the normal value. This reduced physical performance was associated with impaired diffusion capacity and hypocapnia on blood gas analysis in 23.0% and 54.8% of Long COVID patients, respectively. On the Borg category ratio (CR) 10 scale, the degree of perceived exertion ranged from 5 to 7 ("severe" to "very severe") in 36.9% of patients.

[0094] Symptoms at the onset and during the course of the disease. During acute SARS-CoV-2 infection, patients who later developed Long COVID, were significantly more likely to suffer from dyspnea (p<0.001), while headache / myalgia was observed more frequently in convalescent controls (p<0.001). These findings suggested that shortness of breath during acute SARS-CoV-2 infection may be indicative of the development of Long COVID. A total of 10 (16.1%) and 23 (37.1%) patients reported slight and considerable improvement of symptoms, respectively, during the course of the disease, whereas symptoms remained unchanged in 28 patients (45.2%) and even worsened in one (1.6%).

[0095] Elevated IgG antibodies to SARS-CoV-2 spike protein in Long COVID. To characterize humoral immune responses in Long COVID patients, binding of IgG, IgA and IgM antibodies to the immobilized SARS-CoV-2 RDB was determined using an inhouse ELISA (Peterhoff et al., 2021). Although SARS-CoV-2 IgG antibodies were undetectable or very low in 11 Long COVID patients, IgG responses were significantly higher in the entire cohort than in convalescent controls regardless of vaccination (p<0.01). In contrast, IgA and IgM antibodies were similarly low in both cohorts. Notably, IgG antibodies were of comparably low avidity in Long COVID patients and convalescentcontrols (median, 37.3% vs. 32.6%, p=0.33, n.s.). IgG antibodies to the stabilized spike ectodomain, but not to N- and C-terminal nucleocapsid protein, were also significantly elevated in Long COVID patients compared to controls (p<0.0001). Respective avidity indices were higher for the stabilized spike ectodomain in Long COVID patients compared to controls (p<0.05), while they were similarly low for the nucleocapsid. Overall, Long COVID patients had significantly more low-avidity IgG antibodies to the spike domain of SARS-CoV-2 than convalescent controls.

[0096] Reactivity against specific SARS-CoV-2 S1 and S2 peptides in Long COVID.To dissect the IgG response against the SARS-CoV-2 spike protein at the epitope level, a custom array with overlapping peptides has been performed that covered full-length SARS-CoV-2 spike and nucleocapsid protein and were immobilized on a glass slide. Framed by control peptides derived from influenza hemagglutinin and poliovirus, specific epitopes were recognized by sera from Long COVID patients (n=9) and controls (n=9), while others showed up in Long COVID only. Heat maps identified hotspots within S1 , S2, and nucleocapsid protein that were recognized by patients, controls, and in some cases, pre-COVID sera. A peptide in the SARS-CoV-2 RBD (NYNYLYRLFRKSNLK) that is associated to induce IL-6 release (Dhall et al., 2020) was not discriminatory between Long COVID patients and convalescent controls and was even recognized by one of three pre-COVID sera. In contrast, a peptide in S1 (DAVRDPQTLEILDIT) was recognized more frequently in Long COVID patients than in controls (p<0.05), which was no longer the case when the peptide sequence was shifted by a few bases. A peptide in S2 (CGSCCKFDEDDSEPV) was strongly recognized by Long COVID patients, also by two convalescent control subjects, albeith somewhat weaker. Another peptide in S2 (QPELDSFKEELDKYF) was recognized significantly more frequently by Long COVID patients. As shown by Fig. 2 and 3, and - in more detail - Fig. 4, 5 and 6, the Polypeptides according to SEQ ID No 1-11, 16-20, and 23 -28 are recognized frequently in Long COVID patients, and not at all or to a much lesser extend (weaker) by convalescent control or vaccinated subjects. Collectively, these data suggests recognition of specific epitopes in the spike protein by sera from patients suffering from Long COVID. Fig. 2 and 3 further show that serum from different Long COVID patients (Samples 1-14) may recognize different epitopes / antigenic fragments of the polypeptides according to SEQ ID No 1-11, 16-20, and 23 -28. Epitopes / antigenic fragments may thus be e.g. of different lengths, and it is envisaged that more than one epitope / antigenic fragment may be recognized by serum of Long COVID patients in the polypeptides asherein disclosed. Using standard techniques, in view of the data shown in Fig. 2 and 3, exact epitopes / antigenic fragments of Long COVID specific antibodies present in sera of Long COVID patients may easily be determined. The results could be further validated using 3 independent Long-COVID cohorts (n=445) by ELISA assays as shown herein.

[0097] Summary and conclusionsThe results described above demonstrate suitability of the isolated polypeptides or antigenic fragments thereof as herewith presented for e.g. use in a method for diagnosing as disease such as Long COVID or Post-Vac-Syndrome.

[0098] Thus, it is contemplated that for the first time, a few epitopes in the viral spike protein have been identified that may discriminated between Long COVID patients and convalescent controls. These epitopes, represented by peptides within the viral spike protein, were not or rarely recognized by convalescent controls or vaccinated controls and not detected by SARS-CoV-2 naive controls.

[0099] It is further contemplated that a few epitopes in the viral spike protein have been identified that may discriminate between Post-Vac-Syndrome patients and convalescent controls and / or vaccinated controls. These epitopes, represented by peptides within the viral spike protein, were not or rarely recognized by convalescent controls or vaccinated controls and not detected by SARS-CoV-2 naive controls, but these epitopes, represented by the peptides as herein disclosed, were clearly recognized by serum of a patient suffering from Post-Vac-Syndrome.

[0100] These peptides are partially buried within the closed conformation of the viral spike protein and may be exposed upon conformational changes induced by SARS- CoV-2 entry into target cells. The polypeptides as herein presented may be thus be used as a tool for e.g. diagnosing Long COVID or e.g. Post-Vac-Syndrome. In addition, it is also envisaged that the polypeptides as herein presented may be used to predict the development of e.g. Long COVID at an early stage of the disease, which would allow early initiation of treatment.

[0101] Furthermore, the polypeptides as herein presented may be used for a therapeutic approach, in which the polypeptides could be loaded on a matrix (e.g. column resin) and used for a targeted apheresis of the respective antibodies.

[0102] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0103] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0104] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.

[0105] The invention may be further characterized by the following items:Item 1. An isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11, 16-20 and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids.Item 2. The isolated polypeptide or an antigenic fragment thereof according to item 1 , wherein the polypeptide region is having at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 12-15, 21-22 and 29-31.Item 3. The isolated polypeptide or an antigenic fragment thereof according to item 1 , wherein the length of the polypeptide is in a range between 4 and 200 contiguous amino acids, or between 4 and 150 contiguous amino acids, or between 4 and 100 contiguous amino acids, or between 4 and 50 contiguous amino acids.Item 4. The isolated polypeptide according to any one of items 1- 3, wherein the polypeptide consists of any one of the polypeptide sequences selected from the group consisting of of SEQ ID No.: 1 - 31; and / or wherein the peptide is linear or cyclic; and / or wherein the antigenic fragment consists of at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12 amino acids in length.Item 5. The isolated polypeptide or antigenic fragment thereof according to anyone of the items 1-4, wherein the polypeptide region is having an at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the polypeptide sequence as set forth in SEQ ID No. 13, and further wherein the polypeptide is cyclic.Item 6. The isolated polypeptide or antigenic fragment thereof according to any one of the foregoing items for use in preventing, treating or ameliorating of a disease, such as Long COVID or Post-Vac-Syndrome; optionally wherein the use further comprises using the polypeptide or antigenic fragment thereof in adsorbing extracorporeally autoantibodies of a patient being diagnosed with the disease from blood or blood plasma of the patient; preferably wherein the disease is Long COVID, or wherein the disease is Post-Vac Syndrome and wherein the polypeptide region is having at least 60% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No. 6, 14,15, 24, 29, 30 and 31.Item 7. Use of the isolated polypeptide or antigenic fragment thereof according to any one of items 1-6 for diagnosing a disease, such as Long COVID or Post-Vac- Syndrome; and / or for predicting a risk of developing a disease, such as Long COVID or Post-Vac-Syndrome; preferably further comprising the use of the isolated polypeptide or antigenic fragment thereof in a method for determining a presence of an antibody against SARS- CoV-2 in a sample of a subject, wherein the antibody against SARS-CoV-2 is capable of binding to the the isolated polypeptide or antigenic fragment thereof, preferably further wherein the antibody is capable of binding to the Spike protein of SARS-CoV-2.Item 8. The use according to item 7, wherein the disease is long COVID-19; or wherein the disease is Post-Vac Syndrome, and wherein the isolated polypeptide or antigenic fragment thereof comprises a polypetide region having at least 60% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No. 6, 14, 15, 24, 29, 30 and 31.Item 9. A pharmaceutical composition comprising the isolated polypeptide or antigenic fragment thereof according to any one of items 1-6.Item 10. The pharmaceutical composition according to item 9, further comprising an adjucant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.Item 11. A method of detecting a presence of antibodies against SARS-CoV-2 in a subject, comprising(a) contacting a sample of said subject with at least one of the polypeptide or antigenic fragment thereof according to any one of claims 1-6;(b) detecting in said sample the presence of antibodies that specifically bind said polypeptide or antigenic fragment thereof.Item 12. The method according to item 11, further comprising comparing adetected amount or concentration of the antibody with a reference; preferably wherein the reference is a reference for a healthy condition and / or for Long COVID or Post-Vac- Syndrome; optionally wherein an increased amount of detected antibody in the sample of the subject compared to the reference is indicative for Long COVID or Post-Vac- Syndrome; and / or wherein the sample comprises serum, plasma, whole blood, saliva, cerebrospinal fluid, interstitial fluid or urine.Item 13. The method according to item 11 or 12, wherein the sample is contacted with a plurality of the polypeptide or antigenic fragments thereof according to any one of items 1 - 6.Item 14. The method according to item 13, wherein the sample is contacted with at least one of the isolated polypeptides or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 1-15; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID No. 16 - 31 ; or wherein the sample is contacted with the isolated polypeptide or antigenic fragment thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 16-22, and further wherein the sample is contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 23 - 31 ; or wherein the sample is contacted with at least one of the isolated polypeptides or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 1-15; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequencesselected from the group of SEQ ID. No. 16-22; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragment thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 23 - 3115. A kit for diagnosing or treating a disease such a long COVID-19 or Post-Vac-Syndrome in a subject, comprising the isolated polypeptide or antigenic fragment thereof according to item 1-6.

Claims

Claims:What is claimed is:

1. Use of an isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11 , 16-20 and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids for diagnosing Long COVID or Post-Vac-Syndrome or for predicting a risk of developing Long COVID or Post-Vac-Syndrome.

2. The use according to claim 1 , wherein the polypeptide region is having at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 12-15, 21-22 and 29-31.

3. The use according to claim 1 or 2, wherein the length of the polypeptide is in a range between 4 and 200 contiguous amino acids, or between 4 and 150 contiguous amino acids, or between 4 and 100 contiguous amino acids, or between 4 and 50 contiguous amino acids.

4. The use according to any one of claims 1- 3, wherein the polypeptide consists of any one of the polypeptide sequences selected from the group consisting of of SEQ ID No.: 1 - 31; and / or wherein the peptide is linear or cyclic; and / or wherein the antigenic fragment consists of at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12 amino acids in length.

5. The use according to anyone of the claims 1-4, wherein the polypeptide region is having an at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with thepolypeptide sequence as set forth in SEQ ID No. 13, and further wherein the polypeptide is cyclic.

6. An isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11 , 16-20 and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids, for use in preventing, treating or ameliorating Long COVID or Post-Vac-Syndrome.

7. The isolated polypeptide or an antigenic fragment thereof for use according to claim 6, wherein the polypeptide region is having at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 12-15, 21-22 and 29-31.

8. The isolated polypeptide or an antigenic fragment thereof for use according to claim 6 or 7, wherein the length of the polypeptide is in a range between 4 and 200 contiguous amino acids, or between 4 and 150 contiguous amino acids, or between 4 and 100 contiguous amino acids, or between 4 and 50 contiguous amino acids.

9. The isolated polypeptide for use according to any one of claims 6 - 8, wherein the polypeptide consists of any one of the polypeptide sequences selected from the group consisting of of SEQ ID No.: 1 - 31 ; and / or wherein the peptide is linear or cyclic; and / or wherein the antigenic fragment consists of at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12 amino acids in length.

10. The isolated polypeptide or antigenic fragment thereof for use according to anyone of the claims 6-9, wherein the polypeptide region is having an at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the polypeptide sequence as set forth in SEQ ID No. 13, and further wherein the polypeptide is cyclic.

11. The isolated polypeptide or antigenic fragment thereof for use according to any one of claims 6 - 10; wherein the use further comprises using the polypeptide or antigenic fragment thereof in adsorbing extracorporeally autoantibodies of a patient being diagnosed with the disease from blood or blood plasma of the patient.

12. The isolated polypeptide or antigenic fragment thereof for use according to any one of claims 6 -11, wherein the use is for preventing, treating or ameliorating Long COVID, or wherein the use is for preventing, treating or ameliorating Post-Vac Syndrome; and wherein the polypeptide region is having at least 60% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No. 6, 14, 15, 24, 29, 30 and 31.

13. The use according to any one of claims 1 - 5; further comprising the use of the isolated polypeptide or antigenic fragment thereof in a method for determining a presence of an antibody against SARS-CoV-2 in a sample of a subject, wherein the antibody against SARS-CoV-2 is capable of binding to the the isolated polypeptide or antigenic fragment thereof, preferably further wherein the antibody is capable of binding to the Spike protein of SARS-CoV-2.

14. The use according to any one of claims 1 - 5 or 13, wherein the use is for diagnosing long COVID-19; or wherein the use is for diagnosing Post-Vac Syndrome, and wherein the isolated polypeptide or antigenic fragment thereof comprises a polypetide region having at least 60% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No. 6, 14, 15, 24, 29, 30 and 31.

15. A method of detecting a presence of antibodies against SARS-CoV-2 in a subject, comprising(a) contacting a sample of said subject with at least one isolated polypeptide or an antigenic fragment thereof, comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group consisting of SEQ ID No.: 1-11 , 16-20 and 23-28; wherein the isolated polypeptide is having a length of no more than 200 contiguous amino acids;(b) detecting in said sample the presence of antibodies that specifically bind said polypeptide or antigenic fragment thereof;(c) comparing a detected amount or concentration of the antibody with a reference, wherein an increased amount of detected antibody in the sample of the subject compared to the reference is indicative for Long COVID or Post-Vac-Syndrome.

16. The method according to claim 15, wherein the reference is a reference for a healthy condition and / or for Long COVID or Post-Vac-Syndrome; and / or wherein the sample comprises serum, plasma, whole blood, saliva, cerebrospinal fluid, interstitial fluid or urine.

17. The method according to claim 15 or 16, wherein the sample is contacted with a plurality of the polypeptide or antigenic fragments thereof.

18. The method according to claim 17, wherein the sample is contacted with at least one of the isolated polypeptides or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 1-15; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID No. 16 - 31 ; or wherein the sample is contacted with the isolated polypeptide or antigenic fragment thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 16-22, and further wherein the sample is contacted with at least one of the isolated polypeptide orantigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 23 - 31 ; or wherein the sample is contacted with at least one of the isolated polypeptides or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 1-15; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragments thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 16-22; and wherein the sample is further contacted with at least one of the isolated polypeptide or antigenic fragment thereof comprising a polypeptide region having at least 60% sequence identity with any one of the polypeptide sequences selected from the group of SEQ ID. No. 23 - 31.

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