Hepatitis delta virus polypeptides and detection

Abstract

The present invention relates to a hepatitis delta virus (HDV) polypeptide comprising an antigenic domain comprising an amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 60% identical thereto, wherein said polypeptide (i) is devoid of a coiled-coil domain consisting of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 60% identical thereto; or (ii) further comprises said coiled-coil domain, wherein said coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan, and (II) the amino acid at position 50 is not tryptophan. Moreover, the present invention relates to proteins, kits, polynucleotides, host cells, methods, and devices related thereto.

Description

[0001] Hepatitis Delta Virus polypeptides and detection

[0002] Technical Field

[0003] The present invention relates to a hepatitis delta virus (HDV) polypeptide comprising an antigenic domain comprising an amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 60% identical thereto, wherein said polypeptide (i) is devoid of a coiled-coil domain consisting of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 60% identical thereto; or (ii) further comprises said coiled-coil domain, wherein said coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan, and (II) the amino acid at position 50 is not tryptophan. Moreover, the present invention relates to proteins, kits, polynucleotides, host cells, methods, and devices related thereto.

[0004] Background art

[0005] The hepatitis delta virus (HDV is a defective, spherical RNA virus, discovered in 1977 with the identification of a new antigen, the hepatitis delta antigen (HDAg). The virus can only cause infection in hepatocytes already infected with hepatitis B Virus (HBV), as it uses the surface protein of HBV (HBsAg) as its envelope protein (Le Gal et al. (2017) Hepatology, 66(6), 1826). HBsAg is required for assembly, release and transmission of HDV (Sureau & Negro (2016) Journal of Hepatology, 64(1), S102). The only viral protein produced by HDV is HDAg. It exists in two isoforms S- and L-HDAg (small and large HDAg, respectively). Both isoforms share 195 amino acids at the N-terminus, while L-HDAg is 19 amino acids longer. Within the viral life cycle, these two antigens have different roles. L-HDAg is necessary for virion packaging, whereas S-HDAg initiates the RNA replication of the virus (Niro et al. (2021) World Journal of Gastroenterology, 27(24), 3530).

[0006] HDAg is a RNA binding protein. It forms dimers and multimers, even if HDV RNA is absent. The multimerization activity is located in the first third of the protein, starting at the N-terminus. A coiled coil region (aa 16 to 48) stabilizes the formation of dimers. Dimers are further stabilized by a hydrophobic core region (aa 50 to 60), that also stabilizes interactions between dimers and therefore the building of octamers (Lin et al. (2010) Journal of Virology, 84(3), 1406). The last 35 amino acids, which are present in both isoforms, contain a proline and glycine rich region.

[0007] The presence of viral RNA, HDAg and antibodies against the two isoforms of HDAg can be used as diagnostic markers (Mauss et al. (2020) Hepatology - A clinical textbook (10th ed), 215). Frequently, the diagnosis of a HDV infection involves screening for serological markers, such as anti-HDV IgG and IgM. Clinical tests for anti-HDV antibodies are commercially available.

[0008] Problem to be solved

[0009] Despite the above, anti-HDV antibody assays are limited in sensitivity and / or specificity, making improved assays desirable.

[0010] This problem is addressed by the means, methods, and uses described herein, with the features of the independent claims. Advantageous embodiments which might be realized in an isolated fashion or in any arbitrary combinations are listed in the dependent claims as well as throughout the specification.

[0011] Detailed description and embodiments

[0012] In view of the above, the present invention relates to a hepatitis delta virus (HDV) polypeptide comprising an antigenic domain comprising an amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 60% identical thereto, wherein said polypeptide (i) is devoid of a coiled-coil domain consisting of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 60% identical thereto; or (ii) further comprises said coiled-coil domain, wherein said coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan, and (II) the amino acid at position 50 is not tryptophan.

[0013] The present invention also relates to a hepatitis delta virus (HDV) polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:l, 13, or 14. In general, terms used herein are to be given their ordinary and customary meaning to a person of ordinary skill in the art and, unless indicated otherwise, are not to be limited to a special or customized meaning. As used in the following, the terms "have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the expressions "comprising a" and "comprising an" in an embodiment refer to "comprising one or more", i.e. are equivalent to "comprising at least one" In accordance, expressions relating to one item of a plurality, unless otherwise indicated, in an embodiment relate to at least one such item, in a further embodiment a plurality thereof; thus, e.g. identifying "a cell" relates to identifying at least one cell, in an embodiment to identifying a multitude of cells. Also, the term "plurality" relates to a multitude, in an embodiment at least two, in a further embodiment at least three, in a further embodiment at least four, of the indicated items.

[0014] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

[0015] The methods specified herein below, in an embodiment, are in vitro methods. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but in an embodiment are performed in the indicated sequence; also, one or more, in an embodiment all, of said steps may be assisted or performed by automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned above.

[0016] The methods specified herein below, in an embodiment, provide data and / or information about the status of the indicated conditions and / or disease referred to herein, which provides, in an embodiment, a measure of a physiological state of a subject, which as such is not diagnostic of a disease. The data and / or information, however, may be an aid in diagnosing the indicated disease; as the skilled person will understand, establishing a diagnosis may be based on the aforesaid data and / or information in combination with further diagnostic information, such as anamnesis data, general physical and / or mental examination findings, and / or additional metabolic and / or other diagnostic data including in particular clinical chemistry data. Accordingly, the methods described herein may provide information useful for diagnosing the indicated diseases, for detecting deterioration or improvement of an indicated disease, and / or predicting any deterioration or improvement of an indicated disease in the subject, in particular, with respect to signs and symptoms accompanying said diseases. Moreover, it will be understood that if a prediction is made for a health state, typically, the prediction is made for a predictive time window, i.e. period of time for which the prediction is made. Such a predictive time window may e.g. be of from 1 week to 6 months, in a further embodiment of from four weeks to 2 months.

[0017] Basic statistic tools are known in the art. Thus, whether a portion or a feature of a population is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Details are found in textbooks, such as Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, in an embodiment, 0.05, 0.01, 0.005, or 0.0001.

[0018] As used herein, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, in an embodiment relates to the indicated value ± 20%, in a further embodiment ± 10%, in a further embodiment ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, in a further embodiment ± 10%, in a further embodiment ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. In an embodiment, a composition consisting essentially of a set of components will comprise less than 5% by weight, in a further embodiment less than 3% by weight, in a further embodiment less than 1% by weight, in a further embodiment less than 0.1% by weight of non-specified component(s).

[0019] The term “polynucleotide", as used herein, refers to a linear or circular nucleic acid molecule. The polynucleotide of the present invention shall be provided, in an embodiment, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form, in an embodiment comprising at least one heterologous sequence. The term polynucleotide encompasses single- as well as, partially or completely, double-stranded polynucleotides. In an embodiment, the polynucleotide is a DNA polynucleotide, which may also be referred to as "DNA“. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificially modified derivatives such as biotinylated polynucleotides, locked nucleic acids, peptide nucleic acids, and the like. The activity of the polynucleotide referred to herein is to encode a polypeptide as specified herein below.

[0020] Unless specifically indicated otherwise, reference to specific polynucleotides herein in an embodiment includes polynucleotide variants. The term “polynucleotide variant", as used herein, relates to a variant of a polynucleotide referred to herein comprising a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition and / or deletion, wherein the polynucleotide variant shall have the function and / or activity as specified for the specific polynucleotide. Thus, a variant of a polynucleotide may e.g. be an ortholog, a paralog, or another homolog of the specific polynucleotide; the polynucleotide variant may also be a mutant of the specific polynucleotide, in an embodiment a naturally occurring mutant, e.g. identified in a cancer cell. Also in an embodiment, said polynucleotide variant is or is derived from a non-naturally occurring allele of the specific polynucleotide. Further polynucleotide variants include polynucleotides comprising nucleic acid sequences which are at least 70%, in an embodiment at least 80%, in a further embodiment at least 90%, in a further embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%, identical to the specifically indicated nucleic acid sequences. The percent identity values are, in an embodiment, calculated over the entire nucleic acid sequence region, in an embodiment as specified herein elsewhere. The polynucleotides of the present invention either consist of, essentially consist of, or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well.

[0021] The term "protein" is understood by the skilled person; in an embodiment, the protein comprises at least one amino acid chain, i.e. a polypeptide as specified herein below, in a further embodiment comprises a multitude of polypeptides. Thus, the protein may be a multimer, e.g. a dimer, a trimer, or the like, wherein the polypeptides in the multimer may be connected covalently, e g. by at least one disulfide bridge, or non-covalently, e g. by ionic interactions, hydrophobic interactions, and / or van der Waals interactions. The protein may consist of identical polypeptides, e.g. may be a homodimer, a homotrimer, or the like, or may comprise at least two non-identical polypeptides, e g. may be a heterodimer, a heterotrimer, or the like. In a further embodiment, the protein as specified comprises all structural components as indicated comprised in one continuous covalent polypeptide chain, thus, the protein in an embodiment is a or is comprised in a fusion polypeptide. In accordance with common general use of the term, a protein comprises an ordered association of polypeptides, i.e. a protein typically comprises a pre-determined number or pre-determined numbers of polypeptides; thus, unordered aggregates comprising non-pre-determined numbers of polypeptides, as are formed e.g. during denaturation of polypeptides, are not proteins. In accordance, proteins in an embodiment are soluble proteins.

[0022] The term “polypeptide”, as used herein, refers to a molecule consisting of a multitude of amino acids that are covalently linked to each other by peptide bonds. Polypeptides consisting of less than 20 amino acids covalently linked by peptide bonds may also be referred to as "peptides". In an embodiment, the polypeptide comprises of from 25 to 1000, in a further embodiment of from 50 to 500, in a further embodiment of from 75 to 250, in a further embodiment of from 90 to 200 amino acids. The polypeptide may also be comprised in a fusion polypeptide, i.e. may comprise amino acid sequences in addition to those specifically indicated. Also, the polypeptide may comprise additional, non-peptidic structures, such as at least one glycosylation, lipid conjugation, and the like. Thus, unless specifically indicated otherwise, reference to specific polypeptides herein in an embodiment includes polypeptide variants.

[0023] As used herein, the term "polypeptide variant" relates to any chemical molecule comprising at least one polypeptide as specified herein differing in structure from a specifically indicated polypeptide and retaining the activity of the specifically indicated polypeptide. In an embodiment, the polypeptide variant comprises a polypeptide having a contiguous amino acid sequence corresponding to at least 70%, in an embodiment at least 80%, in a further embodiment at least 90%, in a further embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%, of the amino acid sequence of the polypeptide specifically indicated. Moreover, it is to be understood that a polypeptide variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still, in an embodiment, at least 60%, in a further embodiment at least 65%, in a further embodiment at least 68%, in a further embodiment at least 70%, in a further embodiment at least 75%, in a further embodiment at least 80%, in a further embodiment at least 85%, in a further embodiment at least 90%, in a further embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%, identical with the amino acid sequence of the specific polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and as described herein elsewhere. Polypeptide variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Moreover, the polypeptide variants referred to herein include fragments of the specific polypeptides or the aforementioned types of polypeptide variants, in an embodiment as long as these fragments and / or variants have the activity as specified. Such fragments may be or may be derived from, e.g., degradation products or splice variants of the polypeptides. Further included are variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation, or myristylation, by including non-natural amino acids, and / or by being peptidomimetics. The "activity" of a polypeptide as specified herein is, in an embodiment, preserved in the polypeptide variant; as the skilled person will understand in view of the description herein, the activity of a polypeptide does not necessarily have to reflect its main natural function, but may be a further activity relevant in the context of the claimed invention. On a strictly exemplary basis, the L-HDAg of HDV is necessary for virion packaging, whereas S-HDAg initiates the RNA replication of the virus polypeptide has an activity in cell cycle regulation, but the polypeptides referred to herein have the activity of being immunogenic, as specified herein below. The above applies to peptide variants and to protein variants mutatis mutandis.

[0024] The term "fragment" of a biological macromolecule, in an embodiment of a polynucleotide or polypeptide, is used herein in a wide sense relating to any sub-part of the respective biological macromolecule comprising the indicated sequence, structure and / or activity. Thus, the term includes sub-parts generated by actual fragmentation of a biological macromolecule, but also sub-parts derived from the respective biological macromolecule in an abstract manner, e.g. in silico. Thus, as used herein, an Fc or Fab fragment, but also e.g. a single-chain antibody, a bispecific antibody, and a nanobody may be referred to as fragments of an immunoglobulin.

[0025] The term "domain", as used herein, relates to a functionally and / or structurally defined subsection of a polypeptide. Thus, as referred to herein, a domain is covalently connected to another domain within a polynucleotide, typically via a peptide bond. I.e. in an embodiment, the domains of the polypeptides as specified herein are comprised in a continuous polypeptide.

[0026] Unless specifically indicated otherwise herein, the compounds specified, in particular the polynucleotides and polypeptides, may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further sequences.

[0027] The degree of identity (e.g. expressed as "%identity") between two biological sequences, in an embodiment DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. In an embodiment, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment. The percentage is calculated by determining, in an embodiment over the whole length of the polynucleotide or polypeptide as specified herein, the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (e g. BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are in an embodiment employed to determine their optimal alignment and, thus, the degree of identity. In an embodiment, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. In a further embodiment, the Basic Local Alignment Search Tool (BLAST) implementation is used with default parameter values for alignment. In the context of biological sequences referred to herein, the term "essentially identical" indicates a %identity value of at least 90%, in an embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%. As will be understood, the term essentially identical includes 100% identity. The aforesaid applies to the term "essentially complementary" mutatis mutandis.

[0028] The term "hepatitis delta virus", which may be abbreviated as "HDV", is known to the skilled person. In an embodiment, the term includes any and all viruses being members of the genus deltavirus. In an embodiment, the HDV is one of the HDVs described in Usman et al. (2020), Viruses 12:538. Sequences of HDV genomes are known in the art and are publicly available, e g. from Genbank or from the HDV database (hdvdb.bio.wzw.tum.de / hdvdb / ).

[0029] As used herein, the term "hepatitis delta virus polypeptide", which may also be referred to as "HDV polypeptide", relates to a polypeptide comprising at least a subsequence of the large hepatitis delta antigen (L-HDAg) as specified. Since there is considerable sequence variation between hepatitis delta antigens (HDAgs), reference is made herein to the consensus L-HDAg sequence described in WO 2019 / 219840 Al, which is SEQ ID NO:15 according to the present description. Numbering of amino acids of HDV polypeptides throughout this description is relative to said consensus sequence as a reference sequence; thus, reference to an amino acid sequence "corresponding to" an amino acid sequence of the reference sequence relates to amino acids identified as correlating with the indicated amino acids in an amino acid sequence alignment, wherein said alignment in an embodiment is prepared as specified herein above. Similarly, amino acid position numbering, as used herein, is in relation to the aforesaid consensus sequence provided as SEQ ID NO: 15. Since the consensus sequence has an additional amino acid at position 10 compared to e.g. the HDV1 consensus sequence, HD VI amino acids 10 to 194 are numbered as amino acids 11 to 195 according to the consensus sequence. The skilled person is able to provide an alignment of an amino acid sequence of an HDV polypeptide of interest to the aforesaid reference sequence without further ado. HDV polypeptides as specified may be comprised in fusion polypeptides comprising further domains, which may serve e.g. as a tag for purification and / or detection, as a linker, or the like. The term “detectable tag” refers to a stretch of amino acids which are added to or introduced into the fusion polypeptide and the presence of which can be detected; in an embodiment, the detectable tag is added C- or N- terminally to the fusion polypeptide. Said stretch of amino acids in an embodiment allows for detection of the polypeptide by an antibody which specifically recognizes the detectable tag; or it in an embodiment allows for forming a functional conformation, such as a chelator; or it in an embodiment allows for visualization, e.g. in the case of fluorescent tags. Detectable tags are known in the art, e g. the Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST-tag, or a fluorescent protein tag, e.g. a GFP-tag. Further domains which may be comprised in the HDV polypeptides described herein may be signal and / or transport domains, e.g. a bacterial export sequence, or further domains as described elsewhere herein. Additional further sequences which may be present in the polypeptides referred to herein are described herein below. The further domain in a fusion polypeptide may also be an enzymatic domain having an enzymatic property. The term "enzymatic property", as used herein, relates to a property of a polypeptide or domain thereof of producing a detectable product from a substrate by means of biological catalysis. Accordingly, an enzymatic property is typically conferred by the presence of a polypeptide domain having said enzymatic property in said indicator. Typically, the enzymatic property is at least one enzymatic activity selected from the group consisting of: phosphatase activity (e.g. in alkaline phosphatase), peroxidase activity (e.g. in horseradish peroxidase), and glycosidase activity (e.g. in beta-galactosidase). Typical substrates for enzymatic activities are well-known in the art. Typically, said enzymatic activity produces a product having a determinable optical property as specified herein below, or / and said enzymatic activity produces a product being determinable by an electrical instrument.

[0030] Unless specifically indicated otherwise herein, the compounds specified, in particular the polynucleotides and polypeptides, may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further, non-peptidic molecules, in particular to at least one indicator, surface binding moiety, adjuvant, carrier molecule, retardant, and / or other excipient.

[0031] The term "indicator", as used herein, relates to a compound adapted for making the presence of a molecule or complex comprising said indicator detectable; thus, a HDV polypeptide comprising an indicator may also be referred to herein as "detector compound" or "detection antigen", e.g. in the context of the methods described herein below. Typically, the indicator has a detectable property, typically an optical or / and enzymatic property. It is, however, also envisaged that said detectable property is the property of emitting radioactivity. The term "optical property", as used herein, relates to any property which can be detected by an optical instrument. Specifically, the optically determinable property may be or may comprise at least one property selected from the group consisting of: a reflection property, a transmission property, an emission property, a scattering property, a fluorescence property, a phosphorescence property, a diffraction property, and a polarization property. Further optical properties envisaged by the present invention are color, fluorescence, luminescence, or refraction. In an embodiment, an optically determinable property as referred to herein refers to a property of a chemical compound which can be optically detected such as light absorption, light emission, light remission, or properties associated therewith. It will be understood that detecting an optically determinable property as used herein encompasses the detection of the presence of a property which was not detectable before, the detection of the absence of a property which has been detected before, and the detection of quantitative changes of a property, i.e., the detection of the change of the signal strength which correlates to the extent of the change of the at least one optical property. It is understood that the term "optically determinable property", in an embodiment, also relates to electrochemiluminescence, which is also known as electrogenerated chemiluminescence. Thus, the HDV polypeptides referred to herein may in particular be covalently coupled to a complex comprising a Ruthenium ion, e g. a Tris(2,2'-bipyridyl)ruthenium(II)-complex.

[0032] In an embodiment, the HDV polypeptide referred to herein further comprises a surface binding moiety, i.e. is adapted to be bound to a solid surface; in such case, the HDV polypeptide may also be referred to as "capture compound" or "capture antigen", which may be used e.g. in a method as specified herein below. As referred to herein, the term "solid surface" relates to any suitable solid surface adapted for binding the surface binding moiety and adapted for being separated, e.g., by physical means, from a sample. In an embodiment, said solid surface is a surface of a bead, in an embodiment a microbead, e.g. a magnetic or paramagnetic microbead. In an embodiment, said surface is adapted to improve binding of the surface binding moiety, e.g. by attaching, covalently or non-covalently, molecules binding a substructure of the surface binding moiety. Typical molecules binding a substructure of a surface binding moiety are, e g. antibodies in case the surface binding moiety comprises a detectable tag, streptavidin in case the surface binding moiety comprises biotin, complexed heavy metal, e g. nickel, ions in case the surface binding moiety comprises a hexahistidine tag, and the like. In a further embodiment, the solid surface binds said surface binding moiety by covalent and / or non-covalent bonds, e.g. by hydrophobic interaction. Thus, in an embodiment, said solid surface is a surface of a multicluster plate. In an embodiment, the surface of the multi-cluster plate is pretreated to increase affinity and / or capacity for binding of a surface binding moiety. Suitable pretreatments are known in the art.

[0033] The HDV polypeptide referred to herein comprises an antigenic domain, wherein the term "antigenic domain" relates to a domain comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 60% identical to SEQ ID NO: 1; thus the antigenic domain may comprise, in an embodiment consist of, a polypeptide variant of SEQ ID NO:1 as specified herein above. Thus, the antigenic domain may also comprise an amino acid sequence corresponding to amino acids 61 to 160 of SEQ ID NO : 15 or be a polypeptide variant thereof as specified herein above. Also, the antigenic domain may comprise an amino acid sequence corresponding to amino acids 61 to 160 of any one of HDV Genotype 1 to 8 or an amino acid sequence at least 80 % identical thereto. In a preferred embodiment, the antigenic domain comprises, in an embodiment consists of, the amino acid sequence of any one of SEQ ID NO:32 to 38 or an amino acid sequence at least 80% identical thereto. In a further preferred embodiment, the antigenic domain comprises, in an embodiment consists of, the amino acid sequence of any one of SEQ ID NO:32 to 38.

[0034] As referred to herein, the antigenic domain, and thus the HDV polypeptide, has the activity of being immunogenic; therefore, an activity relevant for hepatitis D virus propagation does not have to, but may, be present in the HDV polypeptide. Thus, the antigenic domain is recognized by anti-HDV antibodies, in an embodiment anti-L-HDAg antibodies. As the skilled person is aware of, the antigenic domain referred to herein will not be recognized by each and every anti- HDV antibody. In an embodiment, the antigenic domain shall, however, be recognized by polyclonal anti-HDV sera. Thus, in an embodiment, the antigenic domain is recognized by essentially all serum samples from subjects which have been infected with HDV for at least 3 months. In a further embodiment, the antigenic domain has the activity of enabling detecting anti-HDV antibodies in a double-antigen immunoassay using a polypeptide or protein comprising or consisting of said antigenic domain as a bait in at least 90%, in an embodiment at least 95%, in a further embodiment at least 98% of samples from subjects which have been infected with HDV for at least 3 months. In an embodiment, the antigenic domain comprises a consensus amino acid sequence of hepatitis delta antigens of at least 3, in an embodiment at least 4, in a further embodiment at least 5, in a further embodiment at least 8, HDV strains or genotypes; in an embodiment, said consensus sequence is a consensus sequence of L-HDAg amino acid sequences corresponding to amino acids 61 to 160 of at least 3, in an embodiment at least 4, in a further embodiment at least 5, in a further embodiment at least 8, HDV strains or genotypes. In an embodiment, said consensus sequence comprises the amino acid sequence of SEQ ID NO:31. Thus, by using appropriate consensus sequences, the antigenic domain may be adapted to skew reactivity to antibodies against HDV strains having L-HDAg amino acid sequences corresponding to amino acids 61 to 160 of SEQ ID NO: 1 with less than 90%, in an embodiment less than 80%, in a further embodiment less than 70%, sequence identity to SEQ ID NO:1. Thus, in case detection of the relatively remote Genotype 3 shall be enhanced, e g. a consensus sequence from Genotype 3 strains may be used. Also, in case the HDV polypeptide of a protein comprising said HDV polypeptide comprising a multitude of antigenic domains is used, the antigenic domains may be non-identical. I.e., in a HDV polypeptide comprising four antigenic domains, two of said antigenic domains may comprise the amino acid sequence of SEQ ID NO:1, and two of said antigenic domains may comprise a consensus sequence as described herein above. Or, in a HDV protein comprising two HDV polypeptides, a first HDV polypeptide may comprise one or more antigenic domain(s) comprising the amino acid sequence of SEQ ID NO:1, and a second HDV polypeptide may comprise one or more domain(s) comprising a consensus sequence as described herein above.

[0035] The HDV polypeptide (i) is devoid of a coiled-coil domain consisting of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 60% identical thereto; or (ii) further comprises said coiled-coil domain, wherein said further coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan, and (II) the amino acid at position 50 is not tryptophan.

[0036] The term "coiled-coil domain", as used herein, relates to any domain comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 60% identical to SEQ ID NO:2. Thus, as referred to herein, the term coiled-coil domain relates to a domain having the indicated primary structure (i.e., amino acid sequence) annotated under the aforesaid designation in the art, without implying any secondary or tertiary structure and without implying any function or activity.

[0037] The term "is devoid of' is understood by the skilled person. In an embodiment, the term indicates that the indicated sequence(s) is / are not present in the HDV polypeptide. Thus, in an embodiment, neither the amino acid sequence of SEQ ID NO:2, nor any amino acid sequence at least 60% identical to SEQ ID NO:2 are present anywhere in the HDV polypeptide.

[0038] The HDV polypeptide may, however, comprise an amino acid sequence at least 60% identical to SEQ ID NO:2, i.e. may comprise a coiled-coil domain, provided that said coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan and (II) the amino acid at position 50 is not tryptophan; in an embodiment, the coiled-coil domain comprises both of mutations (I) and (II). In view of the description herein above, the skilled person understands that the expression "amino acid at position X" relates to the amino acid at position X in SEQ ID NO:2; thus, in case a comparison sequence of interest comprises one or more insertions and / or one of more deletions compared to SEQ ID NO:2, the amino acid position in the comparison sequence is the one corresponding to the indicated position in SEQ ID NO:2 as specified herein above, as determined e.g. by sequence alignment. In an embodiment, the coiled-coil domain comprises additional mutations compared to SEQ ID NO:2, in particular mutations that (III) the amino acid at position 4 is not serine and (IV) the amino acid at position 6 is not serine. In an embodiment, the coiled-coil domain comprises at least two of aforesaid mutations (I) to (IV), in a further embodiment comprises three of aforesaid mutations (I) to (IV), in a further embodiment comprises all four of aforesaid mutations (I) to (IV).

[0039] In case the HDV polypeptide comprises a coiled-coil domain as specified elsewhere herein, the amino acid at, i.e. corresponding to, position 4 is proline, glycine, alanine, valine leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine; and / or the amino acid at, i.e. corresponding to, position 6 is glycine, alanine, valine leucine, isoleucine, serine, proline, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine; and / or the amino acid at, i.e. corresponding to, position 20 is alanine, valine leucine, isoleucine, serine, glycine, proline, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine; and / or the amino acid at, i.e. corresponding to, position 50 is alanine, valine leucine, isoleucine, serine, glycine, proline, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine. In a further embodiment, the amino acid at, position 4 is proline, the amino acid at position 6 is glycine, the amino acid at position 20 is alanine, and / or the amino acid at position 50 is alanine. In a further embodiment, the amino acid at, position 4 is proline, the amino acid at position 6 is glycine, the amino acid at position 20 is alanine, and the amino acid at position 50 is alanine.

[0040] In accordance, the amino acid sequence of the coiled-coil domain, if present, in an embodiment comprises an amino acid sequence of any one of SEQ ID NOs:3 to 12, or an amino acid sequence at least 60% identical, or in an embodiment having one of the other %identity values specified herein above, to one of SEQ ID NOs:3 to 12, wherein said amino acid sequence comprises the aforesaid at least one mutation at position 20 and / or 50. In a further embodiment, the amino acid sequence of the coiled-coil domain, if present, comprises, in a further embodiment consists of, an amino acid sequence of any one of SEQ ID NOs:3 to 12 Thus, in an embodiment, the HDV polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 13, and / or 14, in an embodiment consists of the amino acid sequence of SEQ ID NO:1, 13, and / or 14.

[0041] It will be appreciated that SEQ ID NOs: 1, 13, and 14 comprise two mutations compared to SEQ ID NO: 15, i.e. mutations L90S and P159T. Thus, the HDV polypeptide may in particular be a HDV polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:l, 13, and / or 14 or a sequence at least 60% identical thereto, i.e. may be a polypeptide variant thereof as specified herein above comprising the aforesaid mutations. In a further embodiment, the HDV polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 13, and / or 14. In an embodiment, said amino acid sequence(s) of SEQ ID NO: 1, 13, and / or 14 are the only HDV specific amino acid sequences comprised in the HDV polypeptide, wherein the term "HDV specific amino acid sequence" relates to an amino acid sequence of at least ten continuous amino acids occurring exclusively in HDV. Thus, in an embodiment the HDV polypeptide does not comprise further amino acid sequences from the HDV antigen, in an embodiment does not comprise further amino acid sequences of at least ten continuous amino acids encoded by the HDV genome. In a further embodiment, the HDV polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 1, 13, and / or 14.

[0042] In an embodiment, the HDV polypeptide is devoid of a C-terminal domain of an HDAg, wherein said C-terminal domain is the C-terminal domain of the L-HDAg and / or the C-terminal domain of the L-HDAg. Thus, the HDV polypeptide may be devoid of an amino acid sequence shown in SEQ ID NO:21 and / or 22. In a further embodiment, the HDV polypeptide is devoid of an amino acid sequence shown in SEQ ID NO:21 and an amino acid sequence at least 60% identical thereto and / or is devoid of an amino acid sequence shown in SEQ ID NO: 22 and an amino acid sequence at least 60% identical thereto.

[0043] In an embodiment, the HDV polypeptide comprises at least one further domain, which may also be referred to as auxiliary domain. The term "auxiliary domain" is understood by the skilled person to relate to any arbitrary domain deemed desirable by the skilled person to be present in the HDV polypeptide; thus, the auxiliary domain may be e.g. a domain improving secondary and / or tertiary structure of the HDV polypeptide, e.g. a linker domain, a domain improving and / or simplifying production of the HDV polypeptide, e.g. a purification tag domain, a domain improving detection, e g. a detection tag domain, a domain mediating oligomerization, e g. an oligomerization domain, a domain improving solubility of the HDV polypeptide, e.g. a thioredoxin domain, or the like. In an embodiment, the auxiliary domain is selected from the list consisting of a linker domain, a purification tag domain, and an oligomerization domain. In an embodiment, the HDV polypeptide further comprises at least two, in a further embodiment three, in an embodiment non-identical, auxiliary domains selected from the list consisting of a linker domain, a purification tag domain, and an oligomerization domain.

[0044] The term "linker domain", for which also the term "linker" is used in the art, is known to the skilled person. In an embodiment, the term includes each and every amino acid sequence which provides for additional distance between two non-linker domains, wherein the amino acid sequence of the linker domain typically is selected to not assume a well-defined secondary structure and to provide for structural flexibility. Thus, a linker domain typically consists of a short amino acid sequence, such as of from 3 to 15, in an embodiment of from 5 to 10, amino acids, in an embodiment independently selected from glycine, serine, proline, and alanine, in an embodiment from glycine and serine. In an embodiment, the linker domain comprises, in a further embodiment consists of, the amino acid sequence GGGSGGG (SEQ ID NO: 16), (GGGS)nGGG (SEQ ID NO: 17), with n=2 to 10, in an embodiment n=5, and / or GGGSGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18).

[0045] The term "purification tag domain" is understood by the skilled person as well. The term in an embodiment includes each and every domain comprising an amino acid sequence enabling purification of the HDV polypeptide via affinity purification. Thus, the purification tag domain may comprise an amino acid sequence for which specific antibodies are available, e.g. a detectable tag as specified herein above, such as a myc-tag sequence, or a his-tag sequence; may comprise a metal chelator amino acid sequence, such as a his-tag, in particular H6 (SEQ ID NO: 19), a lectin sequence, a sequence of an Fc section of an antibody, which is bound by protein A or protein G, or the like. Appropriate purification tag domains are known in the art. In an embodiment, the purification tag domain comprises the amino acid sequence of SEQ ID NO:19.

[0046] The term "oligomerization domain" is used in its conventional meaning. The term in principle includes each and every polypeptide domain causing two polypeptides comprising said oligomerization domain to associate, i.e. to form oligomers. In an embodiment, the dissociation constant (Ka) of the resulting oligomers is at most 10'4mol / 1, in a further embodiment, at most 10'5mol / 1, in a further embodiment, at most 10'6mol / 1. As will be appreciated, the number of molecules in the resulting oligomer will in particular depend on the type of oligomerization domain selected. In an embodiment, the oligomers are homo-oligomers, in a further embodiment are homotrimers. Suitable oligomerization domains are known in the art, e.g. oligomerization domains from oligomeric chaperones, such as Skp and FkpA, oligomerization domains of C4-binding proteins, encapsulin oligomerization domains, ferritin oligomerization domains, and hybrid oligomerization domains of two different chicken C4-binding proteins, in particular an IMX313 oligomerization domain.

[0047] In an embodiment, the HDV polypeptide consists of at least one each of said antigenic domain, said linker domain, and said oligomerization domain; in a further embodiment, the HDV polypeptide consists of at least one each of said antigenic domain, said linker domain, said oligomerization domain, and said purification tag domain, in a further embodiment, the HDV polypeptide consists four copies of said antigenic domain, four copies of said linker domain, one copy of said oligomerization domain, and one copy of said purification tag domain. In an embodiment, the HDV polypeptide comprises, in an embodiment consists of, the amino acid sequence of any one of SEQ ID NO:23 to 30.

[0048] The HDV polypeptide may be a monomeric polypeptide; it is, however, also envisaged that the HDV polypeptide is covalently connected to at least one further molecule of said HDV polypeptide. I.e. a multitude of HDV polypeptides may be covalently connected; this may be accomplished e.g. by aldehyde or bisaldehyde cross-linking. Corresponding methods are known in the art.

[0049] The term "antibody", as used herein, includes any and all immunoglobulins known to the skilled person under this designation. Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al., Cellular and Mol. Immunology, 4thed., W.B. Saunders, Co. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0050] The term "anti-HDV antibody" is understood by the skilled person and in an embodiment relates to any antibody binding to a HDV polypeptide described herein. Said binding in an embodiment is strong enough to enable detection of anti-HDV antibody / HDV polypeptide complexes by a method deemed appropriate by the skilled person, e.g. in a double antigen immunoassay. In accordance, the dissociation constant (Ka) of anti-HDV antibody / HDV polypeptide complexes is at most 10'6mol / 1, in a further embodiment, at most 10'7mo 1 / 1, in a further embodiment, at most 10'8mol / 1. As the skilled person understands, in case the anti-HDV antibody is comprised in a mixture of anti-HDV antibodies, e.g. in a polyclonal serum, it is sufficient if a complex of one of the antibody types in said mixture with the HDV polypeptide described herein has the aforesaid dissociation constant. The antibody, in an embodiment, is a naturally occurring antibody, e.g. an antibody produced by a subject in response to a HDV infection. Also in an embodiment, the antibody is comprised in a mixture comprising a plurality of non-identical anti-HDV antibodies, e.g. a polyclonal serum. However, the antibody may, in principle also be a monoclonal antibody, a multispecific antibody, or an antibody fragment so long as it exhibits the aforesaid binding activity as specified elsewhere herein. In an embodiment, the antibody is an anti-L-HDAg antibody. In an embodiment, the antibody is HDV specific, i.e. binds the HDV polypeptide described herein with an affinity at least 5fold, in an embodiment at least lOfold, in a further embodiment at least 25fold, higher than any other component of the sample it is comprised in. In a further embodiment, the anti-HDV antibody is not a cross-reacting antibody. The term "subject", as used herein, relates to a mammal, preferably a human. In an embodiment, the subject is known or suspected to be infected with HDV. Symptoms leading to a suspicion of HDV infection are known in the art and include in particular fatigue, fever, loss of appetite, jaundice, nausea, stomach pain, dark urine, pale stool, diarrhea, and / or infection with hepatitis B virus.

[0051] In an embodiment, a multitude of HDV polypeptides forms a HDV protein. In accordance, the present invention also relates to a HDV protein comprising a multitude of HDV polypeptides as specified herein.

[0052] According to the present description, the term "HDV protein" relates to a protein as specified herein above comprising, in an embodiment consisting of, HDV polypeptides as specified herein. The HDV protein in an embodiment is a non-covalent oligomer of HDV polypeptides. In an embodiment, the HDV polypeptide is a homo-oligomer, i.e. consists of HDV polypeptides. The HDV polypeptide in an embodiment is an oligomer, in an embodiment a dimer, trimer, or tetramer, in a further embodiment a trimer In a further embodiment, the HDV protein is soluble, so the HDV may in particular be a soluble homotrimer of HDV polypeptides. In a further embodiment, the HDV protein comprises three copies of said HDV polypeptide, in an embodiment comprises 12 copies of said antigenic domain.

[0053] Advantageously, it was found in the work underlying the present invention that the HDV polypeptides described herein are particularly stable in solution and do not precipitate during storage. Thus, said HDV polypeptides are particularly suitable for use in diagnostic assays.

[0054] The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

[0055] The present invention also relates to a kit comprising an HDV polypeptide described herein and / or a HDV protein described herein, comprised in a housing.

[0056] The term “kit”, as used herein, refers to a collection of the aforementioned compounds, means or reagents which may or may not be packaged together. The components of the kit may be comprised by separate vials (i.e. as a kit of separate parts) or provided in a single vial, e.g. as a composition as specified herein above. The housing of the kit in an embodiment allows translocation of the compounds of the kit, in particular common translocation; thus, the housing may in particular be a transportable container comprising all specified components. Moreover, it is to be understood that the kit of the present invention may be used for practicing the methods referred to herein above. It is, in an embodiment, envisaged that all components are provided in a ready-to-use manner for practicing the methods referred to above. Further, the kit, in an embodiment, contains instructions for carrying out said methods. The instructions can be provided by a user's manual on paper or in electronic form. For example, the manual may comprise instructions for interpreting the results obtained when carrying out the aforementioned methods using the kit. In an embodiment, the kit is storable over extended periods of time, in an embodiment over a time frame of at least one week, in an embodiment at least two weeks, in a further embodiment at least one month, in a further embodiment at least two months, in a further embodiment at least six months, at a temperature of from 2°C to 8°C. In an embodiment, the HDV polypeptide or HDV protein comprised in said kit essentially does not aggregate during said storage periods, i.e. in an embodiment less than 5%, in an embodiment less than 1%, of the HDV polypeptide or HDV protein molecules comprised in the kit are present in a precipitate after the aforesaid time frames.

[0057] The present invention also relates to a HDV polynucleotide comprising a nucleic acid sequence encoding an HDV polypeptide as specified herein.

[0058] The term "polynucleotide" has been specified herein above. The HDV polynucleotide comprises a nucleic acid sequence encoding an HDV polypeptide as specified, i.e. the HDV polynucleotide has the activity of encoding the aforesaid HDV polypeptide. Appropriate HDV polynucleotide are provided by the skilled person based on a known amino acid sequence of an HDV polypeptide without further ado, e.g. taking into account the genetic code. In an embodiment, the polynucleotide is an expression construct and / or is comprised in a vector.

[0059] The term "expression construct", as used herein, relates to a polynucleotide comprising a nucleic acid sequence encoding a HDV polypeptide as specified herein, operatively linked to at least one expression control sequence causing transcription of the coding sequence to occur. In an embodiment, expression occurs in eukaryotic cells, prokaryotic cells, and / or isolated fractions thereof, in an embodiment into a translatable mRNA. Regulatory sequences for bacterial expression constructs, in particular promoters, such as the lac promoter, are known in the art. Regulatory elements ensuring expression in eukaryotic cells, in an embodiment mammalian cells, are also known in the art. They, in an embodiment, comprise regulatory sequences ensuring initiation of transcription, in particular at least one promoter, and, optionally, poly- A signals ensuring termination of transcription and stabilization of the transcript. In an embodiment, regulatory elements are cell- or tissue-type specific and / or are inducible, e.g. by administration of specific inducers. Preferred constitutive promoters are e.g. the CMV-, SV40-, or RSV (Rous sarcoma virus)-promoter, CMV-enhancer, or SV40-enhancer. Additional regulatory elements may include transcriptional as well as translational enhancers.

[0060] In an embodiment, the polynucleotide is comprised in a vector. The term “vector”, in an embodiment, encompasses phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes, including other autonomously replicating polynucleotides. Moreover, the term also relates to targeting constructs which allow for random or site-directed integration of the targeting construct into genomic DNA. Such targeting constructs, in an embodiment, comprise DNA of sufficient length for either homologous or heterologous recombination. The vector encompassing the HDV polynucleotide as specified herein, in an embodiment, further comprises at least one selectable marker for propagation and / or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens. Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing hosts and / or cells. In a further embodiment, in the vector, the HDV polynucleotide is operatively linked to expression control sequences as specified herein above. Methods used to construct recombinant polynucleotides, expression constructs, or vectors are well known to those skilled in the art from standard text books.

[0061] The present invention also relates to a host cell comprising the HDV polypeptide as specified herein and / or the HDV polynucleotide as specified herein. As used herein, the term "host cell" relates to any cell capable of producing aHDV polypeptide as specified; thus, the host cell in an embodiment is capable of receiving and maintaining an expression polynucleotide and / or an expression vector as specified herein. In an embodiment, the host cell is a bacterial cell, in a further embodiment a cell of a common laboratory bacterial strain known in the art, in an embodiment an Escherichia strain, in particular an E. coli strain. In a further embodiment, the host cell is a eukaryotic cell, in an embodiment a fungal, e.g. yeast cell, or is an animal cell. In an embodiment, the host cell is an insect cell or a mammalian cell, in particular a human, mouse, or rat cell. In a further embodiment, the host cell is a mammalian cell, e g. a 293 human embryonic kidney (HEK) cell.

[0062] The present invention also relates to a diagnostic composition comprising an HDV polypeptide as specified herein and / or a HDV protein as specified herein and an excipient, wherein said excipient may in particular be a diluent and / or a detection reagent, such as an ECL reagent.

[0063] The present invention also relates to an HDV polypeptide as specified herein and / or a HDV protein as specified herein for use in diagnosing a hepatitis D vims (HDV) infection in a sample of a subject.

[0064] The present invention also relates to a HDV protein as specified herein, a HDV polynucleotide as specified herein, and / or a host cell as specified herein, in the manufacture of a diagnostic composition or kit for the detection of a hepatitis D vims (HDV) infection.

[0065] The term “diagnosing” as used herein refers to assessing whether a subject suffers from the indicated disease or condition, or not. As will be understood by those skilled in the art, such an assessment, although preferred to be, may usually not be correct for 100% of the investigated subjects. The term, however, requires that a statistically significant portion of subjects can preferably be correctly assessed and, thus, diagnosed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools as specified herein above. The term includes individual diagnosis of HDV infection or its symptoms as well as monitoring of a patient. Monitoring, i.e. diagnosing the presence or absence of HDV infection at various time points, includes monitoring of patients known to suffer from HDV infection as well as monitoring of subjects known to be at risk of developing HDV infection. Furthermore, monitoring can also be used to determine whether a patient is treated successfully against HDV infection. As indicated herein above, the aforesaid, as the case may be, may require additional assessments in order to lead to a clinical diagnosis.

[0066] The present invention further relates to a method for determining anti-hepatitis D virus (HDV) antibodies in a sample, comprising

[0067] (a) contacting said sample with an HDV polypeptide as specified herein and / or a HDV protein as specified herein;

[0068] (b) determining complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein; and

[0069] (c) thereby determining anti-HDV antibodies in said sample.

[0070] The method comprises step (a) contacting said sample with an HDV polypeptide as specified herein and / or a HDV protein. The term “sample”, as used herein, relates to a composition of matter known or suspected to comprise anti-HDV antibodies. In an embodiment, the sample is or comprises a sample of a body fluid, a sample from a tissue or an organ, or a sample of wash / rinse fluid or a swab or smear obtained from an outer or inner body surface. Body fluid samples include samples of blood, plasma, serum, urine, saliva, and lacrimal fluid. Samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse / wash fluids, punch biopsy devices, puncture of cavities with needles or lancets, or by surgical instrumentation. However, samples obtained by well-known techniques including, in an embodiment, scrapes, swabs or biopsies from the urogenital tract, perianal regions, anal canal, or the oral cavity, are also included as samples. Cell-free fluids may be obtained from the body fluids or the tissues or organs by lysing techniques such as homogenization and / or by separating techniques such as filtration or centrifugation. It is to be understood that the sample may be further processed in order to carry out the method. Particularly, cells may be removed from the sample by methods and means known in the art, calcium ions may be complexed to prevent coagulation, coagulation may be induced, and the like. Moreover, anti-HDV antibodies may be enriched, extracted, and / or purified from the sample by methods and means known in the art. Thus, the term sample also may relate to preparations comprising or suspected to comprise anti-HDV antibodies which are diluted, enriched, purified and / or extracted from a sample.

[0071] The term “contacting” as used in the context of the methods specified is understood by the skilled person. In an embodiment, the term relates to bringing at least one compound in physical contact with a sample and / or with a further compound and thereby, e.g. allowing the sample and the compound to interact. In particular, the term relates to bringing a HDV polypeptide and / or a HDV protein in physical contact with a sample. Step (a) may comprise bringing the aforesaid compounds into contact with further compounds and elements, depending on the assay format chosen; e g. in assays relying on removal of non-complexed sample constituents via binding of complexes to a solid surface, the admixture may be further contacted with a solid surface.

[0072] The method comprises further step (b) determining complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein. The term "complex" is understood by the skilled person to relate to the complex formed in step (a) relevant for determining anti-HDV antibodies. Thus, in an embodiment the complex is a complex comprising at least anti-HDV antibodies and the HDV polypeptide or HDV protein. The complex may form at any time during the method before step (b), and may form sequentially or in one step. Sequential complex formation may be induced by providing a capture compound (i.e. a HDV polypeptide or HDV protein comprising a surface binding moiety) bound to a solid surface, admixing a sample, optionally washing away non-bound components of the sample, and adding a detection compound (i.e. a HDV polypeptide or HDV protein comprising an indicator), in an embodiment followed by a further washing step. One-step complex formation may be induced e.g. by admixing a capture compound which is optionally bound to a solid surface, e g. a bead, a sample, and a detection compound, optionally followed by washing away all compounds which are not bound directly or indirectly to said solid surface. For determining anti-HDV antibodies, in an embodiment the amount of the aforesaid complex formed in step (a) is determined. In an embodiment, liquid phase and solid phase components are separated from each other prior to determining the anti-HDV antibodies in any of the liquid or solid phase or both. In an embodiment, the amount is determined directly by determining the amount of complexes comprising the binding compound and the anti-HDV antibodies; in an embodiment by determining the amount of indicator compound comprised in complexes comprising a capture compound, the anti-HDV antibodies, and a detection compound.

[0073] The method further comprises step (c) thereby determining anti-HDV antibodies in said sample. As used herein, the term "determining" includes establishing whether complexes comprising anti-HDV antibodies are present or absent in a sample at a concentration above the detection limit of the method, i.e., in an embodiment, the determining is qualitative. Methods of determining a detection limit are known to the skilled person. In a further embodiment, determining is determining semi-quantitatively or quantitatively the amount or concentration of HDV antibodies in a sample by means of complexes comprising anti-HDV antibodies. For semi-quantitative determining, the amount may be assigned e.g. to two or more pre-defined categories, e.g. above or not above a reference value, or low, medium, or high. For quantitative determination, either the absolute or precise amount of the HDV antibodies will be determined or the relative amount of the HDV antibodies will be determined. The relative amount may be determined in a case were the precise amount of an HDV antibodies can or shall not be determined. In said case, it can be determined whether the amount in which the HDV antibodies is present is increased or diminished with respect to a reference sample comprising said HDV antibodies in a pre-determined amount. For quantitative determination, any parameter correlating with the amount or concentration of complexes comprising anti-HDV antibodies in the admixture of step (a) or any value derived therefrom by standard mathematical and / or evaluation operations, including in particular multiplication, division, reciprocal formation, scaling, normalization, standardization, error correction, background correction, or mean or median calculation, may be determined and / or output.

[0074] The present invention also relates to a method of diagnosing a hepatitis D virus (HDV) infection in a sample of a subject, comprising

[0075] (A) contacting said sample with an HDV polypeptide described herein and / or a HDV protein described herein;

[0076] (B) determining complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein; and

[0077] (C) based on the determining in step (B), diagnosing a HDV infection in said subject.

[0078] The method comprises step (C) based on the determining in step (B), diagnosing a HDV infection in said subject. As the skilled person understands in view of the description herein, HDV infection typically is diagnosed in case complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein are detected, in an embodiment above the detection limit of the method. Thus, presence of anti-HDV antibodies in a sample of a subject is taken as an indication that the subject is infected with HDV. Since HDV infection essentially may resolve spontaneously, presence of anti-HDV antibodies in a sample of a subject is taken as an indication of acute or past infection. Steps (A) and (B) of the aforesaid method essentially correspond to steps (a) and (b) of the method for determining anti-HDV antibodies described herein above. Thus, a method of diagnosing a hepatitis D virus (HDV) infection in a sample of a subject may also comprise steps (a) to (c) of the method for determining anti-HDV antibodies described herein above, followed by step (Cl) based on the determining anti-HDV antibodies in step (c), diagnosing a HDV infection in said subject.

[0079] The aforesaid method may also comprise a further step (D) treating said HDV infection in said subject in case a HDV infection is diagnosed in step (C). Thus, the method may be a method for diagnosing and treating a HDV infection in a subject.

[0080] The terms "treating" and “treatment” refer to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, as specified herein above. Exemplary treatments envisaged include administration of interferon-a, Bulevirtide (CAS NO. 2012558-47-1), phosphor othioate nucleic acid polymers, such as Rep2139, and / or a farnesyltransferase inhibitor, such as Lonafamib (CAS No 193275- 84-2.

[0081] The present invention also relates to a use of an HDV polypeptide described herein, and / or a HDV protein described herein for detecting anti-hepatitis D virus (HDV) antibodies in a sample. Said use, in an embodiment, is an in vitro use.

[0082] Moreover, the present invention also relates to a device for detecting an anti-hepatitis D virus (HDV) antibody in a sample, said device comprising an analyzing unit with a sample treatment unit, said analyzing unit being adapted to perform the following steps:

[0083] (a) contacting a sample applied to said sample treatment unit with an HDV polypeptide described herein and / or a HDV protein described herein, and

[0084] (b) detecting an anti-HDV antibody in said sample.

[0085] The term “device”, as used herein, relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the result of the detection to be obtained. Preferred means for contacting a sample with reagents as described herein are known in the art and are disclosed above in connection with the methods of the invention. How to link the means in an operating manner will depend on the type of means included into the device. In a simple embodiment, the device may be a lateral flow test strip. In an embodiment, the means are comprised by a single device. In an embodiment, the device comprises a controller unit configured to control the units of the analyzing unit to perform the analytical steps of a method described herein, i.e. at least the contacting and determining steps of the methods described herein above.

[0086] In an embodiment, the sample treatment unit comprises a receptacle for a sample. The receptacle may directly contact the sample, or may be a receptacle for a further means receiving the sample, wherein the further means may be e.g. a multi-well plate, to which a sample or a plurality of samples may be applied. Moreover, the sample treatment unit, in an embodiment, comprises at least one, in an embodiment all of, a solution comprising a HDV polypeptide and / or a HDV protein described herein, a positive control solution and a negative control solution, e g. in separate reservoirs connected to a dosing means, e g. a tubing connected to a pump. In a further embodiment, the device comprises a calibrator stock solution having a predetermined concentration of anti-HDV antibodies. In a further embodiment, the sample treatment unit comprises means for mixing and means for adjusting the temperature of a reaction mixture.

[0087] In an embodiment, the result of the determination is obtained by performing a detection measurement on an appropriate detection unit. Thus, in an embodiment, the analyzing unit of the device of the present invention further comprises a detection unit for detecting a measureable, e g. an optical, property of an admixture of the sample with a capture compound and / or a detector compound, both as specified herein above. Means suitable as a detection unit according to the present invention are known to the skilled person and include, e.g. photometric devices.

[0088] In an embodiment, the device of the present invention further comprises an evaluation unit or is part of an analytic system, said analytic system further comprising an evaluation device. As will be understood by the skilled person, the evaluation means may be comprised in the same housing as the device of the invention as an evaluation unit, or may be a separate device, i.e. an evaluation device. In an embodiment, the evaluation unit or device comprises a microprocessor programmed to receive output data from an output unit of the analyzing unit of the present invention and to perform logical operations providing an evaluation of said output data.

[0089] In an embodiment, the device of the present invention further comprises a data output unit, connected to the analysis unit and / or to the evaluation unit or device. The data output unit, in an embodiment, is adapted to output data obtained by the detection unit or generated by the evaluation unit or device. Suitable data output units are known to the skilled person and include simple output units such as an indicator lamp or a display. An output unit may, however, also be an interface to an output device, wherein said interface may be any kind of means of transferring data, including, e.g. cable connections like USB, wireless connections like wireless LAN, bluetooth, and the like, or indirect connections such as data transfer by instant messaging, email, or the like.

[0090] In an embodiment, where means for automatically detecting anti-HDV antibodies are applied, the data obtained by said automatically operating means can be processed by, e.g., a computer program in order to establish or aid in establishing a diagnosis. Typical means for detection are disclosed in connection with embodiments relating to the methods described herein above. In such a case, the means are operatively linked in that the user of the system brings together the result of the determination of the amount and the diagnostic value thereof due to the instructions and interpretations given in a manual. The person skilled in the art will realize how to link the means without further inventive skills. Typical devices are those which can be applied without the particular knowledge of a specialized clinician, e.g., electronic devices which merely require loading with a sample. The results may be given as output of parametric diagnostic raw data, in embodiments, as absolute or relative amounts. It is to be understood that these data may need interpretation by the clinician. However, also envisaged are expert system devices wherein the output comprises processed diagnostic raw data the interpretation of which does not require a specialized clinician.

[0091] The invention further discloses and proposes a computer program including computerexecutable instructions for performing the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one, more than one or even all of method steps a) to d) as indicated above may be performed by using a computer or a computer network, in an embodiment by using a computer program.

[0092] The invention further discloses and proposes a computer program product having program code means, in order to perform the method according to the present invention in one or more of the embodiments enclosed herein when the program is executed on a computer or computer network. Specifically, the program code means may be stored on a computer-readable data carrier.

[0093] Further, the invention discloses and proposes a data carrier having a data structure stored thereon, which, after loading into a computer or computer network, such as into a working memory or main memory of the computer or computer network, may execute the method according to one or more of the embodiments disclosed herein.

[0094] The invention further proposes and discloses a computer program product with program code means stored on a machine-readable carrier, in order to perform the method according to one or more of the embodiments disclosed herein, when the program is executed on a computer or computer network. As used herein, a computer program product refers to the program as a tradable product. The product may generally exist in an arbitrary format, such as in a paper format, or on a computer-readable data carrier. Specifically, the computer program product may be distributed over a data network.

[0095] Finally, the invention proposes and discloses a modulated data signal which contains instructions readable by a computer system or computer network, for performing the method according to one or more of the embodiments disclosed herein.

[0096] In an embodiment, referring to the computer-implemented aspects of the invention, one or more of the method steps or even all of the method steps of the method according to one or more of the embodiments disclosed herein may be performed by using a computer or computer network. Thus, generally, any of the method steps including provision and / or manipulation of data may be performed by using a computer or computer network. Generally, these method steps may include any of the method steps, typically except for method steps requiring manual work, such as providing the samples and / or certain aspects of performing the actual measurements. Specifically, the present invention further discloses:

[0097] A computer or computer network comprising at least one processor, wherein the processor is adapted to perform the method according to one of the embodiments described in this description, a computer loadable data structure that is adapted to perform the method according to one of the embodiments described in this description while the data structure is being executed on a computer, a computer program, wherein the computer program is adapted to perform the method according to one of the embodiments described in this description while the program is being executed on a computer, a computer program comprising program means for performing the method according to one of the embodiments described in this description while the computer program is being executed on a computer or on a computer network, a computer program comprising program means according to the preceding embodiment, wherein the program means are stored on a storage medium readable to a computer, a storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform the method according to one of the embodiments described in this description after having been loaded into a main and / or working storage of a computer or of a computer network, and a computer program product having program code means, wherein the program code means can be stored or are stored on a storage medium, for performing the method according to one of the embodiments described in this description, if the program code means are executed on a computer or on a computer network.

[0098] In view of the above, the following embodiments are particularly envisaged:

[0099] Embodiment 1 : A hepatitis delta virus (HDV) polypeptide comprising an antigenic domain comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 60% identical thereto, wherein said polypeptide (i) is devoid of a coiled-coil domain consisting of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 60% identical thereto; or (ii) further comprises said coiled-coil domain, wherein said coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan and (II) the amino acid at position 50 is not tryptophan.

[0100] Embodiment 2: The HDV polypeptide of embodiment 1, wherein the amino acid at position 4 of the amino acid sequence of said coiled-coil domain is proline, glycine, alanine, valine leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine; wherein the amino acid at position 6 of the amino acid sequence of said coiled-coil domain is glycine, alanine, valine leucine, isoleucine, serine, proline, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine; wherein the amino acid at position 20 of the amino acid sequence of said coiled-coil domain is alanine, valine leucine, isoleucine, serine, glycine, proline, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine; and / or wherein the amino acid at position 50 of the amino acid sequence of said coiled-coil domain is alanine, valine leucine, isoleucine, serine, glycine, proline, threonine, aspartic acid, asparagine, glutamic acid, glutamine, or methionine.

[0101] Embodiment 3 : The HDV polypeptide of embodiment 1 or 2, wherein the amino acid at position 4 of the amino acid sequence of said coiled-coil domain is proline, the amino acid at position 6 is glycine, the amino acid at position 20 is alanine, and / or the amino acid at position 50 is alanine.

[0102] Embodiment 4: The HDV polypeptide of any one of embodiments 1 to 3, wherein the amino acid sequence of said coiled-coil domain comprises an amino acid sequence of any one of SEQ ID NOs:3 to 12, or an amino acid sequence at least 60% identical to one of SEQ ID NOs:3 to 12.

[0103] Embodiment 5: The HDV polypeptide of any one of embodiments 1 to 4, wherein the amino acid sequence of said coiled-coil domain comprises an amino acid sequence of any one of SEQ ID NOs: 3 to 12.

[0104] Embodiment 6: The HDV polypeptide of any one of embodiments 1 to 5, wherein said

[0105] HDV polypeptide comprises the sequence of any one of SEQ ID NOs: 1, 13, or 14, or a sequence at least 60%, in an embodiment at least 68%, in a further embodiment at least 80%, in a further embodiment at least 90%, in a further embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%, identical thereto.

[0106] Embodiment 7: The HDV polypeptide of any one of embodiments 1 to 6, wherein said

[0107] HDV polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:l, 13, or 14. Embodiment s: A hepatitis delta virus (HDV) polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 13, or 14.

[0108] Embodiment 9: The HDV polypeptide of any one of embodiments 1 to 8, wherein said

[0109] HDV polypeptide further comprises at least one auxiliary domain selected from the list consisting of a linker domain, a purification tag domain, and an oligomerization domain, in an embodiment further comprises at least two auxiliary domains selected from the list consisting of linker domain, a purification tag domain, and an oligomerization domain.

[0110] Embodiment 10: The HDV polypeptide of any one of embodiments 1 to 9, wherein said

[0111] HDV polypeptide consists of at least one each of said antigenic domain, said linker domain, and said oligomerization domain.

[0112] Embodiment 11: The HDV polypeptide of any one of embodiments 1 to 10, wherein said

[0113] HDV polypeptide consists of at least one each of said antigenic domain, said linker domain, said oligomerization domain, and said purification tag domain.

[0114] Embodiment 12: The HDV polypeptide of any one of embodiments 1 to 11, wherein said

[0115] HDV polypeptide consists four copies of said antigenic domain, four copies of said linker domain, one copy of said oligomerization domain, and one copy of said purification tag domain. Embodiment 13: The HDV polypeptide of any one of embodiments 1 to 12, wherein said

[0116] HDV polypeptide forms a protein comprising three copies of said HDV polypeptide, in an embodiment wherein said protein comprises 12 copies of said antigenic domain.

[0117] Embodiment 14: The HDV polypeptide of any one of embodiments 1 to 13, wherein said linker domain is a linker domain comprising the amino acid sequence of SEQ ID NO: 16, in a further embodiment consisting of the amino acid sequence of SEQ ID NO: 16.

[0118] Embodiment 15: The HDV polypeptide of any one of embodiments 1 to 14, wherein said purification tag domain is a his-tag domain, in an embodiment comprising the amino acid sequence of SEQ ID NO: 19, in a further embodiment consisting of the amino acid sequence of SEQ ID NO: 19.

[0119] Embodiment 16: The HDV polypeptide of any one of embodiments 1 to 15, wherein said oligomerization domain is a chaperone domain, in an embodiment an Skp domain, in an embodiment comprising the amino acid sequence of SEQ ID NO:20, in a further embodiment consisting of the amino acid sequence of SEQ ID NO:20.

[0120] Embodiment 17: The HDV polypeptide of any one of embodiments 1 to 16, wherein said antigenic domain comprises a consensus amino acid sequence of hepatitis delta antigens of at least 3, in an embodiment at least 4, in a further embodiment at least 5, in a further embodiment at least 8, HDV strains or genotypes.

[0121] Embodiment 18: The HDV polypeptide of any one of embodiments 1 to 17, wherein said polypeptide consists of the amino acid sequence of SEQ ID NO: 1, 13, or 14, or an amino acid sequence at least 80%, in an embodiment at least 90%, in a further embodiment at least 95%, in a further embodiment at least 98%, in a further embodiment at least 99%, identical to SEQ ID NO l, 13, or 14.

[0122] Embodiment 19: The HDV polypeptide of any one of embodiments 1 to 18, wherein said

[0123] HDV polypeptide consists of the amino acid sequence of SEQ ID NO:1, 13, or 14.

[0124] Embodiment 20: The HDV polypeptide of any one of embodiments 1 to 19, wherein said

[0125] HDV polypeptide comprises, in an embodiment consists of, the amino acid sequence of SEQ ID NO:25 to 30, in an embodiment SEQ ID NO:26, 27, 29, or 30, in a further embodiment SEQ ID NO: 29 or 30.

[0126] Embodiment 21: The HDV polypeptide of any one of embodiments 1 to 20, covalently connected to at least one further molecule of said HDV polypeptide.

[0127] Embodiment 22: A HDV protein comprising a multitude of HDV polypeptides according to any one of embodiments 1 to 20.

[0128] Embodiment 23: The HDV protein of embodiment 22, wherein said protein essentially consists of said multitude of HDV polypeptides.

[0129] Embodiment 24: The HDV protein of embodiment 22 or 23, wherein said protein is an oligomer of HDV polypeptides comprising at least one oligomerization domain per HDV polypeptide.

[0130] Embodiment 25: The HDV protein of embodiment 24, wherein said oligomerization domain is a Skp domain.

[0131] Embodiment 26: A kit comprising an 1TDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25 comprised in a housing.

[0132] Embodiment 27: The kit of embodiment 26, wherein said kit comprises (i) a HDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25 as a detector compound; and / or (ii) a HDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25 as a capture compound.

[0133] Embodiment 28: The kit of embodiment 26 or 27, wherein said kit comprises monomeric species of the HDV polypeptide according to any one of embodiments 1 to 21 and comprises the HDV protein according to any one of embodiments 22 to 25.

[0134] Embodiment 29: The kit of any one of embodiments 26 to 28, wherein said kit is a kit for detecting anti-hepatitis D virus antibodies in a sample.

[0135] Embodiment 30: The kit of any one of embodiments 26 to 29, wherein said HDV polypeptide or HDV protein essentially does not aggregate over a time frame of at least one week, in an embodiment at least two weeks, in a further embodiment at least one month, in a further embodiment at least two months, in a further embodiment at least six months, at a temperature of from 2°C to 8°C.

[0136] Embodiment 31: A HDV polynucleotide comprising a nucleic acid sequence encoding an

[0137] HDV polypeptide according to any one of embodiments 1 to 20.

[0138] Embodiment 32: The HDV polynucleotide of embodiment 32, comprised in an expression construct.

[0139] Embodiment 33: The HDV polynucleotide of embodiment 32 or 33, comprised in a vector.

[0140] Embodiment 34: A host cell comprising the HDV polypeptide according to any one of embodiments 1 to 20 and / or the HDV polynucleotide according to any one of embodiments 31 to 33.

[0141] Embodiment 35: A diagnostic composition comprising an HDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25 and an excipient.

[0142] Embodiment 36: An HDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25 for use in diagnosing a hepatitis D virus (HDV) infection in a sample of a subject

[0143] Embodiment 37: Use of a HDV polypeptide according to any one of embodiments 1 to 21, a HDV protein according to any one of embodiments 8 to 10, a HDV polynucleotide according to any one of embodiments 22 to 25, and / or a host cell according to embodiment 34, in the manufacture of a diagnostic composition or kit for the detection of a hepatitis D virus (HDV) infection.

[0144] Embodiment 38: A method for determining anti-hepatitis D virus (HDV) antibodies in a sample, comprising

[0145] (a) contacting said sample with an HDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25;

[0146] (b) determining complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein; and

[0147] (c) thereby determining anti-HDV antibodies in said sample.

[0148] Embodiment 39: A method of diagnosing a hepatitis D virus (HDV) infection in a sample of a subj ect, comprising

[0149] (A) contacting said sample with an HDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25;

[0150] (B) determining complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein; and (C) based on the determining in step (B), diagnosing a HDV infection in said subject. Embodiment 40: A method for diagnosing and treating a hepatitis D virus (HDV) infection in a subject, comprising steps (A) to (C) of the method of embodiment 39; and further step a. (D) treating said HDV infection in said subject in case a HDV infection is diagnosed in step (C).

[0151] Embodiment 41: The method of embodiment 40, wherein said treating comprises administration of interferon-u, Bulevirtide (CAS NO. 2012558-47-1), Rep2139, and / or Lonafamib (CAS No. 193275-84-2).

[0152] Embodiment 42: Use of an HDV polypeptide according to any one of embodiments 1 to

[0153] 21, and / or a HDV protein according to any one of embodiments 22 to 25 for detecting antihepatitis D virus (HDV) antibodies in a sample.

[0154] Embodiment 43 : The use of embodiment 42, wherein said use is an in vitro use.

[0155] Embodiment 44: A device for detecting an anti-hepatitis D virus (HDV) antibody in a sample, said device comprising an analyzing unit with a sample treatment unit, said analyzing unit being adapted to perform the following steps:

[0156] (a) contacting a sample applied to said sample treatment unit with an FIDV polypeptide according to any one of embodiments 1 to 21 and / or a HDV protein according to any one of embodiments 22 to 25, and

[0157] (b) detecting an anti-HDV antibody in said sample.

[0158] Embodiment 45: The subject matter of any one of embodiments 29 to 44, wherein said anti-HDV antibodies are IgG and / or IgM.

[0159] Embodiment 46: The subject matter of any one of embodiments 36 to 45, wherein said subject is a human.

[0160] Embodiment 47: The subject matter of any one of the preceding embodiments, wherein the antigenic domain comprises, in an embodiment consists of, the amino acid sequence of any one of SEQ ID NO:32 to 38 or an amino acid sequence at least 80% identical thereto, in a further embodiment comprises, in an embodiment consists of, the amino acid sequence of any one of SEQ ID NO:32 to 38.

[0161] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0162] Figure Legend Fig. 1 : Schematic representation of exemplary HDV polypeptides according to the invention: (A) the HDV polypeptides may be recombinant HDV polypeptides and may in particular comprise a C-terminal His-tag domain (not shown); from top to bottom: L-HDAg(l-214, C / A; SEQ ID NO:23), S-HDAg(l-195; SEQ ID NO:24), L-HDAg(l-214, W+C / A; SEQ ID NO 25), S-HDAg(l-195, W / A; SEQ ID NO:26), HDAg(l-160, W / A; SEQ ID NO:27), HDAg(61-214, C / A; SEQ ID NO:28), HDAg(61-160; SEQ ID NO:29); C / A: comprises a C211A mutation; W / A: comprises W20A and W50A mutations, W+C / A: comprises C211A, W20A, and W50A mutations; (B) schematic representation of an exemplary HDV polypeptide forming HDV protein molecules: O: oligomerization domain, e.g. SEQ ID NO:20; LL: long linker, e.g. SEQ ID NO: 18, AG: antigenic domain, e.g. SEQ ID NO:1, L: short linker, e.g. SEQ ID NO: 16; H:his-tag, e.g. SEQ ID NO: 19; the complete construct may have e.g. the amino acid sequence of SEQ ID NO 30

[0163] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0164] Examples

[0165] Example 1

[0166] 1.1 Cloning of HDAg variant encoding genes

[0167] Synthetic genes encoding the HDAg variants as listed in Table 1 were purchased from Eurofins MWG Operon (Ebersberg, Germany). On the basis of the pET24a expression plasmid of Novagen (Madison, WI, USA) the following cloning steps were performed. The vector was digested with Ndel and Xhol and a cassette comprising the respective HDAg variant was inserted. The inserts of the resulting plasmids were sequenced and found to encode the desired HDV polypeptides, which may also be referred to as "HDAgs" herein. The amino acid sequences of the resulting polypeptide are shown in the sequence listing included herein. All recombinant HDV polypeptide variants contained a C-terminal hexahistidine tag to facilitate Ni-NTA-assisted purification. SEQ ID NOs are summarized in Table 1.

[0168] Table 1: HDAg variants used in the Examples

[0169] 1.2 Expression and and purification of HDAg variants

[0170] HDAg variants with SEQ ID NO:23 and 24 were purified according to the following protocol. E. coli BLR (DE3) cells harboring the expression plasmid were grown in LB medium plus kanamycin (30 pg / ml) to an ODeoo of 1, and cytosolic overexpression was induced by adding isopropyl-13-D-thiogalactosid (IPTG) to a final concentration of 1 mM at a growth temperature of 37°C. 4 hours after induction, cells were harvested by centrifugation (20 min at 5000 x g), frozen and stored at -20°C. For cell lysis, the frozen pellet was resuspended in 25 mM sodium phosphate pH 8.5, 6 mM MgCh, 10 U / ml Benzonase®, 1 tablet Complete® and 1 tablet Complete® EDTA-free per 50 ml of buffer (protease inhibitor cocktail) and the resulting suspension was lysed by high pressure homogenization. The crude lysate was supplemented up to 50 mM sodium phosphate, 10 mM imidazole. After centrifugation the supernatant was applied onto a Ni-NTA (nickel-nitrilotriacetate) column pre-equilibrated in buffer A (50 mM sodium phosphate pH 8.5, 100 mM sodium chloride, 10 mM imidazole). Prior to elution, the imidazole concentration was raised to 40 mM in order to remove contaminant proteins. The proteins were then eluted by applying an imidazole concentration of 125 mM. Finally, the proteins were subjected to cation exchange chromatography (SP Sepharose) and the proteincontaining fractions was pooled and concentrated.

[0171] HDAg variants with SEQ ID NO:25, 26, 27 and 29 were purified and refolded according to the following protocol. E. coli BLR (DE3) cells harboring the expression plasmid were grown in LB medium plus kanamycin (30 pg / ml) to an OD600 of 1, and cytosolic overexpression was induced by adding isopropyl-B-D-thiogalactosid (IPTG) to a final concentration of 1 mM at a growth temperature of 37°C. 4 hours after induction, cells were harvested by centrifugation (20 min at 5000 x g), frozen and stored at -20°C. For cell lysis, the frozen pellet was resuspended in 25 mM sodium phosphate pH 8.5, 6 mM MgC12, 10 U / ml Benzonase®, 1 tablet Complete® and 1 tablet Complete® EDTA-free per 50 ml of buffer (protease inhibitor cocktail) and the resulting suspension was lysed by high pressure homogenization. The crude lysate was supplemented up to 7 M GuHCl (guanidine hydrochloride), 50 mM sodium phosphate, 5 mM imidazole and stirred for one hour. After centrifugation the supernatant was applied onto a Ni- NTA (nickel-nitrilotriacetate) column pre-equilibrated in buffer A (50 mM sodium phosphate pH 8.5, 7.0 M GuHCl, 5 mM imidazole. After a washing step, the chaotropic buffer A was displaced by 50 mM sodium phosphate pH 8.5, 100 mM sodium chloride, 10 mM imidazole, 1 tablet Complete® EDTA-free per 50 ml of buffer (protease inhibitor cocktail) in order to induce the conformational refolding of the matrix bound protein. Prior to elution, the imidazole concentration was raised to 40 mM in order to remove contaminant proteins. The native fusion proteins were then eluted by applying an imidazole concentration of 125 mM in 50 mM sodium phosphate pH 8.5, 100 mM sodium chloride. Protein containing fractions were assessed for purity by SDS-PAGE and pooled. Finally, the proteins were subjected to cation exchange chromatography (SP Sepharose) and the protein-containing fractions was pooled and concentrated.

[0172] The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide or using the colorimetric BCA method.

[0173] Example 2

[0174] Coupling of biotin and ruthenium moieties to HDAg variants

[0175] The lysine s-amino groups of the recombinant proteins were modified at protein concentrations of ~10 mg / ml withN-hydroxy-succinimide activated biotin and ruthenium labels, respectively. The label / protein molar ratio varied from 3: 1 to 30: 1, depending on the respective protein. The reaction buffer was 50 mM potassium phosphate (pH 8.5), 150 mM KC1, 0.5 mM EDTA. The reaction was carried out at room temperature for 30 minutes and stopped by adding buffered L- lysine to a final concentration of 10 mM. After the coupling reaction, unreacted free label was removed by passing the crude protein conjugate over a gel filtration column (Superdex 200)

[0176] Example 3

[0177] Polymerization of HDAg variants

[0178] The purified HDAg variants optionally were further polymerized with heterobifunctional crosslinkers and applied to a Superdex 200 gel filtration chromatography to collect the optimal size range of the HDAg variants (the principle is described in DE3640412 and EP0331068). A further possibility for the generation of multivalent antigens is the use of proteins which assemble themselves into non-covalent, defined oligomeric tertiary structures and carry fusions of several repetitions of the proteins of interest (the principle and the protein purification are described in EP2617731, EP2893021 and EP2827147). In this way, the non-covalent oligomerized EcSkp-HDAg(61-160)4x (SEQ ID NO:30) was produced.

[0179] The HDAg polymers optionally were labeled with ruthenium complex as described in Example 2.

[0180] Example 4

[0181] Assessment of the immunological reactivity (signal dynamic) and assay blank value of the recombinant HDAg variants in an immunodiagnostic test

[0182] The immunological reactivity and assay blank value of the different proteins was assessed on an automated cobas® e801 analyzer (Roche Diagnostics GmbH). Measurements were carried out in the double antigen sandwich format. Thereby, the biotin-conjugate (i.e. the capture antigen) is immobilized on the surface of a streptavidin- coated magnetic bead, whereas the detection antigen bears a complexed ruthenium cation as the signaling moiety. Signal detection on cobas® e801 is based on electrochemiluminescence.

[0183] In the presence of a specific immunoglobulin analyte, the chromogenic ruthenium complex is bridged to the solid phase and emits light at 620 nm after excitation at a platinum electrode. The signal output is in arbitrary light units. Raw signal is presented in counts. Signal dynamic (differentiation of positive from negative samples) is calculated by dividing the signal of an Anti-HDV positive sample by the mean signal of known negative samples.

[0184] The recombinant HDAg antigen variants according to the invention were assessed pairwise in a double antigen sandwich (DAGS) immunoassay format. For instance, a L-HDAg(l-214, C / A)-biotin conjugate was assessed together with a L-HDAg(l-214, C / A)-ruthenium complex conjugate at a concentration of 100 ng / ml each in assay buffer containing 50 mM TRIS (pH 7.5), 150 mM NaCl, 0.25% polidocanol, 0.5% albumin, 0.01% N-methylisothiazolon, 0.1% Oxy-Pyrion. The used sample volume was 12 pl. Measurements were performed with human serum and plasma samples negative and positive for antibodies against HDV. HDAg variants L-HDAg (1-214, C / A), rec. S-HDAg (1-195) and rec. L-HDAg (1-214, W+C / A) show high blank counts in Anti-HDV negative samples (>1000 counts) and a weak signal dynamic of Anti-HDV positive samples vs negative samples (table 2). HDAg variants rec. S-HDAg (1-195, W / A), rec. HDAg (1-160, W / A), rec. L-HDAg (61-214, C / A) and rec. HDAg (61-160) show lower blank counts in Anti-HDV negative samples (<1000 counts) and considerable stronger signal dynamic of Anti-HDV positive samples vs negative samples (Tables 2 and 3).

[0185] Of these variants rec. L-HDAg (61-214, C / A) shows the least favorable signal dynamic for Anti-HDV positive samples (Table 3). The three best performing variants are S-HDAg (1-195, W / A), rec. HDAg (1-160, W / A) and rec. HDAg (61-160).

[0186] Example 5

[0187] Assessment of immunological reactivity (signal dynamic) of recombinant oligomerized HDAg variants for early low affine antibodies e.g. IgM antibodies during Anti-HDV seroconversion

[0188] The immunological reactivity and assay blank value of chemically cross-linked oligomerized recombinant antigen of the best performing antigens of Example 4 with S-HDAg (1-195, W / A) as longest variant and rec. HDAg (61-160) as shortest variant was assessed on an automated cobas® e801 analyzer (Roche Diagnostics GmbH). Measurements were carried out according to Example 4 with the exception that 6pl of sample volume was applied and oligomerized HDAg was used as biotin conjugate and ruthenium complex.

[0189] Oligomerized HDAg variant S-HDAg (1-195, W / A) shows high blank counts in Anti-HDV negative samples (»1000 counts) and a weak signal dynamic in most Anti-HDV positive samples (table 4). In comparison, oligomerized HDAg (61-160) displays considerably lower blank counts in Anti-HDV negative samples (approx. 1000 counts) and a considerably stronger signal dynamic in most Anti-HDV positive samples (Table 4). The best performing variant is rec. HDAg (61-160).

[0190] Ruthenylated chemically cross-linked oligomerized HDAg (61-160) was tested against ruthenylated monomeric S-HDAg (1-195, W / A) in consecutive blood samples draws from an Anti-HDV seroconverter and on early infections with HDV to evaluate the detection of early low affine antibodies against HDV. Additionally a mixture of chemically cross-linked oligomerized HDAg (61-160) or non-covalent oligomerized EcSkp-HDAg(61 - 160)4x with monomeric S-HDAg (1-195, W / A) was assessed using a ratio of 90% monomer + 10% oligomer. Testing was performed as described in Example 4 with 12pl of sample volume.

[0191] During seroconversion or in early infections, antibodies of the IgM subtype are predominant. However, their concentration quickly declines and antibodies of the IgG subtype take over. Compared to recombinant monomeric S-HDAg (1-195, W / A), recombinant chemically crosslinked oligomerized HDAg (61-160) shows a significant higher signal dynamic in the first reactive sample of seroconverter 6517 (sample 6517-2; 39.5 signal dynamic vs 5.00 signal dynamic) and early infection 6538-1 (sample 6538-1; 148 signal dynamic vs 9.27 signal dynamic). The data demonstrate that oligomerized HDAg (61-160) is more efficient in binding of early weak affine antibodies of the IgM subtype (Table 5).

[0192] Furthermore, for recombinant chemically cross-linked oligomerized HDAg (61-160) in both sample sets, the signal dynamics decreases in the consecutive sample e.g. in 6517-3 vs 6517-2 due to the decreased Anti-HDV IgM titer while for recombinant monomeric S-HDAg (1-195, W / A) the signal dynamic increases in the consecutive samples due to the rising Anti-HDV IgG titer.

[0193] A mixture of 90 ng / mL monomeric S-HDAg (1-195, W / A) with lOng / mL chemically crosslinked oligomerized HDAg (61-160) facilitates detection of early anti-HDV antibodies compared to the monomeric S-HDAg (1-195, W / A) alone (sample 6517-2 9.99 signal dynamic vs 5.00 signal dynamic; sample 6538-1 18.2 signal dynamic vs 9.27 signal dynamic; table 5). Similar results were obtained with non-covalent oligomerized EcSkp-HDAg(61-160)4x.

[0194] A mixture of 90 ng / mL monomeric S-HDAg (1-195, W / A) with lOng / mL non-covalent oligomerized EcSkp-HDAg(61 - 160)4x facilitates detection of early anti-HDV antibodies compared to the monomeric S-HDAg (1-195, W / A) alone (sample 6517-2 19.1 signal dynamic vs 5.00 signal dynamic; sample 6538-1 24.1 signal dynamic vs 9.27 signal dynamic; Table 5). Thus, a mixture of monomeric and oligomerized HDAg antigen promotes the detection of antibodies of different Ig-subtypes ensuring an increased seroconversion sensitivity and ensuring that the assay acts as an Anti-HDV total test. Table 2 Detection of anti-HDV antibodies in human blood samples by using recombinant HDAg variants (rec. L-HDAg (1-214, C / A); rec. S-HDAg (1-195); rec. L-HDAg (1-214, W+C / A); rec. S-HDAg (1-195, W / A));

[0195] Table 2A: values measured as counts.

[0196] Table 2B: Signal dynamic counts (SDC) of positive samples divided by mean counts of negative samples.

[0197] Table 3: Detection of anti-HDV antibodies in human blood samples by using recombinant HDAg variants (rec. HDAg (1-160, W / A); rec L-HDAg (61-214, C / A); rec. HDAg (61-160))

[0198] Table 3A: values measured as counts.

[0199] Table 3B: Signal dynamic counts (SDC) of positive samples divided by mean counts of negative samples.

[0200] Table 4: Detection of anti-HDV antibodies in human blood samples by using recombinant chemically cross-linked oligomerized HDAg variants S- HDAg (1-195, W / A) and HDAg (61-160).

[0201] Table 5: Detection of early anti-HDV antibodies (e.g. IgM subtype) in human blood samples by using recombinant oligomerized HDAg (61-160), recombinant monomeric S-HDAg (1-195, W / A) and a mixture of oligomerized HDAg (61-160) with monomeric S-HDAg (1-195, W / A)

[0202] Table 5 (continued)

Claims

Claims1. A hepatitis delta virus (HDV) polypeptide comprising an antigenic domain comprising an amino acid sequence of SEQ ID NO:1 or an amino acid sequence at least 60% identical thereto, wherein said HDV polypeptide (i) is devoid of a coiled-coil domain consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence at least 60% identical thereto; or (ii) further comprises said coiled-coil domain, wherein said coiled-coil domain comprises at least one of the following mutations: (I) the amino acid at position 20 is not tryptophan and (II) the amino acid at position 50 is not tryptophan.

2. The HDV polypeptide of claim 1, wherein said HDV polypeptide is devoid of said coiled-coil domain.

3. The HDV polypeptide of claim 1 or 2, wherein said HDV polypeptide is devoid of a C- terminal domain comprising an amino acid sequence shown in SEQ ID NO:21 and / or 22.

4. The HDV polypeptide of any one of claims 1 to 3, wherein said antigenic domain consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 70% identical to SEQ ID NO: 1.

5. The HDV polypeptide of any one of claims 1 to 4, wherein said antigenic domain consists of the amino acid sequence of SEQ ID NO: 1.

6. The HDV polypeptide of any one of claims 3 to 5, wherein said HDV polypeptide is devoid of said coiled-coil domain and of said C-terminal domain.

7. The HDV polypeptide of claim 1, further comprising said coiled-coil domain.

8. The HDV polypeptide of claim 1 or 7 wherein the amino acid at position 4 of the amino acid sequence of said coiled-coil domain is proline, the amino acid at position 6 is glycine, the amino acid at position 20 is alanine, and / or the amino acid at position 50 is alanine.

9. The HDV polypeptide of claim 1, 7, or 8, wherein the amino acid sequence of said coiled-coil domain comprises an amino acid sequence of any one of SEQ ID NOs:3 to 11, or an amino acid sequence at least 60% identical to one of SEQ ID NOs:3 to 11.

10. The HDV polypeptide of any one of claims 1 to 9, wherein said HDV polypeptide further comprises at least one auxiliary domain selected from the list consisting of a linker domain, a purification tag domain, and an oligomerization domain.

11. The HDV polypeptide of any one of claims 1 to 10, wherein said HDV polypeptide comprises, in an embodiment consists of, the amino acid sequence of any one of SEQ ID NOs:l, 13, 14, and 23 to 30.

12. A HDV protein comprising a multitude of HDV polypeptides according to any one of claims 1 to 11.

13. The HDV protein of claim 12, wherein said protein is an oligomer of HDV polypeptides comprising at least one oligomerization domain per HDV polypeptide.

14. The HDV polypeptide of claim 10 or 11 or the HDV protein of claim 12 or 13, wherein said oligomerization domain is an Skp domain.

15. The HDV protein of any one of claims 12 to 14, wherein said HDV protein comprises HDV polypeptides comprising the amino acid sequence of SEQ ID NO: 30.

16. A kit comprising an HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15 comprised in a housing.

17. The kit of claim 9, wherein said kit comprises (i) a HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15 as a detector compound; and / or (ii) a HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15 as a capture compound.

18. A HDV polynucleotide comprising a nucleic acid sequence encoding an HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15.

19. A host cell comprising the HDV polypeptide according to any one of claims 1 to 11 and / or the HDV polynucleotide according to claim 18.

20. An HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15 for use in diagnosing a hepatitis D virus (HDV) infection in a sample of a subj ect.

21. A method for determining anti-hepatitis D virus (HDV) antibodies in a sample, comprising(a) contacting said sample with an HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15;(b) determining complexes comprising anti-HDV antibodies and said HDV polypeptide and / or said HDV protein; and(c) thereby determining anti-HDV antibodies in said sample.

22. A device for detecting an anti-hepatitis D virus (HDV) antibody in a sample, said device comprising an analyzing unit with a sample treatment unit, said analyzing unit being adapted to perform the following steps:(a) contacting a sample applied to said sample treatment unit with an HDV polypeptide according to any one of claims 1 to 11 and / or a HDV protein according to any one of claims 12 to 15, and(b) detecting an anti-HDV antibody in said sample.

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