Polypeptides useful for detecting anti-rhabdovirus antibodies

Abstract

The present invention relates to a recombinantly constructed protein that is useful for analytical assay, particularly for determining the presence of rhabdovirus specific antibody in biological samples obtained from individuals.More specifically, the present invention relates to a polypeptide that comprises the ectodomain of rhabdovirus glycoprotein and the heterologous multimerization domain connected to said ectodomain.In one example, there is provided a fusion protein of formula xyz (wherein x consists of or comprises the ectodomain, which may not contain furin cleavage site, y is a linker site, and z is the heterologous multimerization domain that may be selected from the group consisting of immunoglobulin sequence, coiled coil sequence, streptavidin sequence, fibritin sequence and avidin sequence).

Description

[Technical Field]

[0001] The present invention relates to recombinant construct proteins useful in analytical assays, particularly for determining the presence of antibodies specific for rhabdoviruses in biological samples obtained from individuals. [Background technology]

[0002] Rhabdoviruses are membrane-enveloped negative-strand RNA viruses belonging to the Rhabdoviridae family. Rhabdovirus virions contain an outer membrane derived from the cell in which the virus is produced, and internal ribonucleoproteins, including non-segmented genomic RNA and N (nucleocapsid) protein. Rhabdovirus glycoprotein (G) spans the membrane and forms spikes on the surface of the virus particle. Rhabdovirus glycoproteins are known to be essential for viral propagation due to their functions in both receptor binding and membrane fusion during viral entry. Viral matrix (M) protein molecules are located inside the viral envelope and form a layer between the membrane and the nucleocapsid core. Nonstructural proteins include L (large) and P (phosphoprotein) proteins, which form the viral transcriptase-replicase complex. Rhabdoviruses are widespread in nature, infecting vertebrates, invertebrates, and plants. The prototypic rhabdoviruses are rabies virus (RV) and vesicular stomatitis virus (VSV), which are considered the most studied members of this virus family. In 2014, the discovery of a novel rhabdovirus capable of infecting Spodoptera frugiperda (Sf) cells, and therefore designated Sf-rhabdovirus (SfRV), was reported (Ma et al. J Virol. 88(12): 6576-6585). WO2015051255A1 describes the detection of this virus using a PCR assay.

[0003] In practice, for solid phase assays such as ELISA, a certain amount of antigen needs to be immobilized on a solid support, and it is desirable for the antigen to be able to be produced in as high a yield as possible in cell culture in order to reduce the cost per assay and reduce non-economic waste. Therefore, for the production of solid-phase assays for detecting rhabdovirus-specific antibodies, there is a need for polypeptides that can be produced in cell culture with the highest possible yield while retaining the antigenicity necessary to bind sufficiently to the antibodies when the assay is performed. Summary of the Invention

[0004] The solution to the above mentioned technical problem is achieved by the description and embodiments characterized in the claims. Therefore, the invention in its different aspects is implemented according to the appended claims. The present invention is based on the surprising discovery that fusing the C-terminus of the ectodomain of the SfRV glycoprotein, preferably having substitutions at amino acid positions 306 and 333, to an IgG Fc domain allowed for the expression of a significantly higher molar yield of an antigen useful for detecting SfRV-specific antibodies in serum samples when compared to expression of the respective ectodomain with the wild-type sequence. This beneficial effect, in turn, also allowed for the production of a significantly higher number of ELISA tests for the same cost of protein expression compared to the production of equivalent tests using the wild-type sequence. Thus, in a first aspect, the present invention provides a method for producing a composition comprising: the ectodomain of a rhabdovirus glycoprotein; a heteromultimerization domain linked to the ectodomain; The present invention relates to a polypeptide comprising: DETAILED DESCRIPTION OF THE INVENTION

[0005] Said polypeptide, hereinafter also referred to as "polypeptide of the invention", is preferably for determining the presence of antibodies specific for rhabdovirus in a biological sample obtained from an individual. As used herein, the term " ectodomain " is intended to include the part of the protein that is located on the outer surface of virus envelope.For example, the ectodomain of rhabdovirus glycoprotein is the part of rhabdovirus glycoprotein that extends to extraviral space.More specifically, the ectodomain of rhabdovirus glycoprotein is the rhabdovirus glycoprotein that transmembrane helix and cytoplasmic domain are removed.Even more preferably, the ectodomain of rhabdovirus glycoprotein is the rhabdovirus glycoprotein that transmembrane helix, cytoplasmic domain and N-terminal signaling peptide are removed.

[0006] When used in the context of the present invention, the term "multimerization domain" particularly refers to an amino acid sequence that can specifically bind to or associate with one or more additional multimerization domains, for example, to form multimers. In one example, a multimerization domain is an amino acid sequence that can bind to one other multimerization domain with the same amino acid sequence, or homo-associate with each other to form a homodimer. A multimerization domain can contain one or more cysteine ​​residues so that disulfide bonds can be formed between the associated multimerization domains. In the context of the present invention, " heterologous multimerization domain " particularly refers to the multimerization domain that is derived from the entity other than the rhabdovirus that rhabdovirus glycoprotein is derived from, as referred to herein.For example, heterologous multimerization domain is the multimerization domain that is coded by the genome of virus other than rhabdovirus, or preferably by the genome of eukaryotic cell or prokaryotic cell, particularly mammalian cell.

[0007] Preferably, the multimerization domain is linked to said ectodomain via a linker moiety. The linker moiety is preferably a peptide linker as described herein in the context of the present invention. The term "peptide linker" as used herein refers to a peptide comprising one or more amino acid residues. More specifically, the term "peptide linker" as used herein refers to a peptide capable of connecting two variable domains, for example, an ectodomain and a multimerization domain, and its length depends on the type of variable domains to be connected. In a particularly preferred embodiment, the multimerization domain is linked to said ectodomain via a linker moiety, wherein: - the multimerization domain is linked to the linker moiety via a peptide bond between the N-terminal amino acid residue of the linker moiety and the C-terminal amino acid residue of the ectodomain; - the linker moiety is linked to the multimerization domain via a peptide bond between the N-terminal amino acid residue of the multimerization domain and the C-terminal amino acid residue of the linker moiety.

[0008] It may also be preferred that the multimerization domain is linked to the ectodomain via a peptide bond between the N-terminal amino acid residue of the multimerization domain and the C-terminal amino acid residue of the ectodomain. In another preferred embodiment, the present invention provides a polypeptide which is a fusion protein of the formula xyz, in particular a polypeptide as described above. (In the formula, x consists of or comprises the ectodomain of a rhabdovirus glycoprotein; y is a linker moiety, z is the heteromultimerization domain). It is to be understood that the formula xyz in particular means that the C-terminal amino acid residue of the ectodomain is linked to the linker moiety, preferably via a peptide bond with the N-terminal amino acid residue of the linker moiety, and that the N-terminal amino acid residue of the multimerization domain is linked to the linker moiety, preferably via a peptide bond with the C-terminal amino acid residue of the linker moiety. Most preferably, the ectodomains referred to herein in the context of the present invention do not contain a furin cleavage site. "Does not contain a furin cleavage site" particularly means that there is no furin cleavage site present in the amino acid sequence of the ectodomain.

[0009] Furin cleavage sites, as referred to herein, are in particular the following (a), (b), and (c): (a) an amino acid sequence selected from the group consisting of RXKR (SEQ ID NO: 20) and RXRR (SEQ ID NO: 21), where X can be any amino acid residue; (b) RX1KRX2 (SEQ ID NO: 22) and RX1RRX2 (SEQ ID NO: 23), wherein: X1 can be any amino acid residue, X2 is - other than a lysine residue, or - other than an amino acid residue selected from the group consisting of valine, leucine, isoleucine, and tryptophan residues an amino acid sequence selected from the group consisting of: (c) RX1KRX2X3 (SEQ ID NO: 24) and RX1RRX2X3 (SEQ ID NO: 25), wherein: X1 can be any amino acid residue, X2 is - other than a lysine residue, or - an amino acid residue other than an amino acid residue selected from the group consisting of a valine residue, a leucine residue, an isoleucine residue, and a tryptophan residue and X3 may be any amino acid residue other than a lysine residue) The amino acid sequence is selected from the group consisting of:

[0010] The term "any amino acid residue" as used herein is specifically understood to be equivalent to "any genetically encoded amino acid residue." Amino acid residues other than lysine residues, when referred to herein, are in particular naturally occurring amino acid residues, preferably genetically encoded amino acid residues. The term "genetically encoded amino acid residue", when used in the context of the present invention, refers in particular to an amino acid residue (single letter code in brackets) selected from the group consisting of alanine residue (A), aspartic acid residue (D), asparagine residue (N), cysteine ​​residue (C), glutamine residue (Q), glutamic acid residue (E), phenylalanine residue (F), glycine residue (G), histidine residue (H), isoleucine residue (I), lysine residue (K), leucine residue (L), methionine residue (M), proline residue (P), arginine residue (R), serine residue (S), threonine residue (T), valine residue (V), tryptophan residue (W), and tyrosine residue (Y).

[0011] Therefore, for example, when referred to herein, the expression "may be any amino acid residue other than a lysine residue" is understood to be equivalent to, in particular, "an amino acid residue selected from the group consisting of an alanine residue, an aspartic acid residue, an asparagine residue, a cysteine ​​residue, a glutamine residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a leucine residue, a methionine residue, a proline residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, and a tyrosine residue." Most preferably, the rhabdovirus glycoprotein as referred to in the context of the present invention is a S. frugiperda rhabdovirus (SF-rhabdovirus) glycoprotein. In a preferred embodiment, the ectodomains described herein are (i) has one or more mutations selected from the group consisting of a substitution at amino acid position 306, a substitution at amino acid position 303, a substitution at amino acid position 305, a substitution at amino acid position 307, and a substitution at amino acid position 308; and (ii) having one or more mutations selected from the group consisting of a substitution at amino acid position 333, a substitution at amino acid position 330, a substitution at amino acid position 332, a substitution at amino acid position 334, and a substitution at amino acid position 335; SF - is the ectodomain of the rhabdovirus glycoprotein, The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0012] As used herein, the term "mutation" encompasses any change in the amino acid sequence (substitution with a genetically encoded amino acid residue, and its insertion and deletion). The term "substitution at an amino acid position", as used herein, particularly refers to a change in an amino acid residue at a specific position in the amino acid sequence of a protein. Preferably, the ectodomain referred to herein is the ectodomain of an SF-rhabdovirus glycoprotein having one or more of the mutations or amino acid residues described herein, and the N-terminal amino acid residue of the ectodomain preferably corresponds to any one of amino acids 1 to 22 of the wild-type SF-rhabdovirus glycoprotein, most preferably amino acid 22, 21, or 1. According to a further preferred embodiment, the ectodomain described herein comprises or consists of an amino acid sequence that is 529 to 550 amino acid residues in length. In a particularly preferred embodiment, the ectodomain is the ectodomain of the SF-rhabdovirus glycoprotein having a substitution at amino acid position 306 and a substitution at amino acid position 333, the numbering of the amino acid positions referring to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0013] Preferably, the ectodomains described herein in the context of the present invention are an amino acid residue other than the arginine residue at amino acid position 306, and - an amino acid residue other than arginine at amino acid position 333 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0014] In a particularly preferred embodiment, the ectodomain described herein is an ectodomain of the SF-rhabdovirus glycoprotein that comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or particularly at least 95% sequence identity to the sequence of SEQ ID NO:4.

[0015] The ectodomain referred to herein preferably comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or especially at least 95% sequence identity with the sequence of SEQ ID NO: 4, an amino acid residue other than the arginine residue at amino acid position 306, and - an amino acid residue other than arginine at amino acid position 333 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0016] The term " the numbering of amino acid position refers to the amino acid sequence of wild-type SF-rhabdovirus glycoprotein " means that the numbering of amino acid position refers to the amino acid sequence of full-length wild-type SF-rhabdovirus glycoprotein.Therefore, this numbering of amino acid position referred to herein refers to the wild-type SF-rhabdovirus glycoprotein with 610 amino acid residues, in particular including the methionine residue at (N-terminal) amino acid position 1.Therefore, when used in the context of the present invention, this numbering particularly relates to the sequence of wild-type SF-rhabdovirus glycoprotein as set forth in SEQ ID NO: 16.In other words, in this context, when reference is made to amino acid position 306, it means the amino acid residue corresponding to amino acid position 306 of SEQ ID NO: 16, or when reference is made to amino acid position 333, it means the amino acid residue corresponding to amino acid position 333 of SEQ ID NO: 16.However, this does not mean that the ectodomain described in this specification in this manner has the same amino acid sequence as the amino acid sequence of SEQ ID NO: 16. This merely states that the corresponding amino acid is located at a position in the sequence, and that this position corresponds to the explicitly stated position in the sequence of wild-type SF-rhabdovirus glycoprotein. For example, when referring to the ectodomain of SF-rhabdovirus glycoprotein having an amino acid residue other than an arginine residue at amino acid position 306 and an amino acid residue other than an arginine residue at amino acid position 333, and the numbering of amino acid positions refers to the amino acid sequence of wild-type SF-rhabdovirus glycoprotein, in one example, this refers to the ectodomain derived from the wild-type SF-rhabdovirus glycoprotein of SEQ ID NO: 16, which contains substitutions at amino acid positions 306 and 333 (i.e., the arginine residues at amino acid positions 306 and 333 of the sequence corresponding to SEQ ID NO: 16 are each replaced by an amino acid residue other than an arginine residue).

[0017] As an example, positions 285 and 312 of the sequence of the ectodomain of SEQ ID NO: 1 (i.e., the ectodomain of the glycoprotein set forth in SEQ ID NO: 16 (without the N-terminal signaling peptide) having substitutions at amino acid positions 306 and 333, respectively (i.e., glutamine residues instead of arginine residues)) correspond to amino acid positions 306 and 333 of the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein set forth in SEQ ID NO: 16. It should also be understood that, as used herein, the term "signaling peptide" is equivalent to the term "signaling domain."

[0018] According to a further preferred aspect, the ectodomain, as described herein, is an amino acid residue other than an arginine residue at amino acid position 306, and / or an amino acid residue other than an arginine residue at amino acid position 303, and / or an amino acid residue other than the basic amino acid residue at amino acid position 305, and / or an amino acid residue at amino acid position 307 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 308 and an amino acid residue other than the arginine residue at amino acid position 333, and / or an amino acid residue other than the arginine residue at amino acid position 330, and / or an amino acid residue other than the basic amino acid residue at amino acid position 332, and / or an amino acid residue at amino acid position 334 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 335 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0019] As referred to herein, the term "basic amino acid residue" particularly relates to amino acid residues selected from the group consisting of arginine, lysine and histidine residues. Therefore, as used herein, "amino acid residues other than basic amino acid residues" particularly refers to - other than an arginine residue, - other than a lysine residue, or - Non-histidine residues The amino acid residues are: Preferably, the ectodomain is an amino acid residue other than the arginine residue at amino acid position 306, and - an amino acid residue other than arginine at amino acid position 333 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0020] The wild-type SF-rhabdovirus glycoprotein, as referred to herein, preferably consists of or is the amino acid sequence of SEQ ID NO: 16. In this regard, it is understood that the expression "consisting of the amino acid sequence" is equivalent to the expression "is the amino acid sequence." Thus, the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein, as referred to herein, is preferably the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein of SEQ ID NO:16. Furthermore, according to the present invention, it is particularly preferred if the ectodomain is the ectodomain of SF-rhabdovirus glycoprotein having an amino acid residue other than an arginine residue at each of amino acid positions 306 and 333, wherein the numbering of the amino acid positions refers to the amino acid sequence of wild-type SF-rhabdovirus glycoprotein, and wherein said ectodomain consists of an amino acid sequence having at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0021] When referred to herein, amino acid residues other than arginine residues are in particular naturally occurring amino acid residues, preferably genetically encoded amino acid residues. According to another preferred embodiment, the ectodomain referred to herein is an amino acid residue at amino acid position 306 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 303 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and - an amino acid residue at amino acid position 333 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 330 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0022] Amino acid residues having a polar but uncharged side chain, when referred to herein in the context of the present invention, are preferably selected from the group consisting of serine, threonine, tyrosine, asparagine, and glutamine residues. Amino acid residues having a hydrophobic side chain, as used herein, are preferably selected from the group consisting of alanine, valine, leucine, methionine, isoleucine, phenylalanine, and tryptophan residues. According to a more specific preferred embodiment, the ectodomain referred to herein is - an amino acid residue at amino acid position 306 selected from the group consisting of glutamine and asparagine residues, and / or an amino acid residue at amino acid position 303 selected from the group consisting of glutamine and asparagine residues, and, - at amino acid position 333, an amino acid residue selected from the group consisting of glutamine and asparagine residues, and / or at amino acid position 330, an amino acid residue selected from the group consisting of glutamine and asparagine residues, SF - is the ectodomain of the rhabdovirus glycoprotein, The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0023] Even more specifically, in the context of the present invention: - an amino acid residue at amino acid position 306 selected from the group consisting of glutamine and asparagine residues, and an amino acid residue at amino acid position 333 selected from the group consisting of glutamine and asparagine residues and an ectodomain which is the ectodomain of the SF-rhabdovirus glycoprotein, having the formula: The numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein. In another preferred embodiment, the ectodomain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or specifically 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0024] Thus, in one example, an ectodomain as described herein in the context of the present invention comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, an amino acid residue other than the arginine residue at amino acid position 285, and / or an amino acid residue other than the arginine residue at amino acid position 282, and / or an amino acid residue at amino acid position 286 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 287 and and, an amino acid residue other than the arginine residue at amino acid position 312, and / or an amino acid residue other than an arginine residue at amino acid position 309, and / or an amino acid residue at amino acid position 313 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or -Lysine residue at amino acid position 314 an ectodomain having the formula: The numbering of the amino acid positions refers to the amino acid sequence of SEQ ID NO:1.

[0025] In particular, said ectodomain preferably comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; an amino acid residue other than the arginine residue at amino acid position 285, and - an amino acid residue other than arginine at amino acid position 312 and The numbering of the amino acid positions refers to the amino acid sequence of SEQ ID NO:1.

[0026] More specifically, said ectodomain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; - an amino acid residue at amino acid position 285 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 282 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and and, - an amino acid residue at amino acid position 312 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 309 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue. and The numbering of the amino acid positions refers to the amino acid sequence of SEQ ID NO:1.

[0027] Even more particularly, said ectodomain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, - an amino acid residue at amino acid position 285 selected from the group consisting of glutamine and asparagine residues, and / or an amino acid residue at amino acid position 282 selected from the group consisting of glutamine and asparagine residues, and an amino acid residue at amino acid position 312 selected from the group consisting of glutamine and asparagine residues, and / or an amino acid residue at amino acid position 309 selected from the group consisting of glutamine and asparagine residues; and The numbering of the amino acid positions refers to the amino acid sequence of SEQ ID NO:1.

[0028] The term "the numbering of the amino acid positions refers to the amino acid sequence of SEQ ID NO: 1" means that the numbering of the amino acid positions refers to the amino acid sequence of the entire sequence of SEQ ID NO: 1. Thus, the numbering of the amino acid positions referred to herein refers to a sequence having 529 amino acid residues, including the asparagine residue at (N-terminal) amino acid position 1. In other words, in this context, when reference is made to amino acid position 285, it means the amino acid residue corresponding to amino acid 285 of SEQ ID NO: 1, or when reference is made to amino acid position 312, it means the amino acid residue corresponding to amino acid 312 of SEQ ID NO: 1. However, this does not mean that the ectodomain described herein in this manner has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1. It merely states that the corresponding amino acid is located at a position in the sequence, which position corresponds to the explicitly mentioned position in the sequence of SEQ ID NO: 1.

[0029] With regard to the term "at least 90%", when referred to in the context of the present invention, it is understood that said term preferably relates to "at least 91%", more preferably "at least 92%", even more preferably "at least 93%", or especially "at least 94%". With regard to the term "at least 95%", when referred to in the context of the present invention, it is understood that said term preferably relates to "at least 96%", more preferably "at least 97%", even more preferably "at least 98%", or especially "at least 99%". It is understood that the term "having 100% sequence identity" as used herein is equivalent to the term "identical."

[0030] Percent sequence identity has an art-recognized meaning, and several methods exist for determining identity between two polypeptide or polynucleotide sequences. See, for example, Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides have been compiled in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711), which uses the local homology algorithm of Smith and Waterman (Adv. App. Math., 2:482-489 (1981)).For example, the computer program ALIGN, which uses the FASTA algorithm, can be used with an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. For purposes of the present invention, nucleotide sequences are aligned using the Clustal W method in MegAlign software version 11.1.0 (59) by DNASTAR Inc., 419, with the program's default multiple alignment parameters set (gap penalty = 15.0, gap length penalty = 6.66, divergent sequence delay (%) = 30%, DNA transition weight = 0.50, and DNA weight matrix = IUB), and protein / amino acid sequences are aligned using the Clustal W method in MegAlign software version 11.1.0 (59) by DNASTAR Inc., 419, with the program's default multiple alignment parameters set (Gonnet series protein weight matrix, gap penalty = 10.0, gap length penalty = 0.2, and divergent sequence delay (%) = 30%).

[0031] As used herein, the term "sequence identity to the sequence of SEQ ID NO:X" is specifically understood to be equivalent to the terms "sequence identity to the sequence of SEQ ID NO:X over the length of SEQ ID NO:X" or "sequence identity to the sequence of SEQ ID NO:X over the entire length of SEQ ID NO:X," respectively. In this context, "X" is any integer selected from 1 to 25, such that "SEQ ID NO:X" represents any of the SEQ ID NOs referred to herein. According to further preferred embodiments, the ectodomains described herein comprise or consist of an amino acid sequence that is 529, 530 or 550 amino acid residues in length. More specifically, the ectodomain referred to herein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The heterologous multimerization domains described herein are preferably selected from the group consisting of immunoglobulin sequences, coiled-coil sequences, streptavidin sequences, fibritin sequences, and avidin sequences.

[0032] More preferably, the heteromultimerization domain is selected from the group consisting of an immunoglobulin constant region domain, a leucine zipper domain, and an E. coli virus T4 fibritin sequence. Even more preferably, the heteromultimerization domain is a dimerization domain, preferably selected from the group consisting of an IgG Fc domain and a leucine zipper domain. It is particularly preferred if the heteromultimerization domain comprises or consists of an IgG Fc domain.

[0033] When polypeptide of the present invention is for determining the antibody specific to rhabdovirus in biological sample obtained from individual, the amino acid sequence of immunoglobulin constant region domain or IgG Fc domain, respectively, as referred to herein, preferably originates from the organism of biological family other than the biological family of said individual.For example, when polypeptide of the present invention is for determining the antibody specific to rhabdovirus in biological sample obtained from pig, the amino acid sequence of immunoglobulin constant region domain or IgG Fc domain, respectively, preferably originates from the organism of biological family other than Suidae.Therefore, for example, when said biological sample is obtained from pig, immunoglobulin constant region domain or IgG Fc domain, respectively, preferably originates from bovine, dog, goat, sheep, human or guinea pig.

[0034] More specifically, the heteromultimerization domain preferably comprises or consists of a guinea pig IgG Fc domain. In a further preferred embodiment, the heterologous multimerization domain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or specifically 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. The linker moiety, or peptide linker, respectively, referred to herein is an amino acid sequence that is preferably 1 to 50 amino acid residues in length, in particular 3 to 20 amino acid residues in length. According to a preferred embodiment, the linker moiety comprises or consists of an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0035] In a particularly preferred embodiment, the polypeptide of the present invention is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. Preferably, the polypeptide of the present invention is a protein comprising a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. It is further understood that the term "protein consisting of (a) sequence", as used herein, also relates to any co-translational and / or post-translational modifications of the sequence that are particularly affected by the cell in which the polypeptide is expressed. Thus, the term "protein consisting of (a) sequence", as used herein, also refers to a sequence that has one or more modifications that occur by the cell in which the polypeptide is expressed, particularly modifications of amino acid residues that occur in protein biosynthesis and / or protein processing, preferably selected from the group consisting of glycosylation, phosphorylation, and acetylation.

[0036] According to a particularly preferred embodiment of the present invention, in a baculovirus expression system, the yield of the polypeptide of the present invention is preferably at least 2-fold, more preferably at least 3-fold, even more preferably at least 5-fold, and even more preferably at least 8-fold higher than the yield of the polypeptide of SEQ ID NO: 15.

[0037] Thus, the polypeptides of the present invention are preferably recombinant proteins, in particular recombinant baculovirus-expressed proteins. The term "recombinant protein", as used herein, particularly refers to a protein produced by recombinant DNA techniques, in which DNA encoding the protein to be expressed is generally inserted into a suitable expression vector, which is then used to transform a host cell, or in the case of a viral vector, to infect a cell, to produce the heterologous protein. Thus, the term "recombinant protein", as used herein, particularly refers to a protein molecule expressed from a recombinant DNA molecule. A "recombinant DNA molecule", as used herein, refers to a DNA molecule that is composed of segments of DNA joined together using molecular biological techniques. Suitable systems for producing recombinant proteins include, but are not limited to, insect cells (e.g., baculovirus), prokaryotic systems (e.g., Escherichia coli), yeast (e.g., Saccaromyces cerevisiae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary, HEK293), plants (e.g., safflower), avian cells, amphibian cells, fish cells, and cell-free systems (e.g., rabbit reticulocyte lysate).

[0038] The term "yield" when referred to herein is understood to be specifically "molar yield." According to another aspect, the present invention also provides a polynucleotide encoding a polypeptide of the present invention, said polynucleotide being hereinafter also referred to as "a polynucleotide according to the present invention". Preferably, the polynucleotide according to the present invention comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or especially 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0039] Production of the polynucleotides described herein is within the skill of one in the art and can be carried out according to recombinant techniques as described, inter alia, in Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Amusable, et al., 2003, Current Protocols In Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed), 1994, PCR Technology, Oxford University Press, New York, which are incorporated herein by reference. In a still further aspect, the present invention provides a vector comprising a polynucleotide encoding a polypeptide of the present invention.

[0040] "Vector" and "vector comprising a polynucleotide encoding a polypeptide of the invention", for purposes of the present invention, refer to a suitable expression vector, preferably a baculovirus expression vector, used to transfect, or in the case of a baculovirus expression vector, infect a host cell to produce the protein or polypeptide encoded by the DNA. Vectors for expression and methods for making and / or using vectors (or recombinants) are described in, among other places, U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, and 382,425; PCT Publication Nos. WO 94 / 16716, WO 96 / 39491, and WO 95 / 30018; Paoletti, “Applications of pox virus vectors to vaccination: An update,” PNAS USA 93: 11349-11353, October 1996; Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93: 11341-11348, October 1996; Smith et al., U.S. Patent No. 4,745,05 (recombinant baculovirus); Richardson, CD (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.); Smith et al., "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector," Molecular and Cellular Biology, December 1983, Vol. 3, No. 12, pp. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infected Cells with a Baculovirus Vector," Molecular and Cellular Biology, March 1984, Vol. 4, No. 3, p. 406; EPA0 370 573; U.S. Application No. 920,197 filed October 16, 1986; European Patent Publication No. 265785; U.S. Patent No. 4,769,331 (Recombinant Herpesvirus); Roizman, "The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors," PNAS USA 93:11307-11312, October 1996, Andreansky et al., “The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors,” PNAS USA 93: 11313-11318, October 1996, Robertson et al., “Epstein-Barr virus vectors for gene delivery to B lymphocytes”, PNAS USA 93: 11334-11340, October 1996, Frolov et al., "Alphavirus-based expression vectors: Strategies and applications," PNAS USA 93: 11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Patent Nos. 5,591,439, 5,552,143, and WO98 / 00166, both filed July 3, 1996, and allowed U.S. Application Serial Nos. 08 / 675,556 and 08 / 675,566 (recombinant adenovirus); Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) pp. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990, Prevec et al., J. Gen Virol. 70, 42434, PCT WO91 / 11525, Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993, and McClements et al., “Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease”, PNAS USA 93: 11414-11420, October 1996, and U.S. Patent Nos. 5,591,639, 5,589,466, and 5,580,859, as well as WO90 / 11092, WO93 / 19183, WO94 / 21797, WO95 / 11307, WO95 / 20660, Tang et al., Nature, and Furth et al.These vectors can be made or performed by or similar to the methods disclosed in WO98 / 33510, Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression systems), U.S. Pat. No. 4,945,050 to Sanford et al., Fischbachet et al. (IntraCell), WO90 / 01543, Robinson et al., Seminars in Immunology, vol. 9, pp. 271-283 (1997) (DNA vector systems), U.S. Pat. No. 4,394,448 to Szoka et al. (methods for inserting DNA into living cells), U.S. Pat. No. 5,677,178 to McCormick et al. (use of cytopathic viruses), and U.S. Pat. No. 5,928,913 (vectors for gene delivery), as well as other documents cited herein.

[0041] Preferred viral vectors include baculoviruses, particularly BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.), particularly provided that the producer cells are insect cells. While baculovirus expression systems are preferred, those skilled in the art will recognize that other expression systems, including those mentioned above, will also work for the purposes of the present invention, i.e., expression of recombinant proteins.

[0042] Accordingly, the present invention also provides a baculovirus comprising a polynucleotide encoding a polypeptide of the present invention, which baculovirus is hereinafter also referred to as a "baculovirus according to the present invention".

[0043] Further provided by the present invention is a plasmid comprising a polynucleotide encoding a polypeptide of the present invention, said plasmid being hereinafter also referred to as "the plasmid according to the present invention", and is preferably an expression vector.

[0044] The present invention further comprises: a cell comprising a plasmid, in particular an expression vector, comprising a polynucleotide encoding a polypeptide of the invention; or A cell infected with a baculovirus comprising a polynucleotide encoding a polypeptide of the present invention is provided, which cell is also referred to hereinafter as "a cell according to the present invention". In another particular embodiment, the plasmid according to the invention, the cell according to the invention or the baculovirus according to the invention comprises a polynucleotide comprising a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or in particular 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In yet another aspect, the present invention provides a kit comprising a polypeptide of the invention immobilized on a solid support.

[0045] As used herein, the term "immobilized" specifically means that the polypeptides of the invention may be attached to a surface (e.g., a solid support) in any manner or by any method, including, for example, reversible or irreversible binding, covalent or non-covalent binding, etc. The term "solid support" as used herein refers to non-fluidic materials, including chips, vessels, and particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic and ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, and polymer gels; capillaries that can be made from polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. The solid support component of an assay is distinguished from an inert solid surface that the assay may contact in that the "solid support" comprises at least one moiety on its surface that is intended to interact with a capture reagent either directly or indirectly. The solid support may be a fixed component, such as a tube, strip, cuvette, or microtiter plate, or a non-fixed component, such as beads and microparticles. Microparticles can also be used as solid supports in homogeneous assay formats. A variety of microparticles can be used that allow for both non-covalent and covalent binding of proteins and other substances. Such particles include polymer particles, such as polystyrene and poly(methyl methacrylate), gold particles, such as gold nanoparticles and gold colloids, and ceramic particles, such as silica, glass, and metal oxide particles. See, for example, Martin, CR, et al., Analytical Chemistry-News & Features 70 (1998) 322A-327A, which is incorporated herein by reference.

[0046] A "chip" is a solid, non-porous material, such as metal, glass, or plastic. These materials may be coated entirely or in specific areas. On the surface of the material, there is an arbitrary spot array, either visible or coordinated. Each spot may have a predetermined polypeptide immobilized on the surface of the material, with or without a linker or spacer. All documents mentioned herein, both above and below, are incorporated herein by reference. In yet another aspect of the invention, there is provided a method for producing a polypeptide of the invention, comprising the steps of: - transfecting the cell with a plasmid, preferably an expression vector, comprising a polynucleotide comprising a sequence encoding a polypeptide of the invention, or - infecting a cell, preferably an insect cell, with a baculovirus containing a polynucleotide comprising a sequence encoding a polypeptide of the invention. The method includes:

[0047] In a particular embodiment of the method for producing a polypeptide of the invention, said plasmid is a plasmid according to the invention, in particular one of those described above. In another particular embodiment of the method for producing a polypeptide of the invention, said baculovirus is a baculovirus according to the invention, in particular one of those described above. The present invention provides a method for determining the presence or absence of antibodies specific for a rhabdovirus in a biological sample obtained from an individual, comprising: a. contacting the biological sample with a capture reagent immobilized on a solid support, wherein the capture reagent is a polypeptide of the invention; b. determining the presence or absence of said antibody bound to said capture reagent; The method further comprises:

[0048] Preferably, said method for determining the presence or absence of antibodies specific for a rhabdovirus in a biological sample obtained from an individual comprises: c. Separating the biological sample from the immobilized capture reagent; d. contacting the immobilized capture reagent-antibody complex with a detectable agent that binds to the antibody of the reagent-antibody complex; e. determining the level of antibody bound to the capture reagent using a means for detecting the detectable agent, preferably further comprising a comparison to a standard curve to determine the level of antibody bound to the capture reagent; Further includes:

[0049] The detectable agent that binds to the antibody of the reagent-antibody complex is preferably a detectable antibody, more preferably a labeled secondary antibody. The term "biological sample," as used herein, refers to any sample obtained from an individual (e.g., from a pig or bird), including, without limitation, cell-containing bodily fluids, peripheral blood, plasma or serum, saliva, tissue homogenates, lung and other organ aspirates, and lavage and enema fluids, and any other source obtainable from a human or animal subject. With respect to animals, examples of "biological samples" include blood, cells, feces, diarrhea, milk, mucus, sputum, pus, saliva, semen, sweat, tears, urine, tears, ocular fluids, vaginal secretions, and vomit, if present in the animal. Biological samples, as referred to herein, are preferably isolated from mammals or birds, preferably pigs or chickens (Gallus domesticus), and / or are selected from the group consisting of whole blood, plasma, serum, urine, and oral fluid, among others. As used herein, the term "serum" is meant to be equivalent to "blood serum."

[0050] As used herein, the term "oral fluid" refers specifically to one or more fluids found in the oral cavity, individually or in combination. These include, but are not limited to, saliva and mucosal exudates. Oral fluids can include a combination of fluids from several sources (e.g., parotid gland, submandibular gland, sublingual gland, accessory gland, gingival mucosa, and buccal mucosa), and it is specifically understood that the term "oral fluid" includes fluids from each of these sources, individually or in combination. The term "saliva" refers to the combination of oral fluids typically found in the mouth, especially after chewing. The term "mucosal exudate" as used herein refers to a fluid produced by passive diffusion of serum components from the oral mucosal stroma into the oral cavity. Mucosal exudates often form one component of saliva.

[0051] Preferably, the antibodies described herein are polyclonal antibodies. The term "antibody specific for a rhabdovirus" is in particular equivalent to "antibody specific for an antigen of a rhabdovirus", said antigen preferably being a rhabdovirus glycoprotein. The rhabdovirus glycoprotein in the context of the present invention is most preferably the SF-rhabdovirus glycoprotein set forth in SEQ ID NO:16. As used herein, the term "antigen" refers to any molecule, moiety, or entity that is capable of eliciting, inter alia, an immune response, including a cellular and / or humoral immune response.

[0052] As used herein, the term "antibody specific for" a given antigen refers to, inter alia, an antibody specific for, e.g., about 10 5 M -1 That's it, 10 6 M -1 That's it, 10 7 M -1 That's it, 10 8 M -1 That's it, 10 9 M -1 That's it, 10 10 M -1 That's it, 10 11M -1 That's it, 10 12 M -1 or more, or 10 13 M -1 or greater affinity or K a (i.e., the equilibrium binding constant of a particular binding interaction, units 1 / M) refers to an antibody, preferably a polyclonal antibody, that binds to an antigen. Alternatively, binding affinity is measured in units M (e.g., 10 -5 M~10 -13 Equilibrium dissociation constant (K) of a specific binding interaction at M d The binding affinity of an antibody can be readily determined using techniques well known to those of skill in the art (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; U.S. Pat. Nos. 5,283,173; 5,468,614; BIACORE® analysis, or equivalent). The immobilized capture reagent, as described herein, is preferably coated onto a microtiter plate, particularly a microtiter plate that can be read by an ELISA reader.

[0053] "Insect cells," as used herein, refer to cells or cell cultures derived from insect species. In the context of the present invention, insect cells derived from the species Spodoptera frugiperda and Trichoplusia ni are of particular interest. Spodoptera frugiperda (Sf) cells are preferably selected from the group consisting of Sf9 cells and Sf+ cells. When referring to insect cells herein, they are preferably Spodoptera frugiperda (Sf) cells, preferably selected from the group consisting of Sf9 cells and Sf+ cells. The present invention further relates to the use of the polypeptides of the present invention in a method for determining the presence or absence of antibodies specific for a rhabdovirus in a biological sample obtained from an individual, said method preferably being each of the methods described above. The present invention also relates to the use of the polypeptides of the present invention in methods for determining whether an individual has received an immunogenic composition comprising a recombinant protein produced by an expression system in cultured insect cells, said methods being preferably each of the methods described above. [Example]

[0054] The following examples are intended to merely illustrate the invention and in no way limit the scope of the claims. Example 1 Sf rhabdovirus glycoprotein (SFRVG) baculovirus expression construct First, the SFRVG ectodomain was generated by removing the transmembrane helices and cytoplasmic domain of SFRVG. Further modifications were made to fuse wild-type and mutant SFRVG ectodomains to immunoglobulin G crystallizable fragment (IgG Fc) proteins.

[0055] The SfRV glycoprotein ectodomain (SFRVGecto) was cloned and inserted into a baculovirus transfer plasmid to generate recombinant baculoviruses expressing SFRVGecto. Successive iterations of recombinant baculoviruses were generated, resulting in recombinant baculoviruses capable of expressing full-length SFRVGecto, which could then be harvested in high molar quantities.

[0056] Discovery of furin cleavage sites in SFRVG The secondary structure prediction programs Jpred (http: / / www.compbio.dundee.ac.uk / jpred / ) and PSIPRED (http: / / bioinf.cs.ucl.ac.uk / psipred / ) were used to identify the location of the coiled region of SFRVG (SEQ ID NO: 16). Because such regions are assumed to be more susceptible to proteolytic cleavage, Globplot (http: / / globplot.embl.de / ) was used to identify predicted disordered regions. These programs predicted a large coiled region encompassing approximately amino acids 285-335 (numbering according to the immature protein), overlapping with the disordered region encompassing approximately amino acids 285-305. Examination of this region identified a highly charged region with the sequence RERR at amino acids 303-306, which is the furin protease site, with the putative cleavage site following the last arginine. The furin site was then confirmed using the proprotein convertase recognition site prediction program ProP (http: / / www.cbs.dtu.dk / services / ProP / ; Duckert, 2004). Not only was this location predicted with high confidence to be a furin site, but a second site within the large coiled region at amino acids 330–333, with the sequence RHKR, was also predicted to be cleavable by furin.

[0057] Removal of SFRVG furin site Site-directed mutagenesis was performed to remove the furin site from SFRVG. Three different mutations were then introduced into SFRVGecto: R306Q, R333Q, and R306Q / R333Q. These were then evaluated for their ability to express SFRVGecto. The SFRVGecto sequence used for this purpose contained the first 550 amino acids of SFRVG, including the native signaling peptide, fused to a TEV protease site (C-terminus) followed by a 6xHis tag.

[0058] Point mutants were generated as follows: the first 550 amino acids, including the SFRVG ectodomain, were PCR-amplified, gel-purified, and TOPO-cloned. After insert verification by PCR colony screening and DNA sequencing, the R306Q and R333Q point mutations were generated individually using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, catalog no. 000628596). The insert was verified by DNA sequencing, and the R306Q / R333Q double mutant was generated using TOPO-SFRVGecto-R306Q as a template and subsequently verified by DNA sequencing. All three TOPO-SFRVGecto mutants were digested with EcoRI / PstI and gel-purified, while pVL1393 was digested with EcoRI / PstI, dephosphorylated, and gel-purified. Ligation was performed using T4 DNA ligase. The insert into pVL1393 was verified by PCR colony screening and DNA sequencing. Baculovirus was generated by cotransfecting Sf9 cells with FlashBAC ULTRA (FBU). IFA was performed using anti-baculovirus envelope gp64 purified clone AcV1 (eBiosciences, Cat. No. 14-6991-83) or anti-His (C-terminus, Invitrogen, Cat. No. 46-0693) primary antibodies at 1:100, and FITC-conjugated goat anti-mouse (JIR, Cat. No. 115-095-003) secondary antibodies, also at 1:100 dilution. Sf9 cells transfected with the pVL1393-SFRVGecto-R306Q, -R333Q, or -R306Q / R333Q plasmids were positive for both 6xHis and baculovirus gp64 proteins.

[0059] FBU / pVL1393-SFRVGecto-R306Q, -R333Q, and -R306Q / R333Q baculoviruses were expanded in T25 flasks of Sf9 cells for 6 days, and P2 baculoviruses were titrated. P3 expansion and protein expression experiments were performed by inoculating 100 mL of Sf+ cells into 500 mL spinner flasks at 0.1 MOI. Samples of spent medium and cells were harvested 3–5 days post-infection (DPI), and the remaining culture was harvested at 5 DPI. Cell pellets were dissolved in buffer containing 1% Triton X-100, and samples were centrifuged at 20,000 g for 20 min. The resulting samples were run on SDS-PAGE, transferred to nitrocellulose, and probed by Western blot with anti-His (C-terminus, Invitrogen, Cat. No. 46-0693) primary antibody at a dilution of 1:500 and HRP-conjugated goat anti-mouse (JIR, Cat. No. 115-035-146) secondary antibody at a dilution of 1:1000.

[0060] Fusion of the SFRVG ectodomain to porcine immunoglobulin G 2a crystallizable fragment (IgG Fc) To aid in SFRVG expression and provide a means for protein purification, SFRVGecto-R306Q / R333Q was fused to a porcine IgG 2a Fc domain (the IgG Fc domain having the sequence of SEQ ID NO: 6). Concurrently, SFRVGecto with an intact furin site (SFRVGecto-WT) was also fused to IgG to determine whether removal of the two furin sites was required in the construction of the fusion protein. Assembly of the two protein-coding sequences and insertion into the pVL1393 baculovirus transfer plasmid was briefly as follows: primers for amplifying SFRVGecto and IgG Fc were accepted; SFRVGecto-WT, SFRVG-R306Q / R333Q, and IgG Fc were amplified by PCR, gel-purified, and OEPCR was performed to generate the fusion protein insert. The OEPCR product was gel-purified and TOPO-cloned, and the insert was verified by PCR colony screening and DNA sequencing. TOPO clones containing the SFRVGecto-WT-IgG2a and SFRVGecto-R306Q / R333Q-IgG2a inserts were digested with EcoRI / PstI and gel purified, while pVL1393 was digested with EcoRI / PstI, dephosphorylated, and gel purified. Ligation was performed using T4 DNA ligase, and the inserts were confirmed by PCR colony screening and DNA sequencing.

[0061] Baculovirus was generated by cotransfecting Sf9 cells with pVL1393-SFRVGecto-R306Q / R333Q-IgG2a or pVL1393-SFRVGecto-WT-IgG2a using FlashBAC ULTRA (FBU). IFA was performed using anti-baculovirus envelope gp64 purified clone AcV1 (eBiosciences, catalog no. 14-6991-83) primary antibody at a dilution of 1:100 and FITC-conjugated goat anti-mouse (JIR, catalog no. 115-095-003) secondary antibody, also at a dilution of 1:00. Sf9 cells transfected with either pVL1393 plasmid were positive for baculovirus gp64 protein. Both FBU / pVL1393-SFRVGecto-R306Q / R333Q-IgG2a and FBU / pVL1393-SFRVGecto-WT-IgG2a baculoviruses were expanded in T25 flasks of Sf9 cells for 6 days, and P2 baculoviruses were titrated.

[0062] P3 expansion and protein expression experiments were performed by inoculating 100 mL of Sf+ cells into 500 mL spinner flasks with either baculovirus at an MOI of 0.1. Samples of spent medium and cells were collected at 3 and 4 DPI, and the remaining culture was harvested at 4 DPI. Cell pellets were dissolved in buffer containing 1% Triton X-100, and samples were centrifuged at 20,000 g for 20 minutes. The resulting samples were run on SDS-PAGE, transferred to nitrocellulose, and probed by Western blot with HRP-conjugated goat anti-pig (JIR, Cat. No. 115-035-003) antibody at a 1:1000 dilution. Experiments including the SDS Page and Western blot analyses described above confirmed that (i) fusing the SFRVG ectodomain to a porcine immunoglobulin G 2a crystallizable fragment (IgG Fc) or (ii) an SFRVG ectodomain with one of the substitutions (R306Q or R333Q), each resulted in a significantly higher molar yield in this expression system compared to expression of the respective unmodified SFRVG ectodomain. Furthermore, it was found that the combination of both the R306Q and R333Q substitutions within the SFRVG ectodomain resulted in a significantly higher molar yield compared to expression of an SFRVG ectodomain having only one of these substitutions (R306Q or R333Q).

[0063] Finally, and surprisingly, expression of the SFRV ectodomain with both substitutions R306Q / R333Q fused to IgG2a (pVL1393-SFRVGecto-R306Q / R333Q-IgG2a) - the respective SFRVG wild-type ectodomain fused to IgG2a (pVL1393-SFRVGecto-WT-IgG2a), or - SFRVG ectodomain with both substitutions R306Q and R333Q (pVL1393-SFRVGecto-R306Q / R333Q) It was found that the combination of the above mentioned modifications resulted in a synergistic effect, since it was found that much higher yields (at least 8-fold) were found compared to expression of The respective synergistic effects resulting in much higher yields were also observed for the corresponding combination comprising a guinea pig IgG Fc domain, i.e., a fusion protein comprising the sequence of SEQ ID NO: 1. Furthermore, the sequence of SEQ ID NO: 1 can be linked via a linker to a guinea pig IgG Fc domain, for example, the sequence of SEQ ID NO: 5. Thus, in particular, the respective synergistic effects resulting in much higher yields were observed for a fusion protein having the sequence of SEQ ID NO: 12, which comprises the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 5 linked to the sequence of SEQ ID NO: 1 via a peptide linker.

[0064] Example 2 ELISA is used to assess the presence of anti-rhabdovirus antibodies in different fluid samples. For this purpose, the fusion protein of formula xyz mentioned above (wherein x is the ectodomain of the glycoprotein of the rhabdovirus that the antibody to be detected is specific for, y is peptide linker, and z is IgG Fc domain) is immobilized on ELISA plate as antigen.For example, the fusion protein that comprises the sequence of SEQ ID NO: 12 is immobilized.

[0065] The ELISA method used in connection with the present invention is described in the following protocol. 1. Coat plates or strips with 5-500 ng / well of antigen (including plates with different binding capacities, materials (e.g., polystyrene), formats (strips / 96-well plates), etc.). Incubate overnight at 2-8°C to allow binding. 2. Wash the plate and block with blocking buffer containing 2-10% non-fat milk in PBS and 0.5-10% additional protein with BSA / non-relevant serum. 3. After the blocking step, wash the plate in a plate washer and gently tap the plate with a stack of paper towels to remove any remaining wash solution. 4. Dilute the test serum 1:100 and add 100 μL of diluted test serum per well. Add 100 μL of negative control serum (and positive control serum, if desired) diluted 1:100 to control wells. 5. Gently tap the side of the plate to shake and mix. Seal the plate / strip and incubate at 37°C (98.6°F) for 1 hour. 6. Wash the plate in the plate washer and gently tap the plate on a stack of paper towels to remove any remaining wash solution. 7. Add 100 μL of pre-diluted (1:1000 to 1:100000 dilution) HRP conjugate (e.g., anti-pig IgG (whole molecule) conjugated to HRP) to each well. Seal the plate and incubate at 37 °C for 1 hour. 8. Wash the plate in the plate washer and gently tap the plate on a stack of paper towels to remove any remaining wash solution. 9. Add 100 μL of substrate solution to each well. Incubate at room temperature for 10 minutes. Start the timer when the first well is filled. 10. Stop the reaction by adding 50 μL of stop solution to each well and gently mixing by tapping the side. 11. Measure the OD at 450 nm within 15 minutes of adding the stop solution to prevent fluctuations in the OD value.

[0066] The ELISA results show a clear difference between samples containing the anti-rhabdovirus antibodies to be detected (e.g., samples exhibiting an S / P ratio of greater than 0.5) and negative controls (i.e., corresponding samples not containing such antibodies and exhibiting an S / P ratio of approximately 0). In conclusion, the use of the polypeptides of the present invention to detect anti-rhabdovirus antibodies makes it possible to easily distinguish samples containing anti-rhabdovirus antibodies from samples that do not contain such antibodies.

[0067] In the sequence listing: SEQ ID NO: 1 corresponds to the sequence of the ectodomain of the glycoprotein set forth in SEQ ID NO: 16 (without the N-terminal signaling peptide) with substitutions at amino acid positions 306 and 333, respectively (i.e., glutamine residues instead of arginine residues), which amino acid positions in SEQ ID NO: 16 correspond to positions 285 and 312 in the sequence of SEQ ID NO: 1; SEQ ID NO: 2 corresponds to the sequence of SEQ ID NO: 1 extended at the N-terminus by a serine residue (corresponding to the serine residue at amino acid position 21 of SEQ ID NO: 16); SEQ ID NO:3 corresponds to the N-terminal sequence of SEQ ID NO:1 extended by 21 amino acid residues N-terminal to SEQ ID NO:16 (i.e., including the N-terminal signaling peptide); SEQ ID NO: 4 corresponds to the sequence of the glycoprotein set forth in SEQ ID NO: 16, with substitutions at amino acid positions 306 and 333, respectively (i.e., glutamine residues instead of arginine residues); SEQ ID NO: 5 corresponds to the sequence of the guinea pig IgG Fc domain, SEQ ID NO: 6 corresponds to the sequence of the porcine IgG Fc domain; SEQ ID NO: 7 corresponds to the sequence of the GCN4 leucine zipper domain, SEQ ID NO: 8 corresponds to the E. coli virus T4 fibritin sequence; SEQ ID NO: 9 corresponds to the sequence of the linker portion, SEQ ID NO: 10 corresponds to the sequence of the linker portion, SEQ ID NO: 11 corresponds to the sequence of the linker portion, SEQ ID NO: 12 corresponds to the sequence of a polypeptide of the invention, SEQ ID NO: 13 corresponds to the sequence of a polypeptide of the invention, SEQ ID NO: 14 corresponds to the sequence of a polypeptide of the invention, SEQ ID NO: 15 corresponds to the sequence of the ectodomain of the wild-type glycoprotein set forth in SEQ ID NO: 16; SEQ ID NO: 16 corresponds to the sequence of the wild-type Sf-rhabdovirus glycoprotein; SEQ ID NO: 17 corresponds to the sequence of a polynucleotide encoding a polypeptide of the invention; SEQ ID NO: 18 corresponds to the sequence of a polynucleotide encoding a polypeptide of the invention; SEQ ID NO: 19 corresponds to the sequence of a polynucleotide encoding a polypeptide of the invention; SEQ ID NO: 20 corresponds to the sequence of a furin cleavage site, SEQ ID NO: 21 corresponds to the sequence of a furin cleavage site, SEQ ID NO: 22 corresponds to the sequence of a furin cleavage site, SEQ ID NO: 23 corresponds to the sequence of a furin cleavage site, SEQ ID NO: 24 corresponds to the sequence of a furin cleavage site, SEQ ID NO: 25 corresponds to the sequence of a furin cleavage site.

[0068] The following items are also disclosed herein: 1.- The ectodomain of the rhabdovirus glycoprotein; a heteromultimerization domain linked to the ectodomain; A polypeptide comprising: 2. The heterologous multimerization domain is linked to the ectodomain via a linker moiety; or the heterologous multimerization domain is linked to the ectodomain via a peptide bond between the N-terminal amino acid residue of the heterologous multimerization domain and the C-terminal amino acid residue of the ectodomain. 3. A polypeptide which is a fusion protein of the formula xyz, in particular a polypeptide according to item 1 or 2, wherein x consists of or comprises the ectodomain of a rhabdovirus glycoprotein; y is a linker moiety, z is the heteromultimerization domain). 4. The polypeptide according to any one of items 1 to 3, wherein the ectodomain does not contain a furin cleavage site.

[0069] 5. The furin cleavage site is selected from the following: (a), (b), and (c): (a) an amino acid sequence selected from the group consisting of RXKR (SEQ ID NO: 20) and RXRR (SEQ ID NO: 21); (b) RX1KRX2 (SEQ ID NO: 22) and RX1RRX2 (SEQ ID NO: 23), wherein: X1 can be any amino acid residue, X2 is - other than a lysine residue, or - an amino acid residue other than an amino acid residue selected from the group consisting of a valine residue, a leucine residue, an isoleucine residue, and a tryptophan residue an amino acid sequence selected from the group consisting of: (c) RX1KRX2X3 (SEQ ID NO: 24) and RX1RRX2X3 (SEQ ID NO: 25), wherein: X1 can be any amino acid residue, X2 is - other than a lysine residue, or - an amino acid residue other than an amino acid residue selected from the group consisting of a valine residue, a leucine residue, an isoleucine residue, and a tryptophan residue and X3 may be any amino acid residue other than a lysine residue) 5. The polypeptide according to item 4, wherein the amino acid sequence is selected from the group consisting of:

[0070] 6. The polypeptide according to any one of items 1 to 5, wherein the rhabdovirus glycoprotein is a S. frugiperda rhabdovirus (SF-rhabdovirus) glycoprotein. 7. The ectodomain is (i) has one or more mutations selected from the group consisting of a substitution at amino acid position 306, a substitution at amino acid position 303, a substitution at amino acid position 305, a substitution at amino acid position 307, and a substitution at amino acid position 308; and, (ii) having one or more mutations selected from the group consisting of a substitution at amino acid position 333, a substitution at amino acid position 330, a substitution at amino acid position 332, a substitution at amino acid position 334, and a substitution at amino acid position 335; SF - is the ectodomain of the rhabdovirus glycoprotein, 7. The polypeptide of any one of items 1 to 6, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein. 8. The polypeptide according to any one of items 1 to 7, wherein the ectodomain is the ectodomain of the SF-rhabdovirus glycoprotein, comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or particularly at least 95% sequence identity with the sequence of SEQ ID NO: 4.

[0071] 9. The ectodomain is an amino acid residue other than an arginine residue at amino acid position 306, and / or an amino acid residue other than an arginine residue at amino acid position 303, and / or an amino acid residue other than the basic amino acid residue at amino acid position 305, and / or an amino acid residue at amino acid position 307 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 308 and and, an amino acid residue other than the arginine residue at amino acid position 333, and / or an amino acid residue other than the arginine residue at amino acid position 330, and / or an amino acid residue other than the basic amino acid residue at amino acid position 332, and / or an amino acid residue at amino acid position 334 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 335 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: 9. The polypeptide of any one of items 1 to 8, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0072] 10. The ectodomain: an amino acid residue other than the arginine residue at amino acid position 306, and - an amino acid residue other than arginine at amino acid position 333 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: 10. The polypeptide of any one of items 1 to 9, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein. 11. The ectodomain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or especially at least 95% sequence identity with the sequence of SEQ ID NO: 4; an amino acid residue other than the arginine residue at amino acid position 306, and - an amino acid residue other than arginine at amino acid position 333 the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: 11. The polypeptide of any one of items 1 to 10, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0073] 12. The polypeptide according to any one of items 9 to 11, wherein the amino acid residues other than arginine residues are naturally occurring amino acid residues, preferably genetically encoded amino acid residues. 13. The polypeptide according to any one of items 6 to 12, wherein the N-terminal amino acid residue of the ectodomain corresponds to any one of amino acids 1 to 22 of the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein. 14. The polypeptide according to any one of items 6 to 13, wherein the N-terminal amino acid residue of the ectodomain corresponds to any one of amino acids 22, 21, or 1 of the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein.

[0074] 15. The ectodomain is an amino acid residue at amino acid position 306 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or at amino acid position 303, an amino acid residue selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and, - an amino acid residue at amino acid position 333 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 330 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: 15. The polypeptide of any one of items 1 to 14, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein. 16. The polypeptide according to item 15, wherein the amino acid residues having a polar but uncharged side chain are selected from the group consisting of serine, threonine, tyrosine, asparagine, and glutamine residues, and / or the amino acid residues having a hydrophobic side chain are selected from the group consisting of alanine, valine, leucine, methionine, isoleucine, phenylalanine, and tryptophan residues.

[0075] 17. The ectodomain is an amino acid residue at amino acid position 306 selected from the group consisting of glutamine and asparagine residues, and / or an amino acid residue at amino acid position 303 selected from the group consisting of glutamine and asparagine residues; and an amino acid residue at amino acid position 333 selected from the group consisting of glutamine and asparagine residues, and / or an amino acid residue at amino acid position 330 selected from the group consisting of glutamine and asparagine residues; the ectodomain of the SF-rhabdovirus glycoprotein having the following structure: 17. The polypeptide of any one of items 1 to 16, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein. 18. The polypeptide according to any one of items 7 to 17, wherein the amino acid sequence of wild-type SF-rhabdovirus glycoprotein consists of or is the amino acid sequence of SEQ ID NO: 16. 19. The polypeptide according to any one of items 1 to 18, wherein the ectodomain comprises or consists of a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or even more preferably at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0076] 20. The ectodomain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, or even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1; The ectodomain is an amino acid residue other than the arginine residue at amino acid position 285, and / or an amino acid residue other than the arginine residue at amino acid position 282, and / or an amino acid residue at amino acid position 286 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 287 and and an amino acid residue other than the arginine residue at amino acid position 312, and / or an amino acid residue other than an arginine residue at amino acid position 309, and / or an amino acid residue at amino acid position 313 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - Lysine residue at amino acid position 314 and 20. The polypeptide according to any one of items 1 to 19, wherein the numbering of the amino acid positions refers to the amino acid sequence of SEQ ID NO: 1.

[0077] 21. The ectodomain is an amino acid residue other than the arginine residue at amino acid position 285, and - an amino acid residue other than arginine at amino acid position 312 and 21. The polypeptide according to item 20, wherein the numbering of amino acid positions refers to the amino acid sequence of SEQ ID NO: 1. 22. The amino acid residue other than the arginine residue is a naturally occurring amino acid residue, preferably a genetically encoded amino acid residue. and / or the amino acid residues other than basic amino acid residues are naturally occurring amino acid residues, preferably genetically encoded amino acid residues.

[0078] 23. The ectodomain is - an amino acid residue at amino acid position 285 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 282 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and, - an amino acid residue at amino acid position 312 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue; and / or an amino acid residue at amino acid position 309 selected from the group consisting of an amino acid residue having a polar but uncharged side chain, an amino acid residue having a hydrophobic side chain, and a glycine residue. and 23. The polypeptide according to any one of items 20 to 22, wherein the numbering of amino acid positions refers to the amino acid sequence of SEQ ID NO: 1.

[0079] 24. The polypeptide according to item 23, wherein the amino acid residue having a polar but uncharged side chain is selected from the group consisting of serine, threonine, tyrosine, asparagine, and glutamine residues, and the amino acid residue having a hydrophobic side chain is selected from the group consisting of alanine, valine, leucine, methionine, isoleucine, phenylalanine, and tryptophan residues.

[0080] 25. The ectodomain is - at amino acid position 285, an amino acid residue selected from the group consisting of glutamine and asparagine residues, and / or at amino acid position 282, an amino acid residue selected from the group consisting of glutamine and asparagine residues, and - an amino acid residue at amino acid position 312 selected from the group consisting of glutamine and asparagine residues, and / or an amino acid residue at amino acid position 309 selected from the group consisting of glutamine and asparagine residues, 25. The polypeptide according to any one of items 20 to 24, wherein the numbering of amino acid positions refers to the amino acid sequence of SEQ ID NO: 1.

[0081] 26. The polypeptide according to any one of items 1 to 25, wherein the ectodomain comprises or consists of an amino acid sequence that is 529 to 550 amino acid residues in length. 27. The polypeptide according to any one of items 1 to 26, wherein the ectodomain comprises or consists of an amino acid sequence that is 529, 530, or 550 amino acid residues in length. 28. The polypeptide according to any one of items 1 to 27, wherein the ectodomain has the sequence of any one of SEQ ID NOs: 1 to 3. 29. The polypeptide according to any one of items 1 to 28, wherein the heteromultimerization domain is selected from the group consisting of an immunoglobulin sequence, a coiled-coil sequence, a streptavidin sequence, a fibritin sequence, and an avidin sequence. 30. The polypeptide according to any one of items 1 to 29, wherein the heteromultimerization domain is selected from the group consisting of an immunoglobulin constant region domain, a leucine zipper domain, and an Escherichia coli virus T4 fibritin sequence. 31. The polypeptide according to any one of items 1 to 30, wherein the heterologous multimerization domain is a dimerization domain, preferably selected from the group consisting of an IgG Fc domain and a leucine zipper domain.

[0082] 32. The polypeptide according to any one of items 1 to 31, wherein the heteromultimerization domain comprises or consists of an IgG Fc domain. 33. The polypeptide according to any one of items 1 to 32, wherein the heteromultimerization domain comprises or consists of a guinea pig IgG Fc domain. 34. The polypeptide according to any one of items 1 to 33, wherein the heterologous multimerization domain comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and wherein the linker portion comprises or consists of an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. 35. The polypeptide according to any one of items 2 to 34, wherein the linker portion is an amino acid sequence having a length of 1 to 50 amino acid residues. 36. The polypeptide according to any one of items 2 to 35, wherein the linker portion is an amino acid sequence having a length of 3 to 20 amino acid residues. 37. The polypeptide according to any one of items 2 to 36, wherein the linker moiety comprises or consists of an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. 38. The polypeptide according to any one of items 1 to 37, which is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. 39. The polypeptide according to any one of items 1 to 38, which is a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0083] 40. The polypeptide according to any one of items 1 to 39, wherein in a baculovirus expression system, the yield of the polypeptide is preferably at least 2-fold, more preferably at least 3-fold, even more preferably at least 5-fold, and still more preferably at least 8-fold higher compared to the yield of the polypeptide of SEQ ID NO: 15. 41. The polypeptide according to any one of items 1 to 40, which is a recombinant protein, preferably a recombinant baculovirus-expressed protein. 42. A polynucleotide encoding the polypeptide according to any one of items 1 to 41. 43. The polynucleotide according to item 42, comprising a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. 44. A plasmid, preferably an expression vector, comprising a polynucleotide encoding the polypeptide according to any one of items 1 to 41.

[0084] 45. A cell comprising a plasmid, preferably an expression vector, containing a polynucleotide encoding a polypeptide according to any one of items 1 to 41. 46. ​​A baculovirus comprising a polynucleotide encoding the polypeptide according to any one of items 1 to 41. 47. A cell, preferably an insect cell, infected with a baculovirus containing a polynucleotide encoding a polypeptide according to any one of items 1 to 41. 48. The plasmid according to item 44, the cell according to item 45, the baculovirus according to item 46, or the cell according to item 47, wherein the polynucleotide comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or especially 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. 49. A kit comprising a polypeptide according to any one of items 1 to 41 immobilized on a solid support.

[0085] 50. A method for producing a polypeptide according to any one of items 1 to 41, comprising: - transfecting the cell with a plasmid, preferably an expression vector, comprising a polynucleotide comprising a sequence encoding said polypeptide, or - infecting a cell, preferably an insect cell, with a baculovirus containing a polynucleotide comprising a sequence encoding said polypeptide. The method comprising: 51. The method according to item 50, wherein the plasmid is a plasmid according to item 44 or 48. 52. The method according to item 50, wherein the baculovirus is the baculovirus according to item 46 or 48.

[0086] 53. A method for determining the presence or absence of antibodies specific to a rhabdovirus in a biological sample obtained from an individual, comprising: a. contacting a biological sample with a capture reagent immobilized on a solid support, wherein the capture reagent is a polypeptide according to any one of items 1 to 41; b. determining the presence or absence of said antibody bound to said capture reagent; The method comprising: 54.c. Separating the biological sample from the immobilized capture reagent; d. contacting the immobilized capture reagent-antibody complex with a detectable agent that binds to the antibody of the reagent-antibody complex; e. determining the level of antibody bound to the capture reagent using a means for detecting the detectable agent, wherein determining step (e) preferably further comprises comparison to a standard curve to determine the level of antibody bound to the capture reagent; Item 54. The method of item 53, further comprising: 55. Use of a polypeptide according to any one of items 1 to 41 in a method for determining the presence or absence of antibodies specific for a rhabdovirus in a biological sample obtained from an individual, the method being preferably a method according to item 53 or 54.

Claims

1. - the ectodomain of a rhabdovirus glycoprotein; a heteromultimerization domain linked to said ectodomain; A polypeptide comprising: the ectodomain is an ectodomain of SF-rhabdovirus glycoprotein having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and having a mutation that is an amino acid substitution of R306Q at amino acid position 306 or R333Q at amino acid position 333, wherein the numbering of amino acid positions refers to the amino acid sequence of wild-type SF-rhabdovirus glycoprotein (amino acid sequence of SEQ ID NO:16); the heteromultimerization domain comprises or consists of an IgG Fc domain; The polypeptides are capable of expression in significantly higher molar yields of antigens useful for detecting antibodies specific to SfRV. or the ectodomain has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and has a mutation that is an amino acid substitution of R306Q at amino acid position 306 or R333Q at amino acid position 333; moreover, an amino acid residue other than the arginine residue at amino acid position 303, and / or an amino acid residue other than the basic amino acid residue at amino acid position 305, and / or an amino acid residue at amino acid position 307 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - lysine residue at amino acid position 308 and and, an amino acid residue other than the arginine residue at amino acid position 330, and / or an amino acid residue other than the basic amino acid residue at amino acid position 332, and / or an amino acid residue at amino acid position 334 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - lysine residue at amino acid position 335 having SF - the ectodomain of the rhabdovirus glycoprotein; the numbering of amino acid positions refers to the amino acid sequence of the wild-type SF-rhabdovirus glycoprotein (amino acid sequence of SEQ ID NO: 16); the heteromultimerization domain comprises or consists of an IgG Fc domain; The polypeptides are capable of expression in significantly higher molar yields of antigens useful for detecting antibodies specific to SfRV. The polypeptide.

2. the heteromultimerization domain is linked to the ectodomain via a linker moiety, or 2. The polypeptide of claim 1, wherein the heterologous multimerization domain is linked to the ectodomain via a peptide bond between the N-terminal amino acid residue of the heterologous multimerization domain and the C-terminal amino acid residue of the ectodomain.

3. Formula xyz (wherein, x consists of or comprises the ectodomain of a rhabdovirus glycoprotein, y is a linker moiety; 3. The polypeptide of claim 1, wherein z is a heterologous multimerization domain.

4. The ectodomain does not contain a furin cleavage site, The furin cleavage site is (a), (b), and (c) below: (a) an amino acid sequence selected from the group consisting of RXKR (SEQ ID NO:20) and RXRR (SEQ ID NO:21), where X can be any amino acid; (b) RX 1 KRX 2 (SEQ ID NO: 22) and RX 1 RRX 2 (SEQ ID NO: 23) X 1 can be any amino acid residue, X 2 teeth, - other than a lysine residue, or other than amino acid residues selected from the group consisting of valine, leucine, isoleucine and tryptophan residues, (can be any amino acid residue in the an amino acid sequence selected from the group consisting of: (c) RX 1 KRX 2 X 3 (SEQ ID NO: 24) and RX 1 RRX 2 X 3 (SEQ ID NO: 25) X 1 can be any amino acid residue, X 2 teeth, - other than a lysine residue, or - other than an amino acid residue selected from the group consisting of valine, leucine, isoleucine, and tryptophan residues and X 3 may be any amino acid residue other than a lysine residue) The polypeptide according to any one of claims 1 to 3, which has an amino acid sequence selected from the group consisting of:

5. the ectodomain comprises or consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1; The ectodomain is an amino acid residue other than the arginine residue at amino acid position 285, and / or an amino acid residue other than the arginine residue at amino acid position 282, and / or an amino acid residue at amino acid position 286 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - lysine residue at amino acid position 287 and and, an amino acid residue other than the arginine residue at amino acid position 312, and / or an amino acid residue other than the arginine residue at amino acid position 309, and / or an amino acid residue at amino acid position 313 selected from the group consisting of a lysine residue, a leucine residue, an isoleucine residue, a valine residue, and a tryptophan residue, and / or - lysine residue at amino acid position 314 and The polypeptide of any one of claims 1 to 4, wherein the numbering of amino acid positions refers to the amino acid sequence of SEQ ID NO:

1.

6. The polypeptide of any one of claims 1 to 5, wherein the heterologous multimerization domain comprises or consists of an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO:

6.

7. the linker portion is an amino acid sequence having a length of 1 to 50 amino acid residues; and / or the linker portion comprises or consists of an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11. The polypeptide of any one of claims 2 to 6.

8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide is a protein comprising a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:

14.

9. A polynucleotide encoding the polypeptide of any one of claims 1 to 8, wherein the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:

19.

10. A plasmid comprising a polynucleotide encoding the polypeptide according to any one of claims 1 to 8.

11. A cell comprising a plasmid containing a polynucleotide encoding the polypeptide according to any one of claims 1 to 8. or An insect cell infected with a baculovirus containing a polynucleotide encoding the polypeptide of any one of claims 1 to 8.

12. A baculovirus comprising a polynucleotide encoding the polypeptide of any one of claims 1 to 8.

13. A kit comprising the polypeptide according to any one of claims 1 to 8 immobilized on a solid support.

14. A method for producing the polypeptide according to any one of claims 1 to 8 in vitro, comprising: - transfecting a cell with a plasmid comprising a polynucleotide comprising a sequence encoding said polypeptide, said plasmid being a plasmid according to claim 10; or - infecting insect cells with a baculovirus containing a polynucleotide comprising a sequence encoding said polypeptide, said baculovirus being the baculovirus of claim 12. The method comprising:

15. 1. A method for determining the presence or absence of antibodies specific for a rhabdovirus in a biological sample obtained from an individual, comprising: a. contacting a biological sample with a capture reagent immobilized on a solid support, said capture reagent being a polypeptide according to any one of claims 1 to 8; b. determining the presence or absence of said antibody bound to said capture reagent; A method comprising:

16. 16. Use of a polypeptide according to any one of claims 1 to 8 in a method for determining the presence or absence of antibodies specific for a rhabdovirus in a biological sample obtained from an individual, said method being the method according to claim 15.