Labeling system for subunit antigen expressed by baculovirus and its use

Abstract

The present invention is in the field of compliance markers and labeled vaccines that allow differentiation between infected and vaccinated individuals. In particular, it relates to methods for determining whether an individual has received an immunogenic composition comprising a recombinant protein produced by a baculovirus expression system in cultured insect cells.

Description

[0001] The present application is a divisional application of Chinese patent application No. 201680013955.8, filed on March 4, 2016, entitled “Labeling system, in particular a labeling system for subunit antigens expressed by baculovirus”.

[0002] Sequence Listing

[0003] This application contains a Sequence Listing pursuant to 37 CFR 1.821-1.825. The Sequence Listing accompanying this application is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention belongs to the field of compliance markers and labeled vaccines allowing differentiation between infected individuals and vaccinated individuals. In particular, the present invention relates to compliance markers for vaccines comprising subunit antigens, and to the DIVA (Differentiation Infected From Vaccinated Animals) system making it possible to differentiate between animals infected with a pathogen and animals treated with subunit antigens derived from said pathogen, wherein said subunit antigens have been expressed in cultured insect cells, preferably by means of genetically engineered baculoviruses. Background Art

[0005] Baculoviruses are large rod-shaped double-stranded DNA viruses that infect invertebrates, especially insects, but do not replicate in mammalian or other vertebrate cells. They were first used as biopesticides in the field of crops starting in the 1940s. In addition, after publishing the first paper describing the overexpression of human beta interferon in insect cells (Smith et al. Mol Cell Biol. 3: 2156-2165 (1983)), genetically engineered baculoviruses have been widely used to produce complex recombinant proteins in insect cell cultures, including the production of antigens for many approved human and veterinary subunit vaccines (van Oers et al. J Gen Virol. 96: 6-23 (2015)).

[0006] Vaccination is an important tool for managing herd health, particularly in high-density confinement environments where many food animals are raised. When a disease outbreak occurs in supposedly vaccinated animals, the question arises whether the vaccine has failed to protect the animals or whether the vaccine was properly delivered, wherein the latter possibility regarding the proper delivery of the vaccine is referred to as vaccine compliance.

[0007] Therefore, the use of compliance markers for determining whether an animal has been properly vaccinated is highly desired by producers. WO 2009 / 058835 A1 describes, for example, the use of purified xylanase added as a compliance marker for swine flu vaccines. With respect to vaccines comprising baculovirus-expressed subunit antigens, it is possible to use baculovirus antigens as compliance markers, however, currently used systems have limitations in the number of animals that test positive and require large amounts of antigen (Gerber et al. Res Vet Sci. 95: 775-81 (2013); Lehnert. Top Agrar 5: S11-S14 (2011)).

[0008] Vaccines used in programs for controlling viral outbreaks and infections must have an effective system to monitor the persistence of viral infections within a population. However, vaccination complicates large-scale surveillance of the spread of infection by, for example, serological means, because both vaccinated individuals and exposed individuals produce antibodies specific to the virus. Antigenic similarities between infectious strong wild strains of the virus and viral vaccines often hinder the distinction between infected subjects and vaccinated subjects, because vaccination results in the appearance and retention of indistinguishable antibodies between infected individuals and vaccinated individuals.

[0009] There is increasing global interest in the DIVA (differentiation between infected and vaccinated animals) vaccination strategy. For example, a joint WHO / FAO / OIE meeting on the avian influenza strain H5N1 HPAI has recommended that all vaccinations be performed using DIVA so that the spread of infection can be monitored. However, current DIVA methods are difficult to scale up and often have problems distinguishing vaccination from infection with other spreading virus strains.

[0010] Current surveillance methods include physical marking of vaccinated animals, the use of sentinel animals, and virological testing. However, these methods currently have many limitations for logistical and economic reasons.

[0011] Physical marking of vaccinated animals involves time-consuming individual identification of vaccinated individuals by physical means such as ear tags, leg bands or ear tags. In addition, the use of unvaccinated sentinel animals is logistically and economically difficult, and there is also the risk that if the sentinel animals become infected with a virus, for example, poultry infected with the H5N1 virus, there is an increased risk of transmission to humans. Virological testing of individuals via screening and detection of live virus or RT-PCR monitoring tests is a very expensive and basic intensive process that is not suitable for subunit vaccines. It only provides information about the current status of the individual and does not allow analysis of the infection and / or vaccination history of the individual.

[0012] In view of such limitations, the use of marker vaccines that allow serological identification of vaccinated and infected animals is highly preferred, wherein such marker vaccines can be prepared as negative or positive marker vaccines.

[0013] Negative marker vaccines are prepared by using antigenic parts of the pathogen or by removing antigens from the pathogen that elicit specific antibodies in infected animals. Negative marker vaccines are usually subunit vaccines or live attenuated vaccines that contain genetically engineered strains that lack immunogenic antigens. Examples of negative marker vaccines are, for example, the use of baculovirus-expressed classical swine fever virus (CSFV) E2 protein as a subunit antigen for vaccination against classical swine fever, where other antigens of CSFV (e.g., E2) are expressed in the sera of vaccinated pigs. RNS Detection of antibodies specific for NS1 protein or NS3 protein indicates CSFV infection.

[0014] Positive marker vaccines contain additional antigens that induce specific antibodies in vaccinated individuals, but not in infected individuals. An example of a positive marker vaccine approach is described in WO 2007 / 053899 A1, where inactivated H6N2 avian influenza (A1) virus and tetanus toxin, both produced separately, are combined in one injection for vaccinating poultry, after which antibodies specific for tetanus toxin are detected in serum obtained from the poultry as a marker showing that the poultry was vaccinated.

[0015] However, the separate production of both vaccine antigen and marker antigen is relatively expensive and, moreover, requires a mixing step for combining both components in one vaccination, wherein this additionally may also influence the stability of the vaccine components / antigens.

[0016] Therefore, there is a need for simple labeling systems for the inexpensive production of positively labeled vaccines, such as subunit vaccines comprising baculovirus-expressed antigens.

[0017] Furthermore, there is a need for efficient adherence markers that also enable sensitive identification of vaccinations with small amounts of baculovirus-expressed subunit antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Run the amplified products on a gel to verify the size.

[0019] Figure 2 : Run the cut insert and vector on a gel to check the linearization of the vector.

[0020] Figure 3 : Positive clones from wells 2, 5, 7, 10, 11 and 12 were sequenced and contigs were aligned to the construct reference sequence to check for mutations; all clones were perfect matches.

[0021] Figure 4 : Western blot evaluating the expression of G gene constructs.

[0022] Figure 5 : The supernatant, soluble and insoluble cell fractions were probed for protein; here the protein was present in the insoluble fraction.

[0023] Figure 6 : Results of a size exclusion chromatography run using isocratic conditions on an AKTA. The elution of the protein from the column was monitored by UV absorption at 280 nm.

[0024] Figure 7 : Real-time PCR results: Column 1 indicates the well number. Column 2 shows the fluorophore 6-carboxyfluorescein (FAM) used, which is linked to a specific probe. Column 3 indicates the fractions of SfRV antigen derived from size exclusion chromatography (fractions A11, A12, B1, B5, B12, and C6) or standards with known amounts of SfRV-specific nucleic acid used to generate a standard curve (wells 7-14). Well 15 is a negative control (no template), and well 16 is a positive control containing concentrated SfRV antigen before separation by size fractionation (SEC).

[0025] Figure 8 : ELISA; ELISA plates were coated with four different antigens including semi-purified SfRV (panel A), size exclusion fractions A11 (panel B), A12 (panel C) and B1 (panel D). Plates were probed with sera from negative control animals (inverted triangles) or day 28 sera from animals administered an experimental vaccine containing SfRV (circles). Summary of the invention

[0026] The solution to the above technical problem is achieved by the embodiments characterized in the description and claims.

[0027] Thus, the invention in its different aspects is implemented according to the claims.

[0028] The present invention is based on the surprising discovery that the use of rhabdovirus infected SF+ cells for the production of baculovirus expressed antigens enables cheap and efficient production of positively labeled vaccines and easy and production-intrinsic compliance markers, which allows a sensitive method to demonstrate proper delivery of subunit vaccines.

[0029] In a first aspect, the present invention therefore provides a method for determining whether an individual has received an immunogenic composition or vaccine comprising a recombinant protein produced by an expression system, preferably a baculovirus expression system, in cultured insect cells, wherein the method comprises the steps of: obtaining a biological sample from an individual; and determining in the biological sample the presence or absence of one or more markers indicating that the individual has received one or more antigens from a virus, which is an RNA virus capable of infecting insect cells, wherein the presence of the one or more markers in the biological sample indicates that the individual has received the immunogenic composition, or wherein the absence of the one or more markers in the biological sample indicates that the individual has not received the immunogenic composition.

[0030] The term "recombinant protein" as used herein refers in particular to a protein molecule expressed by a recombinant DNA molecule, such as a polypeptide produced by recombinant DNA technology. Examples of such technology include when the DNA encoding the expressed protein is inserted into a suitable expression vector, preferably a baculovirus expression vector (which is in turn used for transfection), or when the baculovirus expression vector infects a host cell to produce a protein or polypeptide encoded by the DNA. Therefore, the term "recombinant protein" as used herein refers in particular to a protein molecule expressed by a recombinant DNA molecule.

[0031] According to a specific example, a recombinant protein is produced by a method having the following steps: a gene encoding a protein is cloned into a baculovirus transfer vector; the transfer vector is used to prepare a recombinant baculovirus containing the gene by homologous recombination in insect cells; and the protein is then expressed in insect cells during infection with the recombinant baculovirus.

[0032] According to an alternative example, the recombinant protein is expressed from a recombinant expression plasmid in insect cells. In the case of this alternative example, a baculovirus is not required.

[0033] It is further understood that the term "recombinant protein consisting of a sequence" also particularly relates to any co-translational and / or post-translational modification or modification of the sequence effected by the cell in which the polypeptide is expressed. Thus, the term "recombinant protein consisting of a sequence" as described herein also relates to a sequence having one or more modifications effected by the cell in which the polypeptide is expressed, in particular modifications of the amino acid residues effected in protein biosynthesis and / or protein processing, preferably selected from glycosylation, phosphorylation and acetylation.

[0034] Preferably, the recombinant protein of the invention is produced or obtainable by a baculovirus expression system, in particular in cultured insect cells.

[0035] The term "expression system" as used herein specifically includes vehicles or vectors used to express genes in host cells, as well as vehicles or vectors that result in stable integration of genes into host chromosomes.

[0036] As used herein, "baculovirus expression system" refers specifically to a system for producing a desired protein in insect cells using a recombinant baculovirus vector designed to express the protein. The baculovirus expression system generally contains all the elements necessary to achieve recombinant protein expression in insect cells, and generally involves engineering a baculovirus vector to express the desired protein, introducing the engineered baculovirus vector into insect cells, culturing the insect cells containing the engineered baculovirus vector in a suitable growth medium so that the desired protein is expressed, and recovering the protein. Typically, engineering baculovirus vectors involves the construction and isolation of recombinant baculoviruses in which the coding sequence of a selected gene is inserted behind the promoter of a non-essential viral gene, with most currently used baculovirus expression systems being based on the sequence of Autographa californica nuclear polyhedrosis virus (AcMNPV) (Virology 202(2), 586-605 (1994), NCBI Accession No.: NC_001623). Baculovirus expression systems are well known in the art and have been described, for example, in "Baculovirus Expression Vectors: A Laboratory Manual", David R. O'Reilly, Lois Miller, Verne Luckow, published by Oxford Univ. Press (1994), "The Baculovirus Expression System: A Laboratory Guide", Linda A. King, RD Possee, published by Chapman & Hall (1992). Illustrative, non-limiting examples of baculovirus systems for producing recombinant proteins are described, for example, in WO 99 / 03668. 2006 / 072065 A2.

[0037] According to said first aspect, the present invention therefore provides a method for determining whether an individual has received an immunogenic composition comprising a recombinant protein produced by an expression system in cultured insect cells, said method being hereinafter also referred to as the “method of the present invention”, wherein said method in particular comprises determining in a biological sample obtained from said individual the presence or absence of one or more markers indicating that said individual has received one or more antigens from a virus, said virus being an RNA virus capable of infecting insect cells, and wherein the presence of said one or more markers in said biological sample indicates that said individual has received said immunogenic composition.

[0038] As used herein, "insect cell" means a cell or cell culture derived from an insect species. Of particular interest with respect to the present invention are insect cells derived from the species Spodoptera frugiperda and Trichoplusia ni.

[0039] As used herein, "virus capable of infecting insect cells" is particularly understood as a virus that carries structures on the viral surface that are capable of interacting with insect cells to such an extent that the virus or at least the viral genome becomes incorporated into the insect cell.

[0040] The infection of insect cells more specifically includes viral attachment to host cells, viral entry into cells, uncoating of virions in the cytoplasm, replication and transcription of the viral genome, expression of viral proteins, and assembly and release of new infectious viral particles.

[0041] Preferably, the immunogenic composition of the present invention is a marker vaccine, in particular a positive marker vaccine.

[0042] The term "marker vaccine" as used herein specifically designates a vaccine that causes immunity in an immune organism that is different from the immunity in the organism caused by the actual pathogen.

[0043] A "positive marker vaccine" relates in particular to a marker vaccine containing an additional antigen which induces the production of specific antibodies which are present in vaccinated individuals but in non-infected individuals.

[0044] The term "marker" as used in the context of the present invention is preferably equivalent to the term "biomarker" and particularly refers to a measurable substance or compound (which indicates that an individual has been exposed to an immunogenic composition), preferably a positive marker vaccine, or more specifically, to additional antigens of a positive marker vaccine that induce the production of specific antibodies found in vaccinated subjects but not in infected subjects.

[0045] As used herein, the term "immunogenic composition" refers in particular to a composition that will elicit an immune response in an individual that has been exposed to the composition. The immune response may include the induction of antibodies and / or the induction of a T-cell response. Depending on the intended function of the composition, one or more antigens may be included. Preferably, the immunogenic composition as described herein is a vaccine.

[0046] The term "vaccine" as used herein is defined in accordance with the relevant art and relates to a composition that induces or enhances an individual's immunity to a particular disease. To this end, a vaccine comprises a compound that is similar to a pathogen or a compound of the pathogen that causes the disease. Upon contact with the compound, the individual's immune system is triggered to recognize the compound as a foreign substance and destroy it. The immune system then memorizes the contact with the compound, so that subsequent contact with the pathogen that causes the disease ensures easy and effective recognition and destruction of the pathogen. According to the present invention, the vaccine may be in any formulation of a vaccine known in the art, such as, for example, a vaccine for intramuscular injection, a mucosal vaccine or a vaccine for subcutaneous or intradermal injection, and a vaccine for inhalation, such as, for example, as an aerosol. Such vaccine formulations are well known in the art and have been described, for example, in Neutra MR et al. 2006 Mucosal vaccines: the promise and the challenge 6(2):148-58 or FP Nijkamp, ​​Michael J. Parnham 2011; Principles of Immunopharmacology ISBN-13:978-3034601351.

[0047] Therefore, the method of the present invention is particularly a method for determining whether an individual has received an immunogenic composition comprising a recombinant protein produced by a baculovirus expression system in cultured insect cells, wherein the method comprises determining the presence or absence of one or more markers indicating that the individual has received one or more antigens from a virus, which is an RNA virus capable of infecting insect cells, in a biological sample obtained from the individual, and wherein the presence of the one or more markers in the biological sample indicates that the individual has received the immunogenic composition.

[0048] Preferably, the biological sample is obtained from the individual at least 14 days and most preferably 14 to 35 days after the day on which the individual has been vaccinated or, respectively, has been presumed to have been vaccinated.

[0049] Preferably, the insect cell as mentioned herein is a Fall Armyworm (Sf) cell or a cell from a cell line derived from Fall Armyworm, and more preferably is selected from Sf9 cells and Sf+ cells. Respectively, the insect cell as mentioned herein is a Fall Armyworm (Sf) cell or a cell from a cell line derived from Fall Armyworm, and more preferably is selected from Sf9 cells and Sf+ cells.

[0050] As mentioned herein, the one or more markers showing that an individual has received one or more antigens from an RNA virus capable of infecting insect cells (hereinafter also referred to as "one or more markers of the present invention") are preferably one or more markers selected from the following: antibodies specific for one or more antigens from a virus that is an RNA virus capable of infecting insect cells; one or more antigens from a virus that is an RNA virus capable of infecting insect cells; and one or more nucleic acid molecules specific for an RNA virus capable of infecting insect cells.

[0051] Most preferably, the one or more markers of the invention are antibodies specific for an antigen from a virus that is an RNA virus capable of infecting insect cells.

[0052] Preferably, the antibodies as described herein are polyclonal antibodies.

[0053] As used herein, the term "antibody specific for a particular antigen" refers specifically to an antibody specific for an antigen with a specificity of, for example, greater than or equal to about 10 5 M -1 , 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 Affinity or K a (i.e., the equilibrium association constant for a specific binding interaction in units of 1 / M) of an antibody, preferably a polyclonal antibody, that binds to the antigen. Alternatively, binding affinity can be defined as having units of M (e.g., 10 -5 M to 10 -13 The equilibrium dissociation constant (K) of a specific binding interaction d The binding affinity of an antibody can be readily determined using techniques well known to those skilled 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; analysis; or equivalent).

[0054] As mentioned herein, the one or more antigens from RNA viruses capable of infecting insect cells (which are also referred to as "one or more antigens of the present invention") are preferably proteins comprising a 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 with the sequence of SEQ ID NO: 1, or consisting of a sequence thereof, and / or proteins comprising a 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 with the sequence of SEQ ID NO: 7, or consisting of a sequence thereof.

[0055] Concerning the term "at least 90%" as mentioned in the context of the present invention, it should be understood that the term preferably relates to "at least 91%", more preferably "at least 92%", still more preferably "at least 93%", or in particular "at least 94%".

[0056] Concerning the term "at least 95%" as mentioned in the context of the present invention, it should be understood that the term preferably relates to "at least 96%", more preferably "at least 97%", still more preferably "at least 98%", or in particular "at least 99%".

[0057] As used herein, the term "having 100% sequence identity" should be understood to be equivalent to the term "identical".

[0058] As used herein, the term "antigen" refers in particular to any molecule, part or entity capable of eliciting an immune response. This includes cellular and / or humoral immune responses.

[0059] Percent sequence identity has a meaning recognized in the art, and there are a variety of methods for measuring the 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 are encoded 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 using the FASTA algorithm can be used, which has an affine gap search, a gap opening penalty of -12, and a gap extension penalty of -2.For the purposes of the present invention, nucleotide sequences were aligned using the Clustal W method in DNASTAR Inc's MegAlign software version 11.1.0 (59), 419, using the default multiple alignment parameter set in the program (gap penalty = 15.0, gap length penalty = 6.66, delayed divergent sequence (%) = 30%, DNA transfer weight = 0.50 and DNA weight matrix = IUB), and protein / amino acid sequences were aligned, respectively, using the Clustal W method in DNASTAR Inc's MegAlign software version 11.1.0 (59), 419, using the default multiple alignment parameter set in the program (Gonnet series protein weight matrix with gap penalty = 10.0, gap length penalty = 0.2 and delayed divergent sequence (%) = 30%).

[0060] As used herein, it is specifically understood that the term "sequence identity to the sequence of SEQ ID NO: X" is equivalent to the term "sequence identity to the sequence of SEQ ID NO: X over the length of SEQ ID NO: X" or the term "sequence identity to the sequence of SEQ ID NO: X over the entire length of SEQ ID NO: X". In this context, "X" is any integer selected from 1 to 24, such that "SEQ ID NO: X" represents any SEQ ID NO mentioned herein.

[0061] The one or more nucleic acid molecules specific for RNA viruses capable of infecting insect cells as mentioned herein (hereinafter also referred to as "one or more nucleic acid molecules according to the present invention") are preferably nucleic acid molecules encoding: a protein comprising a 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 with the sequence of SEQ ID NO: 1, and / or; a protein comprising a 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 with the sequence of SEQ ID NO: 7, or consisting of the same; and / or an RNA having a sequence that is reverse complementary to a nucleic acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or in particular 100% sequence identity with the sequence of SEQ ID NO: 9; and / or a sequence that is reverse complementary to a nucleic acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or in particular 100% sequence identity with the sequence of SEQ ID NO: 10. The sequence of NO: 15 has at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity.

[0062] Preferably, the method of the present invention comprises the steps of: contacting a biological sample with a capture reagent immobilized to a solid support, wherein the immobilized capture reagent is capable of binding to one or more markers of the present invention; and determining the presence or absence of the one or more markers bound to the capture reagent, wherein the presence of the one or more markers bound to the capture reagent indicates the presence of the one or more markers in the biological sample.

[0063] The term "capture agent" as used herein refers in particular to a molecule or multimolecular complex that can bind to a marker. The capture agent is preferably capable of binding to the marker in a substantially specific manner, preferably with an affinity or Ka>10 5 M -1 Or preferably >10 6 M -1 Binding markers. The capture agent may optionally be a naturally occurring, recombinant or synthetic biomolecule. Protein and nucleic acid ligands (aptamers) are very suitable as capture agents. Whole viruses or viral fragments or synthetic peptides may also serve as preferred capture agents because they can bind antibodies.

[0064] As used herein, the term "immobilized" specifically means that the capture agent can be attached to a surface (eg, a solid support) in any manner or by any method; including, for example, reversible or irreversible binding, covalent or non-covalent attachment, and the like.

[0065] The capture agent mentioned herein that is fixed to a solid support and is capable of binding to one or more markers of the present invention (wherein the capture agent is also referred to hereinafter as “capture agent according to the present invention”) is preferably selected from: a protein comprising an amino acid 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 with the sequence of any one of SEQ ID NOs: 1 to 6, or consisting of the same; a protein comprising an amino acid 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 with the sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or consisting of the same; an RNA virus capable of infecting insect cells, wherein the virus is optionally inactivated; an oligonucleotide capable of specifically hybridizing with a characteristic sequence of the sequence of SEQ ID NO: 9; and an oligonucleotide capable of specifically hybridizing with a characteristic sequence of the sequence of SEQ ID NO: 15.

[0066] The term "specific hybridization" as used herein particularly relates to hybridization under stringent conditions. The hybridization conditions can be established according to conventional protocols, such as those described in Sambrook, "Molecular Cloning, A Laboratory Handbook", 2nd edition (1989), CSH Press, Cold Spring Harbor, NY; Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989); or Higgins and Hames (eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC (1985). An example of specific hybridization conditions is hybridization at 65°C in 4×SSC and 0.1% SDS, followed by washing in 0.1×SSC, 0.1% SDS at 65°C. Alternatively, stringent hybridization conditions are, for example, 50% formamide, 4×SSC at 42°C.

[0067] The term "solid support" as referred to herein means a non-fluid substance, and includes chips, containers and particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass and ceramics; gel substances such as silica, alumina and polymer gels; capillaries, which can be made of polymers, metals, glass and / or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes or other spectrometer sample containers. The solid support component of the assay is different from the inert solid surface with which the assay can be contacted in that the "solid support" contains at least one portion on its surface that is intended to interact directly or indirectly with the capture reagent. The solid support can be a fixed component, such as a tube, strip, cuvette or microtiter plate, or can be a non-fixed component, such as beads and microparticles. Microparticles can also be used as solid supports for homogeneous assay formats. Various microparticles that allow non-covalent or covalent attachment of proteins and other substances can be used. 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.

[0068] A "chip" is a solid, non-porous material, such as metal, glass or plastic. The material may optionally be coated throughout or in certain areas. On the surface of the material, there is any array of points, visible or in coordinates. At each point, a restricted polypeptide with or without a linker or spacer may be fixed to the surface of the material. All documents mentioned herein are incorporated herein by reference.

[0069] The RNA virus capable of infecting insect cells as mentioned herein (hereinafter also referred to as "the RNA virus of the present invention") is preferably: a (-)ssRNA virus, and optionally a virus belonging to the family Rhabdoviridiae; and / or a virus comprising 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% or in particular 100% sequence identity with the sequence of SEQ ID NO: 1, and / or; the 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% or in particular 100% sequence identity with the sequence of SEQ ID NO: 7; and / or a virus whose genome comprises a nucleic acid molecule encoding a protein comprising or consisting of a 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 with the sequence of SEQ ID NO: 1, and / or; comprising NO:7 has a sequence with at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity or a protein consisting of the same; and / or, a virus whose genome comprises an RNA molecule, the RNA molecule having a sequence that is reverse complementary to a nucleic acid sequence, the nucleic 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 the sequence of SEQ ID NO:9; and / or, a sequence that is reverse complementary to a nucleic acid sequence, the nucleic 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 the sequence of SEQ ID NO:15.

[0070] All nucleotide sequences of the sequence listing are entered in the 5'-'3 direction. The sequences of SEQ ID NO: 9 and 15 encode cDNAs with positive polarity (+ strand). The term "reverse complement" means that the sequence is antiparallel to the reference sequence.

[0071] The RNA virus according to the present invention is preferably capable of replicating in an insect cell line for at least two weeks or more preferably at least three weeks.

[0072] Preferably, the method of the present invention comprises determining the presence or absence of one or more markers of the present invention in a biological sample, wherein the marker is an antibody specific for a protein comprising a 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 the sequence of SEQ ID NO: 1 or consisting thereof; or an antibody specific for a protein comprising a 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 the sequence of SEQ ID NO: 7 or consisting thereof; and wherein the method comprises the following steps:

[0073] a. contacting the biological sample with a capture agent immobilized to a solid support, wherein the capture agent is selected from a sequence comprising a 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 of any one of SEQ ID NOs: 1 to 6 or a 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 of SEQ ID NO: 7 or SEQ ID NO: 8, optionally an inactivated virus containing 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% or in particular 100% sequence identity to a sequence of SEQ ID NO: 1, and / or 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% or in particular 100% sequence identity to a sequence of SEQ ID NO: 7; a virus whose genome comprises a nucleic acid molecule encoding a sequence comprising a nucleic acid molecule having a sequence of SEQ ID NO: NO:1 or a protein 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% or in particular 100% sequence identity to the sequence of SEQ ID NO:7, wherein the virus is optionally inactivated; a virus whose genome comprises an RNA molecule comprising a sequence that is the reverse complement of a nucleic acid 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 the sequence of SEQ ID NO:9, and / or a virus whose genome comprises an RNA molecule comprising a sequence that is the reverse complement of a nucleic acid 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 the sequence of SEQ ID NO:15, wherein the virus is optionally inactivated;

[0074] b. separating the biological sample from the immobilized capture reagent;

[0075] c. contacting the immobilized capture reagent-antibody complex with a detectable reagent that binds to the antibody of the reagent-antibody complex; and

[0076] d. Measuring the level of antibody bound to the capture reagent using a detection method with a detectable reagent, and wherein the measuring step (d) preferably further comprises comparing with a standard curve to determine the level of antibody bound to the capture reagent.

[0077] Preferably, the detectable agent that binds to the antibody of the agent-antibody complex is a detectable antibody, more preferably a labeled secondary antibody.

[0078] The capture agent as described herein is preferably a baculovirus-expressed protein, and the baculovirus-expressed protein is preferably expressed by the baculovirus of the present invention as described herein below.

[0079] According to another preferred aspect of the present invention, one or more markers of the present invention can also be one or more T cells specific for RNA viruses according to the present invention and / or one or more B cells specific for RNA viruses according to the present invention and / or one or more antigen presenting cells presenting one or more antigens according to the present invention. The presence or absence of the one or more B cells and / or the one or more T cells and / or the one or more antigen presenting cells is preferably determined by flow cytometry analysis, and wherein in particular, one or more fluorescently labeled antigens according to the present invention are used to mark the one or more B cells and / or the one or more T cells and / or wherein one or more fluorescently labeled antibodies specific for RNA viruses according to the present invention are used to mark the one or more antigen presenting cells.

[0080] The recombinant protein produced by the expression system in cultured insect cells as mentioned herein (hereinafter also referred to as "the recombinant protein of the present invention") is preferably a PCV2 ORF2 protein, and the PCV2 ORF2 protein is particularly a protein having at least 90%, preferably at least 91%, more preferably at least 92%, still more preferably at least 93%, in particular at least 94% or at least 95% sequence identity with the sequence of SEQ ID NO: 23.

[0081] According to another preferred aspect, the recombinant protein of the present invention is an influenza hemagglutinin, in particular an avian influenza hemagglutinin, wherein the avian influenza hemagglutinin is preferably the H5 protein of the H5N1 virus, and wherein the H5 protein of the H5N1 virus is more preferably 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% or especially 100% sequence identity with the sequence of SEQ ID NO: 24.

[0082] The method of the present invention preferably also includes a step of determining in a biological sample the presence of one or more analytes selected from the group consisting of: antibodies specific to the recombinant protein of the present invention, polypeptides specific to the recombinant protein of the present invention, polypeptides specific to the DNA sequence encoding the recombinant protein of the present invention.

[0083] In the context of the method of the invention, the immunogenic composition is preferably an immunogenic composition as described below.

[0084] As used herein, the term "biological sample" refers to any sample taken from an individual (e.g., from a pig or bird), and includes, but is not limited to, body fluids containing cells, peripheral blood, plasma or serum, saliva, tissue homogenates, lung and other organ aspirates, and lavage and enema solutions and any other source obtainable from a human or animal subject. For animals, examples of "biological samples" include blood, cells, feces, diarrhea, milk, mucus, sputum, pus, saliva, semen, sweat, tears, urine, tears, eye fluids, vaginal secretions, and vomitus, if present in the animal.

[0085] The biological sample as referred to herein is preferably isolated from a mammal or bird, preferably a pig or chicken (Gallus gallus domesticus), and / or is particularly selected from whole blood, plasma, serum, urine and oral fluid. Herein, the term "serum" is intended to be equivalent to "blood serum".

[0086] The term "oral fluid" as used herein refers in particular to one or more fluids found in the oral cavity, either alone or in combination. These include, but are not limited to, saliva and mucosal exudates. It is particularly understood that oral fluid can comprise a combination of fluids from multiple sources (e.g., parotid, submandibular, sublingual, accessory glands, gingival mucosa, and buccal mucosa), and the term "oral fluid" includes fluids from each of these sources, either alone or in combination. The term "saliva" refers to a combination of oral fluids such as those found typically in the mouth (particularly 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 exudate usually forms a component of saliva.

[0087] The immobilized capture reagent as described herein is preferably coated on a microtiter plate, particularly a microtiter plate that can be read by an ELISA reader.

[0088] According to another aspect, the present invention provides a recombinant baculovirus, wherein the baculovirus comprises a DNA sequence encoding 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% or in particular 100% sequence identity to the sequence of any one of SEQ ID NOs: 1 to 6, and / or, 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% or in particular 100% sequence identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and / or, wherein the baculovirus comprises a DNA sequence comprising a 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 the sequence of any one of SEQ ID NOs: 9 to 14, and / or, 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% or in particular 100% sequence identity to the sequence of SEQ ID NO: 15 or SEQ ID NO: 16. The sequence of NO: 16 has or consists of a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity.

[0089] The present invention also provides a vector, in particular a transfer vector, comprising a DNA sequence encoding 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% or in particular 100% sequence identity to the sequence of any one of SEQ ID NOs: 1 to 6, and / or 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% or in particular 100% sequence identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and / or comprising a DNA 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 the sequence of any one of SEQ ID NOs: 9 to 14, and / or a DNA 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 the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0090] The transfer vector in the context of the present invention is preferably a "baculovirus transfer vector".

[0091] The term "transfer vector" is art-recognized and refers to a first nucleic acid molecule to which a second nucleic acid has been linked, and includes, for example, a plasmid, a cosmid, or a bacteriophage.

[0092] In certain embodiments, the transfer vector can be an "expression vector", which refers to a reproducible DNA construct for expressing a DNA encoding a desired protein, and it includes a transcription unit, which comprises the following assembly: (i) a genetic element with a regulatory effect in gene expression, such as a promoter, an operator or an enhancer, operably linked to a DNA sequence encoding the desired protein that is transcribed into mRNA and translated into protein, and (iii) appropriate transcription and translation start and stop sequences. The selection of promoters and other regulatory elements usually varies according to the intended host cell. Generally, the expression vector used in recombinant DNA technology is usually in the form of a "plasmid", which refers to a circular double-stranded DNA loop that is not combined with a chromosome in its vector form. The present invention is intended to include such other forms of expression vectors, which play equivalent functions and become known in the art subsequently.

[0093] Certain transfer vectors may contain regulatory elements for controlling transcription or translation, which are generally derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and selection genes that facilitate recognition of transformants may also be additionally incorporated.

[0094] Transfer vectors derived from viruses, which may be referred to as "viral vectors," may be employed in certain embodiments of the present invention. Some examples include baculovirus, retrovirus, adenovirus, etc. Viral vectors, particularly baculovirus vectors, such as baculovirus transfer vectors, are particularly preferred according to the present invention. For expression vectors, the viral vector may include regulatory elements.

[0095] The design of any transfer vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers (e.g., ampicillin), may also be considered.

[0096] In another aspect, the present invention provides an immunogenic composition, which is also referred to hereinafter as the "immunogenic composition of the present invention", wherein the composition comprises a recombinant protein produced by a baculovirus expression system in cultured insect cells; and one or more antigens from an RNA virus according to the present invention, wherein the virus is preferably inactivated; and wherein the recombinant protein is preferably selected from the group consisting of a PCV2 ORF2 protein, which preferably comprises or consists of a sequence having at least 90%, preferably at least 91%, more preferably at least 92%, still more preferably at least 93% or in particular at least 94% or at least 95% sequence identity to the sequence of SEQ ID NO: 23; and an influenza hemagglutinin, in particular an avian influenza hemagglutinin, preferably the H5 protein of an H5N1 virus, more preferably comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity to the sequence of SEQ ID NO: 24; and a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity to the sequence of SEQ ID NO: 25. The sequence of any one of NO: 1 to 8 has an amino acid sequence or a protein consisting thereof having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or especially 100% sequence identity.

[0097] As used herein, the term "inactivated" means that the antigen does not cause disease when administered to a mammalian host or does not replicate in the host cells.

[0098] Various physical and chemical methods of inactivation are known in the art. The term "inactivation" refers to a previously toxic or non-toxic virus that has been irradiated (ultraviolet (UV), X-rays, electron beams or gamma radiation), heated or chemically treated to inactivate, kill, while retaining its immunogenicity. In one embodiment, the inactivated virus disclosed herein is inactivated by treatment with an inactivating agent. Suitable inactivating agents include β-propiolactone, dibasic or β- or acetyl imide, glutaraldehyde, ozone and formalin (formaldehyde).

[0099] For inactivation with formalin or formaldehyde, formaldehyde is usually mixed with water and methanol to produce formalin. Methanol is added to prevent degradation or cross-reactions during the activation process.

[0100] More specifically, the term "inactivated" means that the virus cannot replicate in vivo or in vitro. For example, the term "inactivated" can refer to a virus that has been propagated in vitro, for example, and then inactivated using chemical or physical means so that it can no longer replicate.

[0101] Preferably, the virus according to the invention which has been inactivated is a virus inactivated with binary ethyleneimine (BEI).

[0102] The present invention also provides a method for producing the immunogenic composition of the present invention, which comprises the following steps: introducing a recombinant baculovirus encoding the recombinant protein into insect cells, wherein the insect cells are infected with the RNA virus capable of infecting insect cells, culturing the insect cells carrying the recombinant baculovirus and the RNA virus; and recovering the recombinant protein and the virus, preferably in the supernatant; and preferably also comprising an initial step of inserting the DNA sequence encoding the recombinant protein into a transfer vector capable of introducing the sequence into the baculovirus genome, thereby producing a recombinant baculovirus.

[0103] According to another aspect, the present invention provides a kit for determining whether an individual has received an immunogenic composition comprising a recombinant protein produced by a baculovirus expression system in cultured insect cells, wherein the kit contains one or more capture agents immobilized to a solid support, wherein the one or more immobilized capture agents are capable of binding to one or more markers selected from antibodies specific for one or more antigens according to the present invention; one or more antigens from an RNA virus according to the present invention; and one or more nucleic acid molecules according to the present invention; and wherein the one or more capture agents are preferably selected from a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity to the sequence of any one of SEQ ID NOs: 1 to 6; a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or in particular 100% sequence identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; an RNA virus capable of infecting insect cells, wherein the virus has optionally been inactivated, an oligonucleotide capable of specifically hybridizing to a sequence characteristic of SEQ ID NO: 9; and an oligonucleotide capable of specifically hybridizing to a sequence characteristic of SEQ ID NO: 10. An oligonucleotide that specifically hybridizes to the characteristic sequence of NO:15.

[0104] Furthermore, the present invention provides a primer or a primer pair, each selected from sequences having at least 90% or preferably at least 95% sequence identity with the sequence of any one of SEQ ID NOs: 17 to 22.

[0105] According to another aspect, the capture agent of the present invention comprises or consists of virus particles and / or virus-like particles of an RNA virus capable of infecting insect cells, wherein the RNA virus capable of infecting insect cells is preferably an RNA virus according to the present invention, wherein the capture agent can be obtained by a method comprising the following steps:

[0106] i) obtaining a supernatant from a culture of insect cells infected with an RNA virus, preferably with an RNA virus of the present invention, wherein the supernatant comprises viral particles and / or virus-like particles of the RNA virus, and wherein the insect cells are preferably not infected with a baculovirus and / or preferably not transfected with a plasmid,

[0107] ii) separating the cell debris from the viral particles and / or virus-like particles via a separation step comprising microfiltration through at least one filter, preferably two filters, wherein at least one filter preferably has a larger pore size than the viral particles and / or virus-like particles, in particular having a pore size of about 0.1 μm to about 4 μm, preferably about 0.2 μm to about 2 μm, and collecting the filtrate,

[0108] iii) and optionally subjecting the filtrate of ii) containing said viral particles and / or virus-like particles to size exclusion chromatography, wherein the presence of the protein in the eluate is preferably measured by measuring the absorbance of the eluate at 260 nm or 280 nm (A260 or A280), and wherein the eluate exhibiting the first A260 or A280 peak is collected.

[0109] Furthermore, the present invention provides a composition comprising the capture agent, wherein the composition is obtainable by the method.

[0110] In the size exclusion chromatography (SEC) step described herein, molecules are separated according to size in a bed filled with an inert porous medium, particularly an inert gel medium, which is preferably a complex of cross-linked polysaccharides (e.g., cross-linked agarose and dextran) in the form of spherical beads. Molecules larger than the largest pores in the swollen gel beads do not enter the gel beads and therefore move most rapidly through the chromatographic bed. Smaller molecules that enter the gel beads to varying degrees depending on their size and shape are delayed in their passage through the bed. Therefore, molecules are generally eluted in order of decreasing molecular size. A SEC column containing a medium suitable for size exclusion chromatography as described herein is preferably in a HiPrep 26 / 60 Sephacryl S300HR column (GE Healthcare Bio-Sciences).

[0111] It is particularly understood that the eluate exhibiting the first A260 or A280 peak is the fraction of the filtrate of ii) that contains the largest protein structure included in the filtrate of ii). Therefore, the eluate exhibiting the first A260 or A280 peak is the eluate or a portion thereof that contains most of the virus particles and / or virus-like particles included in the filtrate of ii). Example

[0112] The following examples are intended only to illustrate the present invention. They should not limit the scope of the claims in any way.

[0113] Embodiment 1:

[0114] Infection of Sf cells with rhabdovirus, production of semi-purified rhabdovirus, and cloning and expression of rhabdovirus antigens

[0115] To confirm infection of SF+ and Sf9 cells with rhabdovirus (hereinafter also referred to as SfRV or SFRV (Sf cell rhabdovirus)), primers were designed to amplify the SFRV G and N genes with the purpose of inserting unique 5' and 3' restriction sites. In addition, the 3' end primer was designed to add a tobacco etch virus (TEV) protease cleavage site followed by a 6X histidine tag. This was done to enable purification of the expressed protein on a nickel column using the His tag, which was then cleaved off using TEV protease to generate native G or N protein.

[0116] The sequences of the primers used for the G gene construct (comprising the sequence of SEQ ID NO: 1) are the sequences shown in SEQ ID NOs: 17 and 18, the nucleic acid sequence of the G gene construct is provided in SEQ ID NO: 12, and the amino acid sequence of the G gene construct is the sequence of SEQ ID NO: 4.

[0117] The sequences of the primers used for the N gene construct (comprising the sequence of SEQ ID NO:7) are the sequences shown in SEQ ID NOs:21 and 22, the nucleic acid sequence of the N gene construct is provided in SEQ ID NO:16, and the amino acid sequence of the N gene construct is the sequence of SEQ ID NO:8.

[0118] In addition, TMpred (http: / / www.ch.embnet.org / software / TMPRED_form.html) described in K. Hofmann & W. Stoffel (1993) TMbase-A database of membrane spanning proteins segments, Biol. Chem. Hoppe-Seyler 374, 166, was used. S1, Croning MD, Apweiler R., Evaluation of methods for the prediction of membrane spanning regions, Bioinformatics (2001) 17 (7): 646-653 described in the hidden Markov model TMHMM (http: / / www.cbs.dtu.dk / services / TMHMM / ) and SOSUI (http: / / harrier.nagahama-i-bio.ac.jp / sosui / ) predict the transmembrane and intracellular domains of the SFRV G glycoprotein. Based on the results of TMpred and TMHMM, the SFRV G sequence terminates at amino acid 550 and adds a TEV cleavage site, a 6X His tag, and a Pst I site. The sequences of primers that can be used for such G gene constructs (comprising the sequence of SEQ ID NO: 2) are the sequences shown in SEQ ID NOs: 17 and 19, the nucleic acid sequence of the G gene construct is provided in SEQ ID NO: 13, and the amino acid sequence of the G gene construct is the sequence of SEQ ID NO: 5.

[0119] In addition, the sequence of the honey bee melittin secretion signal was fused to the sequence of the truncated SFRV G sequence (Chouljenko et al. J Virol, 84: 8596-8606 (2010); Tessier et al. Gene. 98: 177-83 (1991)), wherein the melittin sequence was added to the full-length SFRV G with TEV cleavage and 6X his by replacing its N-terminus. The sequences of primers that can be used for such G gene constructs (comprising the sequence of SEQ ID NO: 3) are the sequences shown in SEQ ID NOs: 20 and 18, the nucleic acid sequence of the G gene construct is provided in SEQ ID NO: 14, and the amino acid sequence of the G gene construct is the sequence of SEQ ID NO: 6.

[0120] The whole genome sequence of SFRV according to MA et al. (J Virol. 88:6576-6585 (2014)) deposited in GenBank (Accession No. KF947078) was used as the basis for primer design. Similarly, for the TEV cleavage site, the sequence ENLYFQG was used based on available public information.

[0121] Purify SFRV from the spent culture medium used in the growth of Sf9 (adherent cells) and Sf+ (suspension cells): Collect the spent culture medium from Sf9 and Sf+ cells infected with SFRV and routinely propagated, and filter it through a 0.2 micron filter to eliminate cell debris. The filtrate is then loaded onto a 30% sucrose cushion in NaCl-Tris HCl-EDTA (NTE) buffer (pH 7.4) and ultracentrifuged at 32,000rpm for 3 hours at 4°C. Carefully aspirate the supernatant, and rehydrate and resuspend the precipitate in NTE buffer. Measure the total protein content on a nanodrop machine, and assign aliquots to lot numbers and freeze at ≤-70°C until further use. This antigen preparation contains semi-purified virus for coating ELISA plates, as described below.

[0122] The spent SF9 medium was used as a source for SFRV viral RNA extraction. The QIAamp viral RNA extraction kit (Qiagen) was used according to the manufacturer's instructions.

[0123] To amplify the G and N genes, the One-Step Superscript III kit was used according to the manufacturer's instructions. Gradient RT-PCR was used with the following conditions: 1 cycle of 30 minutes at 60°C (RT step), followed by 1 cycle of 2 minutes at 94°C. This was followed by 40 cycles of 15 seconds at 94°C, annealing gradient 75°C-50°C for 60 seconds, followed by extension at 68°C for 2 minutes. Finally, the reaction was subjected to a single cycle of 5 minutes at 68°C and was kept at 4°C.

[0124] The amplified product was run on a gel to verify the size. The gel band of the expected size was excised from the gel and purified using the Qiaquick gel extraction kit using the manufacturer's instructions ( Figure 1 ).

[0125] In the following, only further work using the G gene (comprising the sequence of SEQ ID NO: 1) is described.

[0126] The amplified G gene with an expected size of ∼1.6 Kb was excised as described previously ( Figure 1 The upper band in ( ) was extracted from the gel. This was then cut with Eco RI and Pst I restriction enzymes. The product of the cut is the insert. Similarly, the baculovirus transfer vector plasmid pVL1393 (Pharmingen) was cut with Eco RI and Pst I restriction enzymes to generate the vector. The cut insert and vector were run on a gel (see Figure 2) to check the linearization of the vector. The bands were excised and gel extracted, and the vector was dephosphorylated. The insert (SFRV-G construct cut with Eco RI-PstI) was cloned into the vector (pVL1393 cut with Eco RI-PstI, dephosphorylated) and ligated using standard procedures.

[0127] The ligated product was used to transform E. coli cells (One Shot Max potency DH5α chemically competent cells from Invitrogen) and the cells were plated on LB agar with ampicillin. Colonies were picked the next day and screened for plasmid uptake using colony PCR and assigned clone numbers.

[0128] The reaction conditions for colony PCR were as follows: one cycle at 98° C. for 3 minutes, followed by 34 cycles of denaturation at 98° C. for 30 seconds, annealing at 58° C. for 30 seconds, and extension at 72° C. for 2 minutes. This step was followed by a final extension step at 72° C. for 10 minutes and a final hold at 4° C.

[0129] Run the PCR products on an agarose gel to identify clones containing the plasmid (see Figure 3 ).

[0130] Positive clones were then grown in LB-ampicillin liquid medium and plasmids were purified from the culture using the Qiaprep miniprep plasmid purification kit (Qiagen) using the manufacturer's instructions. The transfer vector thus generated contained the SFRV G gene construct and was aliquoted and assigned lot numbers.

[0131] To generate recombinant baculovirus expressing the SFRV G gene construct, the transfer plasmid was co-transfected into Sf9 cells with linearized flashBAC ULTRAbaculovirus backbone DNA (Genway biotech) using ESCORT transfection reagent (SAFC). One week later, the supernatant from the transfection (p0) was inoculated onto fresh Sf9 cells to amplify any recombinant baculovirus that may have been generated.

[0132] Cell pellets from the transfection were collected and run on an SDS gel and transferred to a nitrocellulose membrane for Western blotting. The protein was probed with an anti-His antibody (Invitrogen). The predicted protein size was ~71.5 KDa. The gel showed ( Figure 4 ) Two close bands at and above the 62 KDa marker for all five clones tested (2, 5, 7, 10 and 11), but no band in the cell control lane (cc).

[0133] Clone 2 and 5 of the recombinant virus were further passaged and grown in spinner flasks to produce the protein on a large scale in Sf+ suspension culture. The supernatant, soluble and insoluble cell fractions were probed for the protein. At this point, the protein was present only in the insoluble fraction ( Figure 5 As a result, a C-terminal truncation of the SFRV G glycoprotein was generated as the next step for use in an ELISA assay.

[0134] Embodiment 2:

[0135] ELISA development

[0136] An ELISA was developed to assess the presence of antibody responses against SFRV in animals vaccinated with PCV2 or other subunit vaccine baculovirus expressed in SFVV-infected Sf cells. Briefly, ELISA plates were coated with 250 ng / well of semi-purified SFRV antigen from Sf9 or Sf+ cell supernatants (as described above).

[0137] • Coating was performed by diluting the antigen in carbonate-bicarbonate buffer pH 9.0 to give a final concentration of 250 ng / well. Coating was performed overnight at 4°C.

[0138] The next day the plates were washed with PBS-Tween (PBST) and blocked with 10% milk for 1 hour at room temperature.

[0139] - Plates were then probed with sera (in blocking buffer) diluted 1:100 from animals vaccinated with PCV2 subunit antigens, baculovirus expressed in SFRV-infected Sf cells, or unvaccinated controls (see Data section).

[0140] • Incubate the plate at 37°C for 1 hour and then wash 5 times with PBST to remove unbound antibody.

[0141] • The plates were then probed with secondary antibody (goat anti-swine IgG H+L-HRP conjugate - Bethyllaboratories) diluted 1:10,000, incubated for 1 hour at 37°C, and then washed 5 times with PBST to remove unbound antibody.

[0142] - Finally, SureBlue TMB substrate (KPL) was added and the plate was incubated for 5 minutes at room temperature before being stopped with TMB stop solution (KPL).

[0143] • The plate was then read at 450 nm.

[0144] The ELISA plate setup is:

[0145] ● Row a, wells 1-10 coated with Sf9-derived SFRV

[0146] ● Row b, wells 1-10 coated with Sf+-derivatized SFRV

[0147] • Pig sera were assessed in duplicate and animals vaccinated with PCV2 (A,B) subunit antigens are shown in bold. These should show a positive readout if the animal has encountered SFRV through vaccination and developed antibodies against SFRV.

[0148] Negative controls are in italics (C and D)

[0149] ● Columns 9 and 10 are buffer controls (no primary antibody)

[0150] The results of ELISA are shown in Table 1.

[0151] Table 1:

[0152]

[0153] Table 1 shows data evaluating SfRV antigens derived from Sf9 cells (row A) and Sf+ cells (row B). Four serum samples were evaluated in duplicate. Columns A and B contain day 28 sera from animals vaccinated with the experimental vaccine, while columns C and D contain sera from negative control animals. The results show that both Sf9 and Sf+ cells contain SfRV and can be used as viral antigens. In addition, specific recognition of the antigen in vaccinated animals, but not in control animals, indicates the usefulness of SfRV as an intrinsic compliance marker.

[0154] Data explanation:

[0155] - Animals vaccinated with PCV2 subunit antigens (Groups A and B) showed good responses against semi-purified SFRV based on ELISA readouts.

[0156] • Negative control animals (Groups C and D) showed no response to semi-purified SFRV.

[0157] • The results demonstrate the usefulness of SFRV for both intrinsic adherence markers and the DIVA approach.

[0158] Embodiment 3:

[0159] Using the above-mentioned SFRV antigen (wherein the antigen is a protein comprising the sequence of any one of SEQ ID NOs: 1 to 6, or ELISA wherein the antigen is a purified or semi-purified virus according to the invention:

[0160] SCOPE: Testing serum (or oral fluid) samples for the presence of antibodies against SFRV antigens

[0161] Materials and methods

[0162] A. Equipment

[0163] ELISA plate washer

[0164] ELISA reader

[0165] ●WFI for cell culture, USP (Gibco, catalog number A12873-02)

[0166] Carbonate-bicarbonate buffer (pH 9.6) tablets (Sigma, catalog number C3041-100CAP)

[0167] 96-well immunoplate (round-bottom or flat-bottom plate, Nunc Maxisorb)

[0168] ●12-channel pipettes, various pipettes ranging from 1μL to 1mL

[0169] ●Pipette tips

[0170] 37℃ incubator

[0171] 4℃ refrigerator

[0172] ●Vortexer

[0173] ●Flat cover (Thermo, catalog number AB-0752)

[0174] S-block 2 mL dilution block (Phenix catalog number M-1810S, or equivalent)

[0175] Reagent reservoir

[0176] ●Timer

[0177] B. Reagents

[0178] 1. Coating buffer: carbonate-bicarbonate buffer

[0179] ■100mL WFI

[0180] ■1 capsule of carbonate-bicarbonate buffer

[0181] ■Open capsule, dispense powder into WFI, mix until dissolved

[0182] ■ Sterilize the solution by filtering it with a 0.2μm filter

[0183] ■Store at 4℃

[0184] ■Expiration time: 1 week

[0185] ■Each assay requires (4 plates): 50 mL

[0186] 2.10X PBS:

[0187] ■1 pack of PBS concentrate, Fisher BP665-1

[0188] ■Use GenPur H 2 O (or equivalent) adjusted to 1L

[0189] ■Store at room temperature

[0190] ■Expiration time: 1 year

[0191] 3. Washing buffer: 0.05% Tween 20 / Dulbeccos PBS.

[0192] ■0.5 mL of Tween 20, Fisher BP337 or equivalent

[0193] 100 mL 10X D-PBS, pH 7.2-7.4

[0194] ■Use GenPur H 2 O (or equivalent) adjusted to 1L

[0195] ■pH to 7.2±0.1

[0196] ■Store at room temperature

[0197] ■Expiration time: 6 months

[0198] ■Each assay requires (4 plates): 2 liters

[0199] 4.PBST:

[0200] 500 mL 1X PBS pH 7.4 (Gibco, catalog number 10010-023)

[0201] ■0.3mL tween 20, Fisher BP337, or equivalent

[0202] ■Store at room temperature

[0203] ■Expiration time: 6 months

[0204] ■Each assay requires (4 plates): 100 mL

[0205] 5. Blocking solution: 10% skim milk powder / PBST solution.

[0206] 20 g blotting grade blocker, Bio-Rad 170-6404, or equivalent

[0207] 200 mL PBST

[0208] ■Store at 4℃

[0209] ■Expiration time: 0 days

[0210] ■Each assay requires (4 plates): 200 mL

[0211] 6.SFRV antigen:

[0212] ■ Uninfected SF or SF+ cell culture supernatant was filtered through a 0.2 micron filter (Thermo cat456-0020). In this context, "uninfected SF or SF+ cell culture supernatant" means the supernatant of SF or SF+ cells in culture, wherein the cells are not infected with baculovirus, but are infected with SFRV. In addition, in the context of the cells described in the present disclosure, the term "SF" is equivalent to the term "Sf", the term "SF+" is equivalent to the term "Sf+", and the term "SF9" is equivalent to the term "Sf9", respectively.

[0213] ■ Load the filtrate onto a 30% sucrose cushion and centrifuge at 28,000-34,000 rpm at 4°C for 2-4 hours

[0214] ■After centrifugation, carefully aspirate the supernatant and resuspend the pellet in NaCl-Tris-EDTA buffer pH 7.4

[0215] ■This is a semi-purified antigen

[0216] ■ Estimate protein concentration by spectrophotometry and aliquot and freeze at -70°C until use.

[0217] 7.2° Antibody: Goat anti-swine IgG h+l HRP conjugated. Bethyl Labs Cat. No. A100-105P, stored at 4°C ± 3.0°C.

[0218] 8. Substrate: SureBlue TMB 1-Component Microwell Peroxidase Substrate.

[0219] Kirkgaard and Perry Laboratories catalog number 52-00-01 or equivalent. Store substrate at 4°C ± 3.0°C, pre-incubate at 25°C ± 2.0°C, and use at 25°C ± 3.0°C.

[0220] 9. Stop Solution: TMB Stop Solution. Kirkgaard and Perry Laboratories Catalog No. 50-85-04 or equivalent. Store at room temperature.

[0221] C. Procedure

[0222] 1. Prepare coating buffer using the recipe listed in B1 above.

[0223] 2. Dilute SFRV antigen to 250 ng / well in coating buffer. Mix by inverting the antigen 10 times. Coat with 250 ng / 100 μl (i.e. 2.5 μg / ml = 250 ng / well).

[0224] 3. Add 100 μL of diluted SFRV antigen to all wells,

[0225] 4. Seal the assay plate with a plate cover and incubate overnight at 4°C on the bottom of a refrigerator to minimize disturbance.

[0226] the next day

[0227] 5. Prepare enough blocking solution for the current assay only. A 200 mL block is recommended for 4 plates. Store at 4°C until needed.

[0228] 6. Wash the assay plate 5 times with wash buffer using an ultrawash plus microtiter plate washer or equivalent.

[0229] 7. Add 100 μL of blocking solution to all wells of the test plate. Cover the test plate and incubate at 37°C ± 2.0°C for 1.0 hour.

[0230] 8. During the blocking incubation, dilute the test serum samples 1:100 in block in an S-type block. For oral fluid, dilute the samples 1:2 in block. Test each sample individually. Dilute the positive and negative controls in the same manner.

[0231] 9. Wash the test plate once. After the last wash, tap the plate onto a paper towel.

[0232] 10. Add 100 μL of pre-diluted test sample per well to the corresponding plate. Avoid full contact with the tip of the pipette. Change tips between each test sample.

[0233] For serum samples, cover the test plate and incubate at 37°C ± 2.0°C for 1.0 hour. For oral fluid, incubate the samples at 4°C ± 2.0°C for 16.0 hours.

[0234] 11. Just before washing the test plate, remove the secondary antibody vial from the refrigerator and dilute to 1:10,000 in block. Serial dilutions are recommended to achieve a 1:10,000 dilution (4 dilutions). Mix the diluted antibody by inverting 10 times.

[0235] 12. Wash the test plate 5 times.

[0236] 13. Add 100 μL of diluted detection antibody to all wells of the test plate. Cover the test plate and incubate at 37°C ± 2.0°C for 1.0 hour.

[0237] 14. Immediately remove SureBlue TMB 1-Component Microporous Peroxidase Substrate from the refrigerator (4°C ± 3°C) and transfer an appropriate volume to a brown or opaque high-density polyethylene (HDPE) container and incubate at 25°C ± 2.0°C for 1 hour ± 15 minutes (benchtop).

[0238] 15. Wash the test plate 5 times. After the last wash, tap the plate onto a paper towel. It is recommended to keep the plate reader on while washing the plate.

[0239] 16. Add 100 μL of substrate to all wells of the assay plate.

[0240] 17. Incubate at 25°C ± 3°C for 5 minutes.

[0241] 18. Stop the reaction by adding 100 μL of stop solution to all wells

[0242] 19. Measure the absorbance at 450 nm.

[0243] D. Acceptance criteria / results

[0244] ■ Positive control: serum (or oral fluid, respectively) from pigs hyperimmunized with SFRV / SRFV G glycoprotein

[0245] ■ Untreated pig serum (or oral fluid, respectively) or serum (or oral fluid, respectively) from unvaccinated pigs showing negligible to no response to SFRV antigens

[0246] Embodiment 4:

[0247] Production of semi-purified rhabdovirus, size exclusion chromatography (SEC), real-time PCR, electron microscopy of SEC fractions ELISA

[0248] Production of Semi-Purified Rhabdovirus:

[0249] Semi-purified rhabdovirus was produced by filtering cell culture supernatant (40 mL) of SfRV-infected Sf+ insect cells, concentrated from 5 liters to 800 mL using hollow fiber filtration, through a 1.2 μm syringe filter before loading onto the column. The resulting filtrate was the "semi-purified rhabdovirus" according to this example.

[0250] Size Exclusion Chromatography (SEC):

[0251] Size exclusion chromatography was run on an AKTA Explorer with a HiPrep 26 / 60 Sephacryl S300HR column (GE Healthcare Bio-Sciences) using isocratic conditions at a flow rate of 1 mL / min. The column was equilibrated with 1.5 column volumes of buffer (1X phosphate buffered saline, pH 7.4, Gibco) followed by injection of a clarified sample of the semi-purified rhabdovirus produced according to (i) (approximately 5% column volume). Separation was performed over 1.5 column volumes of buffer at a flow rate of 1.0 mL / min, and fractions (8 mL) were collected over the entire separation step according to injection time. The elution of protein from the column was monitored by UV absorption at 280 nm ( Figure 6 ).

[0252] After using TCA / acetone precipitation to concentrate the peak fraction, the fraction was analyzed by 4-12% SDS-PAGE (Thermo Fisher). In brief, 1mL of each fraction was precipitated on ice with TCA (200 μL) for 1 hour. The sample was centrifuged at 20,000xg for 2 minutes, and the supernatant was removed. The fraction was washed with 500 μL ice-cold acetone, mixed by vortex, and then centrifuged at 20,000xg for 2 minutes. Repeat the centrifugation and acetone steps, a total of three acetone washes. The precipitate was dried for 20 minutes, suspended in 20 μL gel loading buffer, and loaded onto the gel. The gel was stained with Imperial protein dye (Thermo Fisher) for 1 hour, and decolorized with deionized water for at least 3 hours. After gel analysis, bovine serum albumin was used as a standard, and the protein concentration of the fraction was determined by BCA assay (Thermo Fisher).

[0253] Real-time PCR

[0254] The presence of SfRV RNA in (i) the semi-purified rhabdovirus (filtrate) and (ii) the SEC collected fractions was detected / quantified using the following real-time PCR method and sequence:

[0255] Primer / Probe / G-Block Control:

[0256]

[0257]

[0258] *Based on genomic location of GenBank reference strain: KF947078. All sequences target the region encoding the SfRV glycoprotein

[0259] Loop conditions:

[0260] 1 cycle at 50 °C for 10 min

[0261] 1 cycle at 95 °C for 3 min

[0262] 40 cycles at 95 °C for 15 sec

[0263] 57℃ for 15 seconds, **Data collection (FAM)

[0264] A brief description of the steps performed:

[0265] Amplification was performed using the BioRad iTaq Universal Probes One-Step kit (catalog number 172-5141) according to the recommended manufacturer's recommended protocol. Primers were added to a final concentration of 0.4 μM in a 25 μl reaction, while probes were added to a final concentration of 0.16 μM. In each run, the standard curve composition corresponded to a synthetic double-stranded g-block (IDT) sequence of the expected amplicon. The reaction was performed using a CFX96 real-time PCR detection system (BioRad) under the following conditions: initial reverse transcription at 50 ° C for 10 minutes, followed by initial denaturation at 95 ° C for 3 minutes, followed by 40 cycles of denaturation at 95 ° C for 15 seconds and annealing and extension at 57 ° C for 15 seconds, wherein data were collected in the FAM channel. Optical data were analyzed using CFX Manager software (version 2.1, BioRad). The run was considered valid based on the following: consistency of the standard curve, r square value exceeded 0.99, and the calculated efficiency was between 80-120%. For each determination, the threshold line was automatically calculated using the regression setting of the cycle threshold (Ct) determination mode. Baseline subtraction was performed automatically using the baseline subtraction mode.

[0266] The results of real-time PCR are shown in Figure 7 middle.

[0267] exist Figure 7 middle:

[0268] Column 1 represents the number of holes. Column 2 shows the fluorophore 6-carboxyfluorescein (FAM) connected to the specific probe used in the real-time PCR. Column 3 represents the fractions (fractions A11, A12, B1, B5, B12 and C6) of the SfRV antigen derived from size exclusion chromatography or the standard (holes 7-14) of the SfRV specific nucleic acid with a known amount for generating a standard curve. Hole 15 serves as a negative control (no template), and hole 16 serves as a positive control containing the concentrated SfRV antigen before separation by size fractionation (SEC).

[0269] The quantitative cycle (Cq) is the cycle of detecting fluorescence. A lower Cq value indicates a higher copy number of a specific target in a sample. The data is shown in column 4. The extrapolated genome copy number is shown in column 5 as sequence quantification (SQ) and displays the genome copy number per mL.

[0270] The data show that the starting material has 6 logarithmic SfRV specific genome copies / mL (well 16), and similarly, fractions A11 and A12 have 6 logarithmic genome copies / mL. These two fractions, together with the tail fraction B1, should contain most of the SfRV virus particles / virions and virus-like particles (VLPs). Other fractions B5, B12, and C6 should contain subviral particles in SEC and therefore contain less viral RNA, if any.

[0271] Electron Microscopy:

[0272] (ii) The fractions collected by SEC were stained with 2.5% phosphotungstic acid (PTA) for 3 min (negative staining) for electron microscopy. Figure 6 ), particles of ~30-35 nm were observed, which were considered to be viral particles of SfRV.

[0273] ELISA:

[0274] The ELISA was performed as described in Example 2, using the materials and methods described under Example 3, with the difference that instead of "SFRV antigen" (under point B.6 of Example 3), (i) semi-purified rhabdovirus (filtrate) and (ii) SEC collected fractions A11, A12 and B1 were used, each diluted to a concentration of 250 ng in 100 μl in coating buffer, and 100 μl of each of the antigens was then coated on the wells.

[0275] As test serum samples, serum from animals immunized with an experimental vaccine comprising recombinant proteins produced by a baculovirus expression system in cultured SfRV-infected insect cells was used. Serum was obtained from blood taken from the animals 28 days after administration of the experimental vaccine.

[0276] As negative controls, blood sera from corresponding non-immunized animals were used.

[0277] The results of ELISA are shown in Figure 8 middle.

[0278] ELISA plates were coated with four different antigens, including semi-purified SfRV (panel A), size exclusion fractions A11 (panel B), A12 (panel C), and B1 (panel D). Plates were probed with sera from negative control animals (inverted triangles) or day 28 sera from animals administered an experimental vaccine containing SfRV (circles).

[0279] The results show that sera from vaccinated animals reacted with the coated antigen, while negative control sera had minimal reaction. In addition, vaccinated animals reacted strongly with wells coated with fractions A11, A12, and B1 (panels B, C, and D), as evidenced by increased OD values, and the reactions were more tightly clustered with these fractions compared to semi-purified SfRV (panel A). This indicates a stronger recognition and more specific response to the coated antigens (fractions A11, A12, and B1).

[0280] In the sequence listing:

[0281] SEQ ID NO: 1 corresponds to the sequence of SFRV G protein,

[0282] SEQ ID NO:2 corresponds to the sequence of a truncated SFRV G protein,

[0283] SEQ ID NO:3 corresponds to the sequence of a truncated SFRV G protein with an N-terminal melittin sequence,

[0284] SEQ ID NO:4 corresponds to SEQ ID NO:1 with modifications including a 6x His tag,

[0285] SEQ ID NO:5 corresponds to SEQ ID NO:2 with modifications including a 6x His tag,

[0286] SEQ ID NO:6 corresponds to SEQ ID NO:3 with modifications including a 6x His tag,

[0287] SEQ ID NO:7 corresponds to the sequence of SFRV N protein,

[0288] SEQ ID NO:8 corresponds to SEQ ID NO:7 with modifications including a 6x His tag,

[0289] SEQ ID NO:9 corresponds to the sequence encoding SEQ ID NO:1,

[0290] SEQ ID NO:10 corresponds to the sequence encoding SEQ ID NO:2,

[0291] SEQ ID NO:11 corresponds to the sequence encoding SEQ ID NO:3,

[0292] SEQ ID NO:12 corresponds to the sequence encoding SEQ ID NO:4,

[0293] SEQ ID NO:13 corresponds to the sequence encoding SEQ ID NO:5,

[0294] SEQ ID NO:14 corresponds to the sequence encoding SEQ ID NO:6,

[0295] SEQ ID NO:15 corresponds to the sequence encoding SEQ ID NO:7,

[0296] SEQ ID NO:16 corresponds to the sequence encoding SEQ ID NO:8,

[0297] SEQ ID NO: 17 corresponds to the forward primer of construct SEQ ID NO: 12 or SEQ ID NO: 13,

[0298] SEQ ID NO: 18 corresponds to the reverse primer of construct SEQ ID NO: 12 or SEQ ID NO: 14,

[0299] SEQ ID NO: 19 corresponds to the reverse primer of construct SEQ ID NO: 13,

[0300] SEQ ID NO:20 corresponds to the forward primer of construct SEQ ID NO:14,

[0301] SEQ ID NO:21 corresponds to the forward primer of construct SEQ ID NO:16,

[0302] SEQ ID NO:22 corresponds to the reverse primer of construct SEQ ID NO:16,

[0303] SEQ ID NO:23 corresponds to the sequence of PCV2 ORF2 protein,

[0304] SEQ ID NO: 24 corresponds to the sequence of hemagglutinin H5 protein (influenza virus).

Claims

1. Use of a capture reagent in the preparation of a kit for determining whether an individual has received an immunogenic composition, wherein the capture reagent is selected from: a. a protein comprising or consisting of an amino acid sequence having at least 70% sequence identity with a sequence selected from SEQ ID NOs: 1 to 6; b. an RNA virus capable of infecting insect cells, wherein the RNA virus is a virus belonging to the family Rhabdoviridiae, and wherein the virus has been inactivated; wherein the immunogenic composition comprises a recombinant protein produced by a baculovirus expression system in cultured insect cells, wherein the insect cells are infected with a virus comprising or consisting of an amino acid sequence having at least 70% sequence identity with a sequence of SEQ ID NO: 1; wherein the capture reagent is capable of binding to antibodies obtained from a biological sample of an individual who has received the immunogenic composition; and wherein the presence of the antibodies in the biological sample indicates that the individual has received the immunogenic composition.

2. The use according to claim 1, wherein the RNA virus is selected from: a. an RNA virus capable of infecting insect cells comprising a protein comprising or consisting of: i. an amino acid sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 1; and / or ii. an amino acid sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 7; and / or b. an RNA virus capable of infecting insect cells, whose genome comprises a nucleic acid molecule encoding a protein, wherein the protein comprises or consists of: i. a sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 1; and / or ii. a sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 7; and / or c. an RNA virus capable of infecting insect cells, whose genome comprises an RNA molecule having the following sequence: i. the sequence is reverse complementary to a nucleic acid sequence, the nucleic acid sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 9; and / or ii. the sequence is reverse complementary to a nucleic acid sequence, the nucleic acid sequence having at least 70% sequence identity with the sequence of SEQ ID NO:

15.

3. The use according to claim 1, wherein the insect cell is a Spodoptera frugiperda cell or a cell derived from a cell line of Spodoptera frugiperda.

4. The use according to claim 3, wherein the Spodoptera frugiperda cell or the cell of the cell line derived from Spodoptera frugiperda is selected from: Sf9 cell and Sf+ cell.

5. The use according to claim 1, wherein the capture reagent comprises or consists of viral particles and / or virus-like particles of an RNA virus capable of infecting insect cells, and wherein the capture reagent is obtained by a method comprising the following steps: i) obtaining a supernatant from a culture of insect cells infected with an RNA virus capable of infecting insect cells, wherein the supernatant comprises viral particles and / or virus-like particles of an RNA virus, ii) separating cell debris from the viral particles and / or virus-like particles via a separation step comprising microfiltration through at least one filter, wherein at least one filter has a larger pore size than the viral particles and / or virus-like particles, and iii) optionally, subjecting the filtrate of ii) containing the viral particles and / or virus-like particles to size exclusion chromatography, wherein the presence of proteins in the eluate is measured by measuring the absorbance at 260 nm or 280 nm.

6. The use according to claim 5, wherein in step i), the insect cells infected with the RNA virus capable of infecting insect cells are not infected with baculovirus and / or are not transfected with a plasmid.

7. The use according to claim 5, wherein in step ii), there are two filters.

8. The use according to claim 5, wherein in step ii), the at least one filter has a pore size of 0.1 μm to 4 μm.

9. The use according to claim 5, wherein in step ii), the at least one filter has a pore size of 0.2 μm to 2 μm.

10. The use according to claim 5, wherein in step iii), the presence of the protein in the eluate is measured by measuring the absorbance at 260 nm or 280 nm by collecting the eluate showing a first peak of absorbance at 260 nm or absorbance at 280 nm.

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