PCV2 ORF2 carrier platform

Abstract

The present invention relates to immunogen carriers, wherein said immunogen carriers are preferably virus-like particles (VLPs) comprising a plurality of modified PCV2 ORF2 proteins. In particular, the present invention belongs to the field of compliance markers and marker vaccines allowing to distinguish between infected and vaccinated individuals. In particular, it relates to compliance markers for vaccines comprising subunit antigens and DIVA (distinguishing infected from vaccinated animals) systems, which enable to distinguish between animals infected with a pathogen and animals treated with subunit antigens derived from said pathogen.

Description

[0001] sequence list

[0002] This application contains sequence listings according to 37C.FR1.821–1.825. The sequence listings appended to this application are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to immunogen vectors, preferably virus-like particles (VLPs) comprising a plurality of modified PCV2ORF2 proteins. In particular, this invention belongs to the field of compliance markers and marker vaccines that allow differentiation between infected and vaccinated individuals. Specifically, it relates to compliance markers for vaccines comprising subunit antigens, and DIVA (Distinguishing Infected Animals from Vaccinated Animals) systems that enable differentiation between animals infected with a pathogen and animals treated with subunit antigens derived from said pathogen. Background Technology

[0004] Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter), icosahedral, non-enveloped DNA virus containing a single-stranded circular genome. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV-1). However, unlike the generally non-virulent PCV1, pigs infected with PCV2 exhibit a syndrome commonly known as post-weaning multisystemic wasting syndrome (PMWS). Clinical features of PMWS include emaciation, pale skin, weakness, respiratory distress, diarrhea, icterus, and jaundice. In some infected pigs, a combination of all manifestations becomes apparent, while others exhibit only one or two of these clinical signs. On necropsy, microscopic and macroscopic lesions are also present in multiple tissues and organs, with lymphoid organs being the most common site of lesion. A high correlation has been observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. Mortality rates in pigs infected with PCV2 can reach 80%. Besides PMWS, PCV2 is associated with several other infections, including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, post-weaning colibacillosis, nutritional liver dysfunction, and purulent bronchopneumonia.

[0005] Currently, three subtypes of PCV2 (PCV2a, PCV2b, and PCV2c) are known, classified according to the unified nomenclature of PCV2 genotypes (Segales, J. et al., 2008, PCV-2 genotype definition and nomenclature, Vet Rec 162:867-8). Two additional subtypes (PCV2d and PCV2e) were proposed (Wang et al., Virus Res. 2009, 145(1):151-6), but they were later confirmed to belong to the PCV2a and PCV2b clusters (Cortey et al., Vet Microbiol. 2011, 149(3-4):522-32011). Based on this unified nomenclature of PCV2 genotypes, the orf2 gene is used for genotyping of pcv-2, where the genotyping is based on the proportion of different nucleotide sites (p-distance) of two compared sequences. This value is obtained by dividing the number of nucleotide differences by the total number of nucleotides compared (Kumar et al., 2001, Bioinformatics 17, 1244-1245). Subsequently, the construction of a p-distance / frequency histogram allows for the determination of possible cutoff values ​​to distinguish different genotypes (Rogers and Harpending, 1992, Molecular Biology and Evolution 9, 552-569; Biagini et al., 1999, Journal of General Virology 80, 419-424). Using this method, ORF2 PCV-2 sequences were assigned to different genotypes when the genetic distance between them was 0.035.

[0006] WO2011116094A2 discloses an infectious DNA clone of chimeric porcine circovirus and a live attenuated chimeric virus with the PCV2b subtype of PCV2, as well as the capsid gene of the PCV2b subtype integrated into the genome of a non-pathogenic PCV1 virus. The attenuated chimeric virus can be used as a live vaccine as well as an inactivated (killed) vaccine.

[0007] Vaccination is an important tool for managing livestock health, especially in high-density, confined environments where many food animals are raised. When a disease outbreak is suspected in vaccinated animals, questions arise about whether the vaccine failed to protect the animals or whether it was properly delivered; the latter possibility regarding proper vaccine delivery is known as vaccine compliance.

[0008] Therefore, producers have a strong desire to use compliance markers to determine whether animals have been properly vaccinated. WO 2009 / 058835 A1 describes, for example, the use of purified xylanase, which is added as a compliance marker to swine influenza vaccines.

[0009] Vaccines used in procedures to control viral outbreaks and infections must have effective systems in place to monitor the persistence of viral infection within a population. However, vaccination complicates large-scale surveillance of infection transmission through means such as serological methods, because both vaccinated and exposed individuals develop virus-specific antibodies. The antigenic similarity between highly infectious wild-type strains of the virus and viral vaccines often hinders differentiation between infected and vaccinated individuals, as vaccination results in the indistinguishable presence and persistence of antibodies between infected and vaccinated individuals.

[0010] There is growing global interest in DIVA (distinguishing between infected and vaccinated animals) vaccination strategies. For example, the joint WHO / FAO / OIE meeting on the H5N1 HPAI avian influenza strain has recommended using DIVA in all vaccination practices to monitor infection transmission. However, current DIVA methods are difficult to scale up and often have the problem of distinguishing between vaccination and infection by other circulating viral strains.

[0011] Current surveillance methods include adding physical tags to vaccinated animals, using sentinel animals, and virological testing. However, these methods have many limitations due to logistical and economic reasons.

[0012] Adding physical tags to vaccinated animals involves time-consuming individual identification of vaccinated individuals using physical means such as ear tags, leg bands, or wing tags. Furthermore, the use of unvaccinated sentinel animals is logistically and economically difficult, and there is an increased risk of transmission to humans if the sentinel becomes infected with a virus, such as H5N1 virus in poultry. Virological testing of individuals via screening and detection of live virus or RT-PCR surveillance testing is a very expensive and infrastructure-intensive process, unsuitable for subunit vaccines, and only provides information relevant to the individual's current status, without allowing analysis of the individual's infection and / or vaccination history.

[0013] In view of the aforementioned limitations, the use of marker vaccines that allow serological differentiation between vaccinated animals and infected animals is highly preferred, wherein such marker vaccines can be prepared as negative or positive marker vaccines.

[0014] Negative marker vaccines are prepared by using an antigenic portion of a pathogen or by removing an antigen from the pathogen (which elicits specific antibodies in infected animals). Negative marker vaccines are typically subunit vaccines or live attenuated vaccines containing genetically engineered strains lacking immunogenic antigens. An example of a negative marker vaccine is, for instance, the use of the classical swine fever virus (CSFV) E2 protein, expressing a baculovirus, as a subunit antigen for vaccination against CSFV, where the serum of vaccinated pigs shows antibodies against other CSFV antigens (e.g., E2). RNS Specific antibody detection of CSFV infection (either NS3 protein or NS3 protein) indicates CSFV infection.

[0015] Positive marker vaccines contain additional antigens that induce specific antibodies in vaccinated individuals rather than infected individuals. An example of a positive marker vaccine method is described in WO 2007 / 053899 A1, in which inactivated H6N2 avian influenza (A1) virus and tetanus toxin (produced separately) are combined in a single injection to vaccinate birds, and subsequently, antibodies specific to tetanus toxin are detected in serum obtained from said birds as a marker indicating that the birds have been vaccinated.

[0016] However, the separate production of vaccine antigens and marker antigens is relatively expensive.

[0017] In light of the above, there is a need for a simple vector system that can be used as a platform for the inexpensive production of positive marker vaccines and effective compliance markers, and in addition, allows for strong immunization of animals against PCV2 and, if applicable, at least one further pathogen. Attached Figure Description

[0018] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in combination with the detailed description of the specific embodiments presented herein.

[0019] Figure 1The PCV2b ORF2-c-myc band was detected in stained protein gels by comparing its size with that of PCV2b ORF2. Lane 1: Baculo / PCV2b ORF2 c-myc particles; Lane 2: Purified PCV2b ORF2 VLPS; Lane 3: Baculo / pVL1393 without insert control. A. Coomassie blue stained protein blot; B. Anti-PCV2B porcine polyclonal antibody stained protein blot; C. Anti-c-myc monoclonal antibody 9E10 stained protein blot; D. Anti-c-myc monoclonal antibody 9E101 stained protein blot; E. Anti-c-myc polyclonal rabbit antibody stained protein blot; PCV2b ORF2-c-myc was detected by both anti-PCV2b antibody and anti-c-myc antibody, while the PCV2b ORF2 antigen was detected only by PCV2b antibody.

[0020] Figure 2 The harvest of BaculoG / PCV2b ORF2 c-myc was centrifuged at 100,000 g at 4 °C for 2 h to precipitate the VLP. The resuspended particles were separated by centrifugation at 100,000 g at 4 °C for 2 h on a discontinuous sucrose gradient of 10%–60% to partially purify the PCV2b ORF2-cmyc protein for quantification and VLP confirmation by electron microscopy (EM). The sucrose gradient was fractionated into 9 fractions and separated by SDS-PAGE, with the majority of PCV2b ORF2-c-myc detected in the fraction expected for PCV2b ORF2 VLP.

[0021] Figure 3 Merge fractions containing PCV2b ORF2-c-myc (from...) Figure 2 (The boxed section) and the sucrose gradient purified material was concentrated and submitted for VLP confirmation by EM, using phosphotungstic acid as negative staining.

[0022] Figure 4 ELISA plates were coated with PCV2b ORF2-c-myc VLP, PCV2b ORF2 VLP, or baculovirus control antigen, and detected using serial dilutions of antibody A. porcine anti-PCV2b antibody or B. mouse anti-c-myc mAb 9E10. The anti-PCV2b antibody recognized both PCV2b ORF2 VLP and PCV2b ORF2-c-myc VLP, while the anti-c-myc antibody recognized only PCV2b ORF2-c-myc VLP.

[0023] Figure 5Rabbits were vaccinated with purified PCV2b ORF2-c-myc VLP formulated with Freund's adjuvant. Serum samples were obtained and their IgG responses against the c-myc peptide and PCV2b ORF2 VLP were assessed by ELISA. A. IgG responses against c-myc and PCV2b ORF2 after three vaccinations. All rabbits developed IgG responses against c-myc peptide and PCV2b ORF2 VLP after three vaccinations. B. IgG responses against c-myc and PCV2b ORF2 after a single vaccination. Five of the six rabbits developed IgG responses against the c-myc peptide after a single dose of the vaccine, while all six rabbits developed IgG responses against PCV2b ORF2 VLP after a single dose. Detailed Implementation

[0024] The solutions to the above-mentioned technical problems are achieved through the embodiments described in the specification and claims.

[0025] Therefore, the invention is implemented in various aspects as described in the claims.

[0026] This invention is based on the surprising discovery that replacing amino acid residues in the BC loop of the PCV2 ORF2 protein with a target epitope allows the generation of a VLP presenting the target epitope, thereby triggering an immune response against the epitope while preserving its antigenic properties in terms of providing active acquired immunity against PCV2.

[0027] In the first aspect, the present invention therefore relates to polypeptides hereinafter also referred to as "polypeptides of the present invention", selected from the following (a), (b) and (c):

[0028] a. PCV2 ORF2 protein, characterized in that at least one amino acid residue in the BC ring is replaced by the target amino acid sequence;

[0029] b. PCV2 ORF2 protein, characterized by the insertion of the target amino acid sequence into the BC loop;

[0030] c. A combination of (a) and (b).

[0031] As described in this article, the BC ring is specifically understood as the region of amino acid positions 58 to 66, where the amino acid positions are numbered with reference to the amino acid sequence of the wild-type PCV2 ORF2 protein.

[0032] To further understand, the term "amino acid residues that replace the BC ring" is specifically equivalent to the term "amino acid residues that replace the BC ring".

[0033] As described herein, the amino acid positions are numbered with reference to the amino acid sequence of the full-length wild-type PCV2 ORF2 protein (SEQ ID NO:2 or SEQ ID NO:3). Therefore, the amino position numbering mentioned herein refers to the wild-type PCV2 ORF2 protein sequence having 234 or 233 amino acid residues, including the methionine residue at (N-terminal) amino acid position 1.

[0034] Preferably, the target amino acid sequence is an amino acid sequence containing at least two or three amino acid residues or, more preferably, at least eight amino acid residues, or consisting of at least two or three amino acid residues or, more preferably, at least eight amino acid residues.

[0035] The target amino acid sequence preferably contains or is composed of heterologous amino acid sequences. As used herein, the term "heterologous amino acid sequence" refers to any amino acid sequence other than the PCV2 ORF2 sequence. More specifically, the term "heterologous amino acid sequence" refers to an amino acid sequence not found in the protein of the virus, such as PCV2.

[0036] Preferably, the target amino acid sequence is selected from target epitopes, biological response regulators, growth factors, recognition sequences, and fusion proteins.

[0037] Preferably, the target amino acid sequence contains or consists of a target epitope, and the target epitope is preferably an amino acid sequence containing or consisting of 8 to 25 amino acid residues.

[0038] The target epitope is preferably derived from an antigen, veterinary pathogen, or toxin, and more preferably is a peptide containing a c-myc-tagged peptide or a peptide encoded by the orf5 gene of the PRRS virus.

[0039] The peptide encoded by the orf5 gene of the PRRS virus preferably contains or is composed of the amino acid sequence of SEQ ID NO:6, or preferably contains or is composed of at least 8 consecutive amino acid residues of the sequence shown in SEQ ID NO:6.

[0040] Preferably, the polypeptide of the present invention according to aspect (a) is a PCV2 ORF2 protein, characterized in that at least one amino acid residue in the region of amino acid positions 58 to 64 is replaced by a target amino acid sequence, and wherein the amino acid positions are numbered with reference to the amino acid sequence of the wild-type PCV2 ORF2 protein.

[0041] The polypeptide of the present invention according to aspect (a) is preferably a PCV2 ORF2 protein, characterized in that at least two or three amino acid residues in the BC ring are replaced by a target amino acid sequence, and wherein preferably, two, three, four, five, six or seven amino acid residues in the BC ring are replaced by a target amino acid sequence.

[0042] Preferably, the polypeptide according to aspect (a) is the polypeptide of the present invention, wherein:

[0043] - Six amino acid residues at positions 58 to 63 are replaced by the target amino acid sequence, wherein the amino acid positions are numbered with reference to the amino acid sequence of the wild-type PCV2 ORF2 protein; and / or

[0044] - The target amino acid comprises or is composed of an amino acid sequence, said amino acid sequence consisting of 11 amino acid residues, and / or

[0045] - The target amino acid contains or is composed of the sequence of SEQ ID NO:5 or SEQ ID NO:7.

[0046] The polypeptide of the present invention is preferably a recombinant protein, and more preferably a protein expressed by a recombinant baculovirus.

[0047] As used herein, the term "recombinant protein" specifically refers to a protein molecule expressed by a recombinant DNA molecule, such as a polypeptide produced by recombinant DNA technology. Examples of such technologies include situations where DNA encoding the expressed protein is inserted into a suitable expression vector, preferably a baculovirus expression vector, which is then used to transfect host cells, or, in the case of a baculovirus expression vector, to infect host cells, to produce a DNA-encoded protein or polypeptide. As used herein, the term "recombinant protein" therefore specifically refers to a protein molecule expressed by a recombinant DNA molecule.

[0048] According to a specific example, recombinant proteins are produced by a method comprising the following steps: cloning the gene of the polypeptide of the present invention into a baculovirus transfer vector; using the transfer vector to prepare a recombinant baculovirus containing the gene via homologous recombination in insect cells; and then expressing the protein in insect cells during infection with the recombinant baculovirus.

[0049] In this alternative example, the recombinant protein is expressed in insect cells by a recombinant expression plasmid. In this alternative example, baculovirus is not required.

[0050] To further understand, the term "recombinant protein comprising or consisting of a sequence" also specifically refers to any co-translational and / or post-translational modifications of the sequence that are affected by cells expressing a polypeptide therein. Therefore, as described herein, the term "recombinant protein comprising or consisting of a sequence" also refers to a sequence having one or more modifications that are affected by cells expressing a polypeptide therein, particularly modifications of amino acid residues that affect protein biosynthesis and / or protein processing, preferably selected from glycosylation, phosphorylation, and acetylation.

[0051] Preferably, the recombinant protein according to the invention is produced by or can be obtained through a baculovirus expression system, particularly in cultured insect cells.

[0052] Preferably, the polypeptide of the present invention is

[0053] a. PCV2 ORF2 protein, characterized in that at least one amino acid residue in the BC ring is replaced by the target amino acid sequence;

[0054] b. PCV2 ORF2 protein, characterized by the insertion of the target amino acid sequence into the BC loop;

[0055] c. A combination of (a) and (b)

[0056] The PCV2 ORF2 protein is a PCV2b subtype (PCV2b) ORF2 protein or a PCV2a subtype (PCV2a) ORF2 protein, and / or the PCV2 ORF2 protein comprises or is composed of an amino acid sequence, the amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0057] The polypeptide of the present invention preferably comprises or is composed of an amino acid sequence, wherein the amino acid sequence has at least 90%, preferably at least 92%, more preferably at least 94%, even more preferably at least 96%, even more preferably at least 98%, or particularly 100% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0058] In another preferred aspect of the invention, as described herein, the wild-type PCV2 ORF2 protein is the protein shown in SEQ ID NO:2 or SEQ ID NO:3.

[0059] According to another aspect, the present invention further provides an immunogenic composition containing the polypeptide of the present invention.

[0060] According to another preferred aspect, the present invention further provides an immunogenic composition containing the polypeptide of the present invention and a PCV2a ORF2 polypeptide, wherein the PCV2a ORF2 polypeptide is preferably a polypeptide that is at least 94% or preferably at least 95% identical to the sequence of SEQ ID NO:3.

[0061] According to a further aspect, the present invention also provides a polynucleotide comprising a sequence encoding a polypeptide of the present invention, wherein the polynucleotide according to the present invention is preferably an isolated polynucleotide.

[0062] For illustrative purposes and in a non-limiting example, the polynucleotide according to the invention is a polynucleotide comprising the sequence shown in SEQ ID NO:4.

[0063] The production of polynucleotides described herein is within the art and can be performed according to the recombinant techniques described in the following: Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring 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 (eds.), 1994, PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference.

[0064] In addition, the present invention specifically provides a baculovirus containing a polynucleotide, the polynucleotide comprising a sequence encoding the polypeptide of the present invention, wherein the baculovirus according to the present invention is preferably an isolated baculovirus.

[0065] Furthermore, the present invention also provides plasmids containing polynucleotides, preferably expression vectors, wherein the polynucleotides contain sequences encoding polypeptides of the present invention, wherein the plasmids according to the present invention are particularly isolated plasmids.

[0066] The present invention also provides cells comprising a baculovirus or plasmid preferably an expression vector, the baculovirus containing a polynucleotide containing a sequence encoding a polypeptide of the present invention, the plasmid containing a polynucleotide containing a sequence encoding a polypeptide of the present invention, wherein the cells according to the present invention are preferably isolated cells.

[0067] In another aspect, the present invention also relates to the polypeptides of the present invention; baculoviruses according to the present invention; immunogenic compositions according to the present invention; polynucleotides according to the present invention; plasmids according to the present invention; and / or cells according to the present invention for the preparation of pharmaceuticals, preferably vaccines.

[0068] In this context, the present invention also provides a method for generating the polypeptides of the present invention, wherein the method includes the step of infecting cells, preferably insect cells, with the baculovirus of the present invention.

[0069] Furthermore, the present invention also provides a method for generating the polypeptides of the present invention, wherein the method includes the step of transfecting cells with plasmids according to the present invention.

[0070] The peptides of the present invention are preferably expressed in large quantities sufficient to stably self-assemble virus-like particles (VLPs), which can then be used for single-shot vaccination, particularly if they are included in an immunogenic composition, thereby allowing for the reduction and prevention of clinical signs caused by infection with PCV2 (e.g., infection by PCV2b and / or PCV2a).

[0071] The present invention is therefore particularly based on the polypeptides of the present invention or the immunogenic compositions of the present invention, wherein the polypeptides of the present invention or the immunogenic compositions comprising the polypeptides of the present invention can be used for a specific purpose.

[0072] In one aspect, the present invention therefore relates to the polypeptides of the present invention or immunogenic compositions comprising the polypeptides of the present invention in methods for treating or preventing PCV2 infection, reducing, preventing or treating clinical signs caused by PCV2 infection, or preventing or treating diseases caused by PCV2 infection.

[0073] The present invention also provides a method for treating or preventing PCV2 infection, reducing, preventing or treating clinical signs caused by PCV2 infection, or preventing or treating diseases caused by PCV2 infection, comprising administering the polypeptide of the present invention or an immunogenic composition comprising the polypeptide of the present invention to animals, particularly animals in need of such treatment.

[0074] In addition, the present invention provides the use of the polypeptide of the present invention or an immunogenic composition comprising the polypeptide of the present invention for the preparation of a medicament for the treatment or prevention of PCV2 infection, reducing, preventing or treating clinical signs caused by PCV2 infection, or treating or preventing diseases caused by PCV2 infection.

[0075] In a preferred aspect, as described herein, infection with PCV2 is infection with the PCV2b subtype (PCV2b) and / or infection with a PCV2 subtype other than subtype 2b.

[0076] As used in this article, the term “PCV2 infection” is equivalent to the term “PCV2 infection”.

[0077] Specifically, as mentioned in this article, infection with PCV2 subtypes other than subtype 2b is infection with PCV2a (PCV2a) and / or PCV2c (PCV2c), with PCV2a infection being preferred.

[0078] As described herein, the term “PCV2b subtype (PCV2b) ORF2 protein” refers to the protein encoded by the ORF2 gene of PCV-2b as defined by the standardized nomenclature of the PCV2 genotype definition (Segales, J. et al., 2008, PCV-2 genotype definition and nomenclature, Vet Rec 162:867-8, which is incorporated herein by reference).

[0079] According to another preferred aspect, as described herein, infection with PCV2 subtypes other than subtype 2b is co-infection with (i) PCV2 subtypes other than subtype 2b, and (ii) PCV2b, particularly co-infection with PCV2a and PCV2b.

[0080] As described in this article, based on the standardized nomenclature of the PCV2 genotype definition, the terms “PCV2a”, “PCV2b”, and “PCV2c” refer to PCV-2a, PCV-2b, and PCV-2c, respectively (Segales, J. et al., 2008, PCV-2 genotype definition and nomenclature, Vet Rec 162:867-8, which is incorporated herein by reference).

[0081] Specifically, as mentioned herein, infection with PCV2b is infection with (i) PCV2 containing at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to the sequence of SEQ ID NO:2, or (ii) PCV2 containing a polynucleotide containing a sequence encoding a polypeptide that is at least 94%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to the sequence of SEQ ID NO:2.

[0082] As used herein, the term "identical to the sequence of SEQ ID NO:X" should be understood in particular as equivalent to the terms "identical to the sequence of SEQ ID NO:X in length" or "identical to the sequence of SEQ ID NO:X over its entire length." Similarly, as used herein, the term "sequence identity with the amino acid sequence of SEQ ID NO:X" should be understood in particular as equivalent to the terms "sequence identity with the amino acid sequence of SEQ ID NO:X in length" or "sequence identity with the amino acid sequence of SEQ ID NO:X over its entire length."

[0083] In this context, “X” is any integer selected from 1 to 3 such that “SEQ ID NO:X” means any SEQ ID NO mentioned herein in the context of sequence identity.

[0084] Preferably, as described herein, infection with PCV2a is infection with (i) PCV2 containing at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to the sequence of SEQ ID NO:3, or (ii) PCV2 containing a polynucleotide containing a sequence encoding a polypeptide that is at least 94%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to the sequence of SEQ ID NO:3.

[0085] Preferably, in the context of this invention, treatment or prevention of PCV2 infection is based on or includes or consists of: inducing an immune response against said PCV2, clinical manifestations and / or disease, as mentioned herein, wherein said clinical signs are selected from: lymphatic depletion, lymphatic inflammation, positive IHC of PCV2 antigen in lymphoid tissue, viremia, nasal shedding, fever, reduced mean daily weight gain, pulmonary inflammation, positive IHC of PCV2 antigen in lung tissue, and / or disease of PMWS as mentioned herein.

[0086] Specifically, in the context of this invention, treatment or prevention of infection with PCV2 subtypes other than 2b is based on, includes, or consists of: inducing an immune response against the PCV2 subtypes other than 2b, or simultaneously inducing an immune response against both the PCV2 subtypes other than 2b and PCV2b.

[0087] As used herein, the terms “prevention,” “reduction,” “preventing,” or “reducing” mean, but are not limited to, methods comprising administering a PCV2 antigen, i.e., the polypeptide of the present invention, to an animal, which is included in the composition of the present invention, wherein, when administered to the animal, the PCV2 antigen induces or is capable of inducing an immune response against PCV2 in the animal. In summary, such treatment results in a reduction of clinical manifestations of PCV2-induced disease or clinical manifestations associated with PCV2 infection, respectively. More specifically, as used herein, the terms “prevention” or “preventing” generally mean a method of prevention in which an animal is exposed to the immunogenic composition of the present invention prior to the induction or occurrence of a PCV2-induced disease process.

[0088] In this document, "reduction in clinical manifestations associated with PCV2 infection" means, but is not limited to, a reduction in the number of infected subjects in the group compared to wild-type infection, a reduction or elimination of the number of subjects exhibiting clinical manifestations of infection, or a reduction in the severity of any clinical manifestations present in the subjects. For example, it should refer to any reduction in pathogen load, pathogen shedding, pathogen transmission, or any reduction in clinical symptoms of PCV2 infection. Preferably, these clinical manifestations are reduced by at least 10% in subjects receiving the composition of the present invention compared to subjects who have not received the composition and may be infected. More preferably, clinical manifestations are reduced by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50% in subjects receiving the composition of the present invention.

[0089] The term "reduced viremia" refers to, but is not limited to, a reduction in PCV2 virus entering the bloodstream of an animal, wherein the viremia level, i.e., PCV2 RNA copy number / mL serum or plaque-forming colony number / dL serum, is reduced by at least 50% in the serum of a subject receiving the composition of the present invention compared to a subject who has not received the composition and may be infected. More preferably, the viremia level is reduced by at least 90%, preferably at least 99.9%, more preferably at least 99.99%, and even more preferably at least 99.999% in a subject receiving the composition of the present invention.

[0090] As used herein, the term “viremia” is specifically understood to refer to the condition in which PCV2 particles replicate and circulate in the bloodstream of an animal.

[0091] As used herein, the terms “animal” or “object” specifically refer to mammals, preferably pigs, more preferably domestic pigs, and most preferably piglets.

[0092] According to a particularly preferred aspect of the invention, the polypeptide of the invention or the immunogenic composition of the invention is administered only once.

[0093] Preferably, in the context of this invention, the polypeptide of this invention or the immunogenic composition according to this invention is specifically intended to be administered or administered only once to an animal, preferably a pig, more preferably a domestic pig, and particularly preferably a piglet.

[0094] This invention overcomes the inherent problems in the prior art and provides significant progress. According to another aspect, the invention also provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in animals, preferably animals having anti-PCV2 antibodies, said method comprising the step of administering an effective amount of the polypeptide of the invention or the immunogenic composition of the invention to the animal requiring such treatment.

[0095] As used herein, the terms "vaccine" or "immunogenic composition" (both terms used synonymously) refer to any pharmaceutical composition containing the polypeptides of the present invention, which can be used to prevent or treat PCV2 infection-related diseases or conditions in subjects. Preferred immunogenic compositions can induce, stimulate, or enhance an immune response against PCV2. The term therefore covers subunit immunogenic compositions as described below, as well as compositions containing completely killed or attenuated and / or inactivated PCV2 mutants.

[0096] It should be understood in particular that, as described herein, the term "PCV2 mutant" refers to a PCV2 mutant comprising the polypeptide of the present invention and / or the polynucleotide of the present invention.

[0097] According to another aspect, the present invention also provides a method for treating or preventing PCV2 infection or reducing clinical signs caused by or associated with PCV2 infection in animals, preferably animals having anti-PCV2 antibodies (especially maternally derived anti-PCV2 antibodies), the method comprising the steps of: administering to the animal requiring such treatment an effective amount of the polypeptide of the present invention or an immunogenic composition comprising the polypeptide of the present invention, wherein the immunogenic composition is a subunit immunogenic composition comprising a composition of completely killed, attenuated and / or inactivated PCV2.

[0098] As used herein, the term "subunit immunogenic composition" refers to a composition containing at least one immunogenic polypeptide or antigen (but not all antigens) derived from an antigen from a PCV2 mutant or homologous to an antigen from a PCV2 mutant. Such compositions are substantially free of the intact PCV2 mutant. Therefore, a "subunit immunogenic composition" is prepared from at least partially purified or fractionated (preferably substantially purified) immunogenic polypeptides or recombinant analogs thereof from PCV2 mutants. Subunit immunogenic compositions may contain subunit antigens or target antigens that are substantially free of other antigens or polypeptides from PCV2 mutants, or in fractionated form. Preferred immunogenic subunit compositions contain polypeptides of the invention as described herein.

[0099] "Immune response" means, but is not limited to, the development of a cellular and / or antibody-mediated immune response in a host against a target composition or vaccine. Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells specifically targeting antigens or multiple antigens included in the target composition or vaccine. Preferably, the host exhibits a therapeutic or protective immune (memory) response, thereby enhancing resistance to new infections and / or reducing the clinical severity of the disease. Such protection is demonstrated by a reduction in the number or severity of signs associated with PCV2 infection, or the absence of one or more signs associated with PCV2 infection, particularly infection with PCV2b subtype (PCV2b) and / or infection with PCV2 subtypes other than 2b, delayed onset of viremia, reduced viral persistence, reduced total viral load, and / or reduced viral secretion.

[0100] As used herein, the term “antigen” refers to an amino acid sequence that elicits the immune response described above.

[0101] According to a further aspect, as used herein, the immunogenic composition most preferably comprises the polypeptide of the invention expressed by the polypeptide according to the invention, or a fragment thereof. The preferred polypeptide of the invention is the polypeptide of SEQ ID NO:1. However, those skilled in the art will understand that this sequence can vary by up to 1-5% in sequence homology and still retain the antigenic characteristics that make it usable in the immunogenic composition according to the invention.

[0102] As is known in the art, "sequence identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., the relationship between a reference sequence and a given sequence against which the reference sequence is compared. Sequence identity is determined by comparing a given sequence with a reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity (as determined by matching strings of such sequences). In such alignments, sequence identity is determined on a position-to-position basis; for example, if nucleotides or amino acid residues are identical at a particular position, the sequence is "identical" at that position. The total number of such positional similarities is then divided by the total number of nucleotides or residues in the reference sequence to give the % sequence identity. Sequence identity can be readily computed by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M. Stockton Press, New York (1991); and those described in Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. The preferred method for determining sequence identity is designed to provide the maximum match between the test sequences. This method is incorporated into publicly available computer programs for determining sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG package (Devereux, J. et al., NucleicAcids Research, 12(1):387(1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J.Molec.Biol., 215:403-410(1990)).The BLASTX procedure is publicly available from NCBI and other sources (BLASTX et al., Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These procedures utilize default vacancy weights to optimize the alignment sequence to produce the highest level of sequence identity between the given and reference sequences. As an example, for a polynucleotide having a nucleotide sequence with at least, for example, 85%, preferably 90%, or even more preferably 95% "sequence identity" with a reference nucleotide sequence, this means that, except that the given polynucleotide sequence may include up to 15, preferably up to 10, or even more preferably up to 5 point mutations / reference nucleotide sequences per 100 nucleotides, the nucleotide sequence of the given polynucleotide is identical to the reference sequence. In other words, in a polynucleotide having a nucleotide sequence with at least 85%, preferably 90%, or even more preferably 95% identity with a reference nucleotide sequence, up to 15%, preferably 10%, or even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 15%, preferably 10%, or even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence can occur at the 5' or 3' end of the reference nucleotide sequence or anywhere between those end positions, each scattered among the nucleotides in the reference sequence or scattered among one or more adjacent groups within the reference sequence. Similarly, for a polypeptide having a given amino acid sequence with at least, for example, 85%, preferably 90%, or even more preferably 95% sequence identity with a reference amino acid sequence, it means that, except that the given polypeptide sequence may include up to 15, preferably up to 10, or even more preferably up to 5 amino acid alterations / reference amino acid sequences per 100 amino acids, the given amino acid sequence of the polypeptide is identical to the reference sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, and even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, and even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or up to 15%, preferably up to 10%, and even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence can occur at the amino or carboxyl terminus of the reference amino acid sequence, or anywhere between those terminus positions, each scattered among residues in the reference sequence or scattered among one or more adjacent groups within the reference sequence. Preferably, the difference in residue positions lies in conserved amino acid substitutions. However, conserved substitutions are not included as matching when determining sequence identity.

[0103] As used herein, "sequence homology" refers to a method for determining the relevance of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and vacancies are introduced if necessary. However, compared to "sequence identity," conserved amino acid substitutions are counted as matches when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide with 95% sequence homology to a reference sequence, 85%, preferably 90%, and even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or contain a conserved substitution with another amino acid or nucleotide, or up to 15%, preferably up to 10%, and even more preferably up to 5% of the total amino acid residues or nucleotides in the reference sequence (excluding conserved substitutions) can be inserted into the reference sequence. Preferably, the homologous sequence comprises at least a segment of 50, even more preferably at least 100, even more preferably at least 250, and even more preferably at least 500 nucleotides.

[0104] "Conservative substitution" refers to replacing an amino acid residue or nucleotide with another amino acid residue or nucleotide that has similar characteristics or properties (including size, hydrophobicity, etc.) without significantly changing the overall function.

[0105] "Separated" means "artificially" altered from its natural state; that is, if it exists naturally, it is altered or removed from its original environment, or both. For example, when this term is used in this document, a polynucleotide or polypeptide that exists naturally in a living organism is not "separated," but the same polynucleotide or polypeptide that is separated from its natural state from a coexisting substance is "separated."

[0106] Therefore, according to a further aspect, the present invention also provides a method for treating or preventing PCV2 infection or reducing clinical signs caused by or associated with PCV2 infection in animals, preferably animals having anti-PCV2 antibodies (especially maternally derived anti-PCV2 antibodies), the method comprising the steps of: administering to the animal requiring such treatment an effective amount of the polypeptide of the present invention or an immunogenic composition comprising the polypeptide of the present invention, wherein the polypeptide of the present invention is any of those described herein. Preferably, the polypeptide of the protein of the present invention is: (i) a polypeptide comprising or consisting of the sequence of SEQ ID NO:1; or (ii) any polypeptide that is at least 95% homologous to the polypeptide of (i).

[0107] According to a further aspect, the polypeptide of the invention is provided in an immunogenic composition at a protein content level that effectively induces a desired immune response, namely, reducing the occurrence of one or more clinical manifestations caused by or associated with PCV2 infection, mitigating the severity of said clinical manifestations, or preventing or reducing said clinical manifestations. Preferably, the polypeptide of the present invention comprises at least 0.2 μg protein / ml of the final immunogenic composition (μg / ml), more preferably about 0.2 to about 400 μg / ml, still more preferably about 0.3 to about 200 μg / ml, even more preferably about 0.35 to about 100 μg / ml, still more preferably about 0.4 to about 50 μg / ml, still more preferably about 0.45 to about 30 μg / ml, still more preferably about 0.5 to about 18 μg / ml, even more preferably about 0.6 to about 15 μg / ml, even more preferably about 0.75 to about 8 μg / ml, even more preferably about 1.0 to about 6 μg / ml, still more preferably about 1.3 to about 3.0 μg / ml, even more preferably about 1.4 to about 2.5 μg / ml, even more preferably about 1.5 to about 2.0 μg / ml, and most preferably about 1.6 μg / ml.

[0108] According to a further aspect, the protein comprises a final immunogenic composition at a level of at least 0.2 μg / dose of PCV2b ORF-2 protein as described above, more preferably about 0.2 to about 400 μg / dose, still more preferably about 0.3 to about 200 μg / dose, even more preferably about 0.35 to about 100 μg / dose, still more preferably about 0.4 to about 50 μg / dose, still more preferably about 0.45 to about 30 μg / dose, still more preferably about 0.5 to about 18 μg / dose, even more preferably about 0.6 to about 15 μg / ml, even more preferably about 0.75 to about 8 μg / dose, even more preferably about 1.0 to about 6 μg / dose, still more preferably about 1.3 to about 3.0 μg / dose, even more preferably about 1.4 to about 2.5 μg / dose, even more preferably about 1.5 to about 2.0 μg / dose, and most preferably about 1.6 μg / dose. Furthermore, the inclusion level (antigen content) of the polypeptide of the present invention at less than 20 μg / dose, preferably about 0.5-18 μg / dose, is suitable for conferring immunity in young animals and / or animals that are positive for PCV2 antibodies (especially maternally positive anti-PCV2 antibodies). Therefore, according to a further aspect, the present invention also provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in animals, preferably animals with anti-PCV2 antibodies, especially maternally anti-PCV2 antibodies, said method comprising the step of administering to the animal requiring such treatment less than 20 μg / dose, preferably about 0.5-18 μg / dose, of the polypeptide of the present invention or an immunogenic composition containing the polypeptide of the present invention. The polypeptide of the present invention is any of those described in this patent application.

[0109] The polypeptides of the present invention used in the immunogenic compositions according to the present invention can be obtained in any manner, including the isolation and purification of the polypeptides of the present invention, the synthesis of standard proteins, and recombinant methods. Preferred methods for obtaining the polypeptides of the present invention are provided in WO06 / 072065, the teachings and contents of which are incorporated herein by reference in their entirety, as it has been surprisingly found that the methods described therein for obtaining the PCV2a ORF-2 polypeptide can be correspondingly used to obtain the polypeptides of the present invention. In short, susceptible cells are infected with a recombinant viral vector containing a DNA coding sequence encoding the polypeptide of the present invention, the polypeptide of the present invention is expressed by the recombinant virus, and the expressed polypeptide of the present invention is recovered from the supernatant by filtration and inactivation using any conventional method, preferably diethyleneimine (BEI), followed by neutralization to terminate the inactivation process.

[0110] As used herein, an immunogenic composition also refers to a composition comprising: i) any of the polypeptides of the present invention described above, preferably at the concentrations described above, and ii) a viral vector, preferably a recombinant baculovirus, expressing at least a portion of the polypeptide of the present invention. Furthermore, an immunogenic composition may comprise i) any of the polypeptides of the present invention described above, preferably at the concentrations described above, ii) a viral vector, preferably a recombinant baculovirus, expressing at least a portion of the polypeptide of the present invention, and iii) a portion of cell culture supernatant.

[0111] Therefore, according to a further aspect, the present invention also provides a method for treating or preventing PCV2 infection or reducing clinical signs caused by or related to PCV2 infection in animals, preferably animals having anti-PCV2 antibodies (especially maternally derived anti-PCV2 antibodies), the method comprising the step of administering to the animal requiring such treatment an effective amount of the polypeptide of the present invention or an immunogenic composition comprising the polypeptide of the present invention, wherein the polypeptide of the present invention is a recombinant, preferably baculovirus-expressed polypeptide of the present invention. Preferably, those recombinant or baculovirus-expressed polypeptides of the present invention have the sequences described above.

[0112] As used herein, an immunogenic composition also refers to a composition comprising: i) any of the polypeptides of the present invention described above, preferably at the concentrations described above; ii) a viral vector expressing at least a portion of the polypeptides of the present invention, preferably a recombinant baculovirus; and iii) a portion of a cell culture wherein about 90% of the components have a size of less than 1 μm.

[0113] As used herein, an immunogenic composition also refers to a composition comprising: i) any of the polypeptides of the present invention described above, preferably at the concentrations described above; ii) a viral vector expressing at least a portion of the polypeptides of the present invention; iii) a portion of a cell culture; iv) and an inactivating agent to inactivate the recombinant viral vector, preferably a BEI, wherein about 90% of components i)-iii) have a size of less than 1 μm. Preferably, the BEI is present at a concentration that effectively inactivates baculoviruses, preferably at an amount of 2 to about 8 mM BEI, preferably about 5 mM BEI.

[0114] As used herein, an immunogenic composition also refers to a composition comprising: i) any of the polypeptides of the present invention described above, preferably at the concentrations described above; ii) a viral vector expressing at least a portion of the polypeptides of the present invention; iii) a portion of a cell culture; iv) an inactivating agent to inactivate the recombinant viral vector, preferably BEI; and v) a neutralizing agent to terminate the inactivating agent-mediated inactivation, wherein about 90% of components i)-iii) have a size of less than 1 μm. Preferably, if the inactivating agent is BEI, the composition comprises sodium thiosulfate in an amount equal to that of BEI.

[0115] The protein is added to the composition, which can be administered to animals susceptible to PCV2 infection. In a preferred form, the composition may also include additional components known to those skilled in the art (see also Remington's Pharmaceutical Sciences. (1990). 18th edition Mack Publ., Easton). Additionally, the composition may include one or more veterinary-acceptable carriers. As used herein, "veterinary-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delay agents, etc. In a preferred embodiment, the immunogenic composition comprises a polypeptide of the invention as provided herein, preferably at the concentration described above, with an adjuvant (preferably...) (Lubrizol Corporation) and physiological saline.

[0116] Those skilled in the art will understand that the compositions used herein can be incorporated into known injectable, physiologically acceptable sterile solutions. For preparing ready-to-use solutions for parenteral injection or infusion, aqueous isotonic solutions such as saline or corresponding plasma protein solutions are readily available. Furthermore, the immunogenic and vaccine compositions of the present invention may include diluents, isotonic agents, stabilizers, or adjuvants. Diluents may include water, saline, glucose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, glucose, mannitol, sorbitol, and lactose, etc. Stabilizers include albumin and alkali metal salts of ethylenediaminetetraacetic acid.

[0117] As used herein, "adjuvants" may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsions may be particularly based on light liquid paraffin oils (European Pharmacopoeia type); isoprene-like oils such as squalane or squalene oils resulting from the oligomerization of olefins (especially isobutylene or decene); esters of acids or alcohols containing straight-chain alkyl groups, more specifically vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, particularly isostearates. Oils are used in combination with emulsifiers to form emulsions. The emulsifier is preferably a nonionic surfactant, particularly sorbitan, dimannitol (e.g., anhydrous mannitol oleate), glycol, polyglycerol, propylene glycol, and esters (optionally ethoxylated) of oleic acid, isostearic acid, castor oil acid, or hydroxystearic acid, as well as polyoxypropylene-polyoxyethylene copolymer blocks, especially... Products, especially L121 (BASF Corp.). See Hunter et al., The Theory and Practical Application of Adjuvants (edited Stewart-Tull, DES). John Wiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0118] For example, the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book, can be used.

[0119] A further example of an adjuvant is a compound selected from polymers of acrylic acid or methacrylic acid, as well as copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid, particularly crosslinked with polyolefin ethers of sugars or polyols. These compounds are known by the term carbomer (Pharmeuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three (preferably no more than eight) hydroxyl groups, the hydrogen atoms of which are replaced by unsaturated aliphatic groups having at least two carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl, and other vinyl-type unsaturated groups. The unsaturated group itself may contain other substituents such as methyl. Products sold by The Lubrizol Corporation are particularly suitable. They can be crosslinked with allyl sucrose or with allyl pentaerythritol. It can be mentioned that... 974P, 934P, and 971P. The optimal choice is to use... Especially using 971P is preferably administered in amounts of about 500 μg to about 5 mg per dose, even more preferably in amounts of about 750 μg to about 2.5 mg per dose, and most preferably in amounts of about 1 mg per dose.

[0120] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophospholipid A, avridine lipid-amine adjuvant, heat-labile enterotoxins from E. coli (recombinant or otherwise), cholera toxin, IMS 1314, or muramyl dipeptide, etc.

[0121] Preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 μg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[0122] Additionally, the composition may include one or more pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delay agents, etc. Most preferably, the composition provided thereby comprises the polypeptide of the invention recovered from the supernatant of autologous in vitro cultured cells, wherein the cells are infected with a recombinant viral vector containing DNA encoding the polypeptide of the invention and expressing the polypeptide of the invention, and wherein the cell culture is treated with about 2 to about 8 mM BEI, preferably about 5 mM BEI to inactivate the viral vector, and with a final concentration of about 2 to about 8 mM, preferably about 5 mM, of a neutralizing agent of equal concentration, preferably sodium thiosulfate solution.

[0123] The present invention also relates to an immunogenic composition comprising i) any of the polypeptides of the present invention described above, preferably at the concentrations described above; ii) a viral vector expressing at least a portion of the polypeptides of the present invention; iii) a portion of a cell culture; iv) an inactivating agent to inactivate the recombinant viral vector, preferably BEI; v) a neutralizing agent to terminate the inactivating agent-mediated inactivation, preferably sodium thiosulfate in an amount equal to BEI; and vi) a suitable adjuvant, preferably in the amounts described above. 971; wherein approximately 90% of components i)-iii) have a size of less than 1 μm. According to a further aspect, this immunogenic composition also comprises a pharmaceutically acceptable salt, preferably a physiologically acceptable concentration of phosphate. Preferably, the pH of the immunogenic composition is adjusted to a physiological pH, meaning approximately 6.5-7.5.

[0124] As used herein, an immunogenic composition also refers to a composition per 1 ml containing: (i) at least 1.6 μg of the polypeptide of the present invention described above, preferably less than 20 μg; (ii) at least a portion of a baculovirus expressing the polypeptide of the present invention; (iii) a portion of a cell culture; (iv) about 2-8 mM BEI; (v) sodium thiosulfate in an amount equal to BEI; and (vi) about 1 mg 971; and (vii) a physiologically acceptable concentration of phosphate; wherein approximately 90% of components (i)-(iii) have a size of less than 1 μm, and the pH of the immunogenic composition is adjusted to approximately 6.5-7.5.

[0125] The immunogenic composition may also include one or more other immunomodulators, such as interleukins, interferons, or other cytokines. The immunogenic composition may also include gentamicin and thimerosal. While those skilled in the art can readily determine the amounts and concentrations of adjuvants and additives that may be used in the context of this invention, this invention covers compositions comprising a vaccine composition containing about 50 μg to about 2000 μg of adjuvant and preferably about 250 μg / ml. Therefore, as used herein, an immunogenic composition also refers to a composition containing about 1 μg / ml to about 60 μg / ml of antibiotic, and more preferably less than about 30 μg / ml of antibiotic.

[0126] As used herein, an immunogenic composition also refers to a composition comprising: (i) any of the polypeptides of the present invention described above, preferably at the concentrations described above; (ii) a viral vector expressing at least a portion of the polypeptides of the present invention; (iii) a portion of a cell culture; (iv) an inactivating agent to inactivate the recombinant viral vector, preferably BEI; and (v) a neutralizing agent to terminate the inactivating agent-mediated inactivation, preferably sodium thiosulfate in an amount equal to BEI; and (vi) a suitable adjuvant, preferably in the amounts described above. 971; (vii) a saline buffer of pharmaceutically acceptable concentration, preferably phosphate; and (viii) an antimicrobial active agent; wherein approximately 90% of components (i)-(iii) have a size of less than 1 μm.

[0127] To investigate the potential interference of the peptides of this invention with maternal antibodies, a study can be conducted in which antibody titers in study animals are determined at the time of vaccination and then grouped into low, intermediate, and high antibody categories: a geometric mean titer <1:100 is considered low antibody titer, a titer between 1:100 and 1:1000 is considered intermediate antibody titer, and a titer >1:1000 is considered high antibody titer. This grouping pattern is comparable to that used in a Canadian field study, where an antibody titer of 1:80 was considered low, 1:640 was considered intermediate, and >1:1280 was considered high (Larochelle et al., 2003, Can. J. Vet. Res.; 67:114-120). To analyze the effects of low, intermediate, and high antibody titers at the time of vaccination on viremia, the occurrence, termination, duration, number of days to positive sampling, and viral load of viremia are compared between vaccinated and placebo-treated animals. The presence of anti-PCV2 antibodies, particularly maternal antibodies, preferably has no significant effect on any of those parameters. In other words, the efficacy of the peptides of the present invention in animals for the prevention and treatment of PCV2 infection or for reducing clinical manifestations caused by or associated with PCV2 infection is preferably unaffected by the presence of anti-PCV2 antibodies on the day of vaccination, preferably at an anti-PCV2 antibody titer of up to 1:100, preferably greater than 1:100, even more preferably greater than 1:250, even more preferably greater than 1:500, even more preferably greater than 1:640; even more preferably greater than 1:750, and most preferably greater than 1:1000. This effect can be demonstrated in a single-injection vaccination study, meaning that the peptides of the present invention are administered only once, without any subsequent administration of the peptides of the present invention.

[0128] Methods for detecting and quantifying anti-PCV2 antibodies are well known in the art. For example, the detection and quantification of PCV2 antibodies can be performed by indirect immunofluorescence as described in Magar et al., 2000, Can. J. Vet Res.; 64:184-186 or Magar et al., 2000, J. Comp. Pathol.; 123:258-269. Other assays for quantifying anti-PCV2 antibodies are described in Opriessnig et al., 2006, 37th Annual Meeting of the American Association of Swine Veterinarians. Furthermore, indirect immunofluorescence assays that can be used by those skilled in the art involve the following steps: [The text abruptly ends here, likely due to an incomplete translation or a missing section.] R1 cells were seeded per well in a 96-well plate; when the monolayer was approximately 65-85% confluent, the cells were infected with the PCV2 isolate; the infected cells were incubated for 48 hours; the culture medium was removed and the cells were washed twice with PBS; the wash buffer was discarded and the cells were fixed with a cold 50 / 50 methanol / acetone solution at approximately -20°C for approximately 15 minutes; the fixative was discarded and the plates were air-dried; serial dilutions of porcine serum samples in PBS and serial dilutions of anti-PCV2 positive and negative control samples were prepared; the serial dilutions were added to the plates and incubated to allow the antibody (if present in the serum samples) to reach 36.5 ± 1 μmol / L. Bind at ℃ for approximately 1 hour; wash the plate 3 times with PBS and discard the PBS; stain the plate with a 1:100 dilution of a commercial goat anti-pig FITC conjugate in PBS and incubate at 36.5±1℃ for approximately 1 hour; remove the microplate from the incubator, discard the conjugate, and wash the plate twice with PBS; read the plate using UV microscopy and report individual wells as positive or negative, with positive and negative control samples used to monitor the test system; and calculate the serum antibody titer using the highest dilution exhibiting specific IFA reactivity and the number of positive wells / dilution, or calculate the 50% endpoint using the appropriate Reed-Muench formula.

[0129] This assay method is described in Example 2 of WO 2008 / 076915A2, which is incorporated herein by reference.

[0130] In cases where results are disputed or in any questionable matter, the anti-PCV2 titer mentioned herein refers to / can be estimated by this assay.

[0131] Therefore, according to a further aspect, the present invention also provides a method for treating or preventing PCV2 infection or reducing clinical signs caused by or related to PCV2 infection in animals, preferably animals having anti-PCV2 antibodies (especially maternal antibodies), the method comprising the steps of: administering an effective amount of the polypeptide of the present invention, preferably less than 20 μg / dose, to the animal requiring such treatment, wherein the animal has a detectable anti-PCV2 antibody titer up to 1:100, preferably more than 1:100, even more preferably more than 1:250, even more preferably more than 1:500, even more preferably more than 1:640, even more preferably more than 1:750, and most preferably more than 1:1000. Preferably, as exemplarily described in Example 2 of WO2008 / 076915 A2, the anti-PCV2 antibody titer is detectable and quantifiable in a specific anti-PCV2 immunoassay, preferably in an assay as described above. More preferably, those anti-PCV-2 antibodies are maternal antibodies. Most preferably, the polypeptide of the present invention is administered only once, preferably at a dose of less than 20 μg / dose.

[0132] Piglets with only low (<1:100) or medium (<1:1000) titers of maternal anti-PCV2 antibodies are insufficient to protect against PCV2 infection occurring before 3 weeks of age. Therefore, vaccination at a very early stage of life is desirable. In the context of this invention, it is preferred to vaccinate / treat animals at or before 3 weeks of age. Furthermore, anti-PCV2 antibody titers exceeding 1:1000 preferably have no effect on the efficacy of the PCV2 vaccine, regardless of the existing initial antibody titer. For example, vaccination of high-titer animals (anti-PCV2 antibody titer >1:1000) preferably results in a shorter duration of viremia, earlier termination of viremia, fewer days of viremia sampling, and a reduced total genomic equivalents / ml compared to unvaccinated animals. When comparing “high,” “medium,” and “low-titer” vaccinated animals, no significant differences were preferably observed regarding the different parameters of PCV2 viremia. Furthermore, in the presence of high anti-PCV2 antibody titers, the peptides of the present invention used for vaccination preferably still significantly reduce viremia in the blood (e.g., termination of viremia, duration of viremia, viral load). Preferably, no difference in live body weight was found when comparing low- and high-titer animals in the vaccination group. Moreover, animals vaccinated with high anti-PCV2 antibody titers (>1:1000) at the time of vaccination / treatment preferably exhibit significantly higher body weight after the onset of viremia, compared to placebo-treated animals with initial high antibody titers. Therefore, according to a preferred aspect, it is feasible to vaccinate / treat animals 1 day old or older with the peptides of the present invention. However, vaccination should be performed within the first 8 weeks of age, preferably within the first 7 weeks. Therefore, according to a further aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or related to PCV2 infection in animals, the method comprising the step of administering an effective amount of the peptides of the present invention to animals requiring such treatment at 1 day old or later, but preferably no later than 8 weeks of age. According to a preferred embodiment, less than 20 μg / dose of the polypeptide of the present invention is required to confer immunity in such animals. According to a more preferred embodiment, the polypeptide of the present invention, preferably less than 20 μg / dose, is administered to the animal requiring such treatment only once.

[0133] According to a further, more general aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals, the method comprising the step of administering an effective amount of the polypeptide of the present invention to the animal requiring such treatment.

[0134] As used herein, the term "young animal" refers to an animal aged 1-22 days. Preferably, the term "young animal" refers to an animal aged 1-20 days. More preferably, the term "young animal" refers to an animal aged 1-15 days, even more preferably an animal aged 1-14 days, even more preferably an animal aged 1-12 days, even more preferably an animal aged 1-10 days, even more preferably an animal aged 1-8 days, even more preferably an animal aged 1-7 days, even more preferably an animal aged 1-6 days, even more preferably an animal aged 1-5 days, even more preferably an animal aged 1-4 days, even more preferably an animal aged 1-3 days, even more preferably an animal aged 1 or 2 days, and most preferably an animal aged 1 day.

[0135] Therefore, according to a further aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals, the method comprising the steps of administering an effective amount of the polypeptide of the present invention to an animal requiring such treatment, said animal being 1-22 days old, preferably 1-20 days old, more preferably 1-15 days old, even more preferably 1-14 days old, even more preferably 1-12 days old, even more preferably 1-10 days old, even more preferably 1-8 days old, even more preferably 1-7 days old, even more preferably 1-6 days old, even more preferably 1-5 days old, even more preferably 1-4 days old, even more preferably 1-3 days old, even more preferably 1 or 2 days old, and most preferably 1 day old. For example, immunization / treatment at 19-22 days of age preferably exhibits highly effective immunization. Furthermore, immunization / treatment at 12-18 days of age, preferably 12-14 days of age, is highly effective in reducing clinical manifestations associated with PCV2 infection, reducing total viral load, reducing the duration of viremia, delaying the onset of viremia, and promoting weight gain. Additionally, immunization at 1 week of age is also highly effective in reducing clinical manifestations associated with PCV2 infection, reducing total viral load, reducing the duration of viremia, delaying the onset of viremia, and promoting weight gain. Preferably, less than 20 μg / dose of the polypeptide of the present invention is required to confer immunity in those young animals. According to a more preferred embodiment, the polypeptide of the present invention, preferably less than 20 μg, is administered to the young animals requiring such treatment only once.

[0136] Because PCV2 is prevalent in this field, most young piglets are seropositive for PCV2. Therefore, according to a further aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or related to PCV2 infection in young animals, preferably animals with anti-PCV2 antibodies on the day of immunization, the method comprising the steps of administering an effective amount of the polypeptide of the present invention to the animal requiring such treatment, the animal being 1-22 days old, preferably 1-20 days old, more preferably 1-15 days old, even more preferably 1-14 days old, even more preferably 1-12 days old, even more preferably 1-10 days old, even more preferably 1-8 days old, even more preferably 1-7 days old, even more preferably 1-6 days old, even more preferably 1-5 days old, even more preferably 1-4 days old, even more preferably 1-3 days old, even more preferably 1 or 2 days old, and most preferably 1 day old.

[0137] Preferably, on the day of immunization / treatment, the young animals have a detectable anti-PCV2 antibody titer, which is as high as 1:100, preferably greater than 1:100, even more preferably greater than 1:250, even more preferably greater than 1:500, even more preferably 1:640, even more preferably greater than 1:750, and most preferably greater than 1:1000. Preferably, less than 20 μg / dose of the polypeptide of the present invention is required to confer sufficient immunity in those young animals. According to a more preferred embodiment, the polypeptide of the present invention, preferably less than 20 μg, is administered to the young animals requiring such treatment only once.

[0138] As described above, immunization / treatment of young animals with the peptides of the present invention preferably results in a shorter period of viremia compared to unimmunized control animals. The average reduction in duration compared to unimmunized control animals of the same species can preferably be, for example, 9.5 days. Therefore, according to a further aspect, the present invention also provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals, the method comprising the step of administering an effective amount of the peptides of the present invention to the animal requiring such treatment, wherein the treatment or prevention results in a shorter period of viremia of 5 days or more, preferably 6 days or more, even more preferably 7 days or more, even more preferably 8 days or more, even more preferably 9 days, even more preferably 10 days, even more preferably 12 days, even more preferably 14 days, and most preferably more than 16 days, compared to untreated control animals of the same species. In some cases, the viremia period is preferably shortened by more than 20 days. Generally, immunization of young piglets preferably results in reduced loss of weight gain, shorter duration of viremia, earlier termination of viremia, and lower viral load. Therefore, according to a further aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals, the method comprising the step of administering an effective amount of the polypeptide of the present invention to an animal requiring such treatment, wherein the treatment or prevention of PCV2 infection results in an improved vaccine efficacy parameter compared to an untreated control group of the same species, the vaccine efficacy parameter being selected from reduced loss of weight gain, shorter duration of viremia, earlier termination of viremia, lower viral load, or combinations thereof. Preferably, less than 20 μg / dose of the polypeptide of the present invention is required to cause any of the above-mentioned improved vaccine efficacy parameters. Furthermore, such improved vaccine efficacy parameters are achieved by a single administration of only one dose.

[0139] As used herein, the term "effective amount" means, but is not limited to, the amount of the polypeptide of the invention that elicits or is capable of eliciting an immune response in an animal administered the effective amount of the polypeptide of the invention. Preferably, the effective amount is defined as the amount of the polypeptide of the invention that confers immunity for at least 10 weeks (DOI), preferably at least 12 weeks (DOI), more preferably at least 15 weeks (DOI), and most preferably at least 20 weeks (DOI).

[0140] The effective dose depends on the vaccine's composition and the timing of administration. Typically, when inactivated virus or modified live virus products are used in combination vaccines, the dose contains approximately 10... 2.0 - Approximately 10 9.0 TCID 50 / Dosage, preferably about 10 3.0 - Approximately 10 8.0 TCID50 / Dosage, preferably about 10 4.0 - Approximately 10 8.0 TCID 50 / Dosage. Specifically, when modified live PCV2 is used as a vaccine, the recommended dose administered to susceptible animals is preferably 10. 3.0 TCID 50 (50% endpoint of tissue culture infection dose) / dose - approximately 10 6.0 TCID 50 / dose, and more preferably about 10 4.0 TCID 50 / Dosage - approximately 10 5.0 TCID 50 / Dosage. Generally, when using purified antigens, the amount of antigen is 0.2-5000 micrograms, and 10 2.0 -10 9.0 TCID 50 10 preferred 3.0 -10 6.0 TCID 50 , more preferably 10 4.0 -10 5.0 TCID 50 .

[0141] Subunit vaccines are typically administered at a protein content level of at least 0.2 μg protein per dose, preferably from about 0.2 to about 400 μg per dose, more preferably from about 0.3 to about 200 μg per dose, even more preferably from about 0.35 to about 100 μg per dose, more preferably from about 0.4 to about 50 μg per dose, more preferably from about 0.45 to about 30 μg per dose, more preferably from about 0.5 to about 18 μg per dose, more preferably from about 0.6 to about 16 μg per dose, even more preferably from about 0.75 to about 8 μg per dose, even more preferably from about 1.0 to about 6 μg per dose, and even more preferably from about 1.3 to about 3.0 μg per dose.

[0142] Preferably, the prophylactic use of the above-described immunogenic compositions effectively reduces clinical manifestations caused by or associated with PCV2 infection, preferably in young animals and / or animals with passive immunization against PCV2 on the day of treatment. In particular, the prophylactic use of the immunogenic compositions described herein, especially compositions containing the peptides of the present invention, preferably effectively reduces lymphadenopathy, lymphatic depletion, and / or multinucleated / giant histiocytes in animals infected with PCV2 on the day of treatment / vaccination and possessing maternal anti-PCV-2 antibodies. For example, it has been found that the prophylactic use of the immunogenic compositions described herein effectively reduces lymphatic depletion, lymphatic inflammation, positive IHC for PCV2 antigen in lymphoid tissue, viremia, nasal shedding, fever, reduced mean daily weight gain, pulmonary inflammation, and positive IHC for PCV2 antigen in lung tissue.

[0143] Furthermore, the prophylactic use of the immunogenic compositions described herein is preferably effective in reducing (1) interstitial pneumonia with interlobular edema, (2) pale skin or jaundice, (3) atrophic liver with spots, (4) gastric ulcers, (5) nephritis, (6) reproductive disorders such as abortion, stillbirth, mummification, etc., (7) pia-like lesions, which are generally known to be associated with Lawsonia intracellularis infection (ileitis), (8) lymphadenopathy, (9) lymphatic depletion, and / or (10) multinucleated / giant histiocytes, (11) porcine dermatitis and nephropathy syndrome (PDNS), (12) PCVAD-related death, (13) PCVAD-related weight loss, (14) reduced growth variability, (15) reduced frequency of “dwarfism”, and (16) reduced co-infection with porcine reproductive and respiratory disease syndrome (PRRSV). Such immunogenic compositions also effectively improve economically important growth parameters such as slaughter time, ketone body weight, and lean meat percentage. Therefore, as used herein, the term "clinical manifestations" refers to, but is not limited to, (1) interstitial pneumonia with interlobular edema, (2) pale skin or jaundice, (3) atrophic liver with spots, (4) gastric ulcers, (5) nephritis, (6) reproductive disorders such as abortion, stillbirth, mummification, etc., (7) pia mater-like lesions, commonly known to be associated with Lawsonia intracellularis infection (ileitis), (8) lymphadenopathy, (9) lymphatic depletion, and / or (10) multinucleated / giant histiocytes, (11) porcine dermatitis and nephrotic syndrome (PDNS), (12) PCVAD-related mortality, (13) PCVAD-related weight loss, (14) reduced growth variability, (15) reduced frequency of “dwarfism”, (16) reduced co-infection with porcine reproductive and respiratory disease syndrome (PRRSV), (17) lymphoid inflammation, (18) positive IHC for PCV2 antigen in lymphoid tissue, (19) viremia, (20) nasal shedding, (21) fever, (22) reduced mean daily weight gain, (23) pulmonary inflammation, (24) positive IHC for PCV2 antigen in lung tissue. Furthermore, the immunogenic compositions described herein reduce total circovirus load, including delayed onset, shorter duration, earlier termination of viremia, and reduced viral load and its immunosuppressive effects in young animals (especially those with anti-PCV2 antibodies on the day of vaccination), resulting in higher levels of overall disease resistance and a reduced incidence of PCV2-related diseases and clinical manifestations.

[0144] Therefore, according to a further aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals and / or animals, preferably animals with anti-PCV2 antibodies, said method comprising the steps of administering an effective amount of the polypeptide of the present invention or an immunogenic composition comprising the polypeptide of the present invention to the animal requiring such treatment, wherein those clinical manifestations are selected from: (1) interstitial pneumonia with interlobular edema, (2) pale skin or jaundice, (3) atrophic liver with spots, (4) gastric ulcers, (5) nephritis, (6) reproductive disorders, such as abortion, stillbirth, mummification, etc., (7) pia mater-like lesions, commonly known to be associated with Lawsonia intracellularis infection. (8) Ileitis, (9) Lymphadenopathy, and / or (10) Multinucleated / giant histiocytes, (11) Porcine dermatitis and nephropathy syndrome (PDNS), (12) PCVAD-related death, (13) PCVAD-related weight loss, (14) Reduced growth variability, (15) Reduced frequency of “dwarfism”, (16) Reduced co-infection with porcine reproductive and respiratory disease syndrome (PRRSV), (17) Lymphatic inflammation, (18) Positive IHC for PCV2 antigen in lymphoid tissue, (19) Viremia, (20) Nasal shedding, (21) Fever, (22) Reduced mean daily gain, (23) Pulmonary inflammation, (24) Positive IHC for PCV2 antigen in lung tissue. According to a further aspect, the present invention provides a method for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals, the method comprising the steps of administering an effective amount of the polypeptide of the present invention to an animal requiring such treatment, wherein the clinical manifestations are selected from: (1) interstitial pneumonia with interlobular edema, (2) pale skin or jaundice, (3) atrophic liver with spots, (4) gastric ulcer, (5) nephritis, (6) reproductive disorders, such as abortion, stillbirth, mummification, etc., (7) pia mater-like lesions, generally known to be associated with Lawsonia intracellularis infection (ileitis), (8) lymphadenopathy, (9) lymphadenopathy. Porcine exhaustion, and / or (10) multinucleated / giant histiocytes, (11) porcine dermatitis and nephropathy syndrome (PDNS), (12) PCVAD-related death, (13) PCVAD-related weight loss, (14) reduced growth variability, (15) reduced frequency of “dwarfism”, (16) reduced co-infection with porcine reproductive and respiratory disease syndrome (PRRSV), (17) lymphoid inflammation, (18) positive IHC for PCV2 antigen in lymphoid tissue, (19) viremia, (20) nasal shedding, (21) fever, (22) reduced mean daily weight gain, (23) pulmonary inflammation, (24) positive IHC for PCV2 antigen in lung tissue.

[0145] The compositions of the present invention can be administered orally, intradermally, intratracheally, or vaginally. The compositions are preferably administered intramuscularly or intranasally, with intramuscular administration being the most preferred. In animals, it has been demonstrated that administration of the pharmaceutical compositions described above via intravenous or direct injection into the target tissue is advantageous. For systemic administration, intravenous, intravascular, intramuscular, intranasal, intra-arterial, intraperitoneal, oral, or intrathecal routes are preferred. More local administration can be achieved subcutaneously, intradermally, intradermally, intracardiacly, intralobarly, intramedullaryly, intrapulmonaryly, or directly in or near the tissue to be treated (e.g., connective tissue, bone, muscle, nerve tissue, epithelial tissue). Depending on the desired duration and effectiveness of treatment, the compositions of the present invention can be administered once or several times, or intermittently, for example, daily for several days, weeks, or months at different doses.

[0146] Preferably, a dose of the immunogenic composition as described above is administered intramuscularly to the subject in need. According to a further aspect, the polypeptide of the present invention as described herein, or an immunogenic composition containing any such polypeptide of the present invention, is bottled and administered at a dose of one (1) mL. Thus, according to a further aspect, the present invention also provides a 1 ml immunogenic composition containing the polypeptide of the present invention as described herein, for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection in young animals, comprising the step of administering an effective amount of the polypeptide of the present invention to the animal requiring such treatment. According to a further aspect, the present invention also provides a 1 ml immunogenic composition containing the polypeptide of the present invention as described herein, in animals, preferably in animals with anti-PCV2 antibodies, for treating or preventing PCV2 infection or reducing clinical manifestations caused by or associated with PCV2 infection, comprising the step of administering an effective amount of the polypeptide of the present invention or an immunogenic composition containing the polypeptide of the present invention to the animal requiring such treatment.

[0147] According to a further aspect, the immunogenic composition as described above is administered at least once more to the subject in need, wherein the second or any additional administration is given at least 14 days after the initial or any previous administration. Preferably, the immunogenic composition is administered together with an immunostimulant. Preferably, the immunostimulant is administered at least twice. Preferably, there is at least 3 days, more preferably at least 5 days, and even more preferably at least 7 days between the first and second or any additional administration of the immunostimulant. Preferably, the immunostimulant is administered at least 10 days, preferably 15 days, even more preferably 20 days, and even more preferably at least 22 days after the initial administration of the immunogenic composition provided herein. Preferred immunostimulants are, for example, keyhole hemocyanin (KLH), preferably emulsified with incomplete Freund's adjuvant (KLH / ICFA). However, it should be understood that any other immunostimulant known to those skilled in the art may also be used. As used herein, the term "immunostimulant" means any substance or composition that can trigger an immune response, preferably without inducing or increasing a specific immune response, such as an immune response against a specific pathogen. Further instructions were given to administer the immunostimulant at an appropriate dose.

[0148] In a further aspect, the present invention provides a method for determining whether an individual has received an immunogenic composition, particularly a vaccine, containing the polypeptide of the present invention, wherein the method comprises the following steps:

[0149] - Obtaining biological samples from individuals,

[0150] - The presence or absence of one or more markers in the biological sample is determined, wherein the one or more markers indicate that the individual has received the target amino acid sequence included in the polypeptide of the present invention.

[0151] Furthermore, the presence of one or more markers in the biological sample indicates that the individual has received the immunogenic composition, or the absence of one or more markers in the biological sample indicates that the individual has not received the immunogenic composition.

[0152] According to the first aspect, the present invention therefore provides a method for determining whether an individual has received an immunogenic composition comprising the polypeptide of the present invention, the method being referred to hereinafter as "the method of the present invention", wherein the method specifically includes determining the presence or absence of one or more markers in a biological sample obtained from the individual, the one or more markers indicating that the individual has received the target amino acid sequence, and wherein the presence of the one or more markers in the biological sample indicates that the individual has received the immunogenic composition.

[0153] Preferably, the immunogenic composition of the present invention is a marker vaccine, particularly a positive marker vaccine.

[0154] As described in this article, the term “marker vaccine” specifically refers to a vaccine that induces immunity in an immune organism, which is different from the immunity induced in an organism by a real pathogen.

[0155] "Positive marker vaccines" specifically refer to marker vaccines containing additional antigens that induce the production of specific antibodies present in vaccinated individuals rather than infected individuals.

[0156] As used in the context of this invention, the term "marker" is preferably equivalent to the term "biomarker," and specifically refers to a measurable substance or compound that indicates an individual's exposure to an immunogenic composition, preferably a positive marker vaccine, or more particularly, an additional antigen of a positive marker vaccine that induces the production of specific antibodies found in a vaccinated subject rather than an infected subject.

[0157] As used herein, the term "immunogenic composition" specifically refers to a composition that will elicit an immune response in an individual who has been exposed to the composition. An 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, it may include one or more antigens. Preferably, the immunogenic composition described herein is a vaccine.

[0158] As used herein, the term "vaccine" is specifically defined according to the relevant field and relates to a composition that induces or enhances an individual's immunity to a particular disease. For this purpose, a vaccine contains a compound similar to 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 "remembers" the contact with the compound, ensuring easy and effective recognition and destruction of the pathogen upon subsequent contact. According to the invention, a vaccine can be any formulation of vaccines known in the art, such as vaccines for intramuscular injection, mucosal vaccines, or vaccines for subcutaneous or intradermal injection, as well as vaccines for inhalation, such as 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 FPNijkamp, ​​Michael J. Parnham 2011; Principles of Immunopharmacology ISBN-13:978-3034601351.

[0159] Preferably, the biological sample is obtained from the individual 14 to 35 days after the individual has been vaccinated or is presumed to have been vaccinated.

[0160] One or more markers indicating that an individual has received a target amino acid sequence (hereinafter also referred to as "one or more markers of the present invention") are antibodies specific to the target amino acid sequence.

[0161] Preferably, the antibody described herein is a polyclonal antibody.

[0162] As used herein, the term "antibody for a defined antigen" specifically refers to antibodies (preferably polyclonal antibodies) that, for example, have a specific antigen content of 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 binding constant of a specific binding interaction in units of 1 / M) binds to the antigen. Alternatively, binding affinity can be defined in units of M (e.g., 10). -5 M to 10 -13 The equilibrium dissociation constant (K) of the specific binding interaction of M) d The binding affinity of an antibody can be readily determined using techniques well known to those skilled in the art (see, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; U.S. Patent Nos. 5,283,173; 5,468,614; Analysis; or equivalent).

[0163] Preferably, the method of the present invention includes the following steps:

[0164] -Contact the biological sample with a capture agent immobilized to a solid support, wherein the immobilized capture agent is capable of binding one or more of the markers, and

[0165] - Determine the presence or absence of the one or more markers that bind to the capture reagent, wherein the presence of the one or more markers that bind to the capture reagent indicates the presence of the one or more markers in the biological sample.

[0166] As used herein, the term "capture agent" specifically refers to a molecule or multimolecular complex capable of binding a labeled substance. The capture agent preferably binds to the labeled substance in a substantially specific manner, and preferably has affinity or K+. a >10 5 M -1 Or preferably >10 6 M -1 The capture agent can optionally be a naturally occurring, recombinant, or synthetic biomolecule. Proteins and nucleic acid ligands (aptamers) are highly suitable as capture agents. Whole viruses, viral fragments, or synthetic peptides can also serve as preferred capture agents because they are capable of binding antibodies.

[0167] The capture reagent mentioned herein, which is immobilized to a solid support and capable of binding one or more markers of the present invention, and which is also referred to hereinafter as “capture reagent according to the invention”, is preferably (i) a protein containing a target amino acid sequence, or (ii) a peptide containing or composed of a target amino acid sequence, such as a synthetic peptide.

[0168] As used herein, the term “fixed” specifically means that the capturing agent can 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 attachment, etc.

[0169] As mentioned herein, the term "solid support" refers to 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; gelling materials such as silica, alumina, and polymer gels; capillaries, which may be made of 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 component of the solid support being measured differs from the inert solid surface that may come into contact with it because the "solid support" contains at least one portion on its surface that is intended to interact directly or indirectly with the capturing reagent. The solid support can be a stationary component, such as a tube, strip, cuvette, or microtiter plate, or it can be a non-stationary component, such as beads and microparticles. Microparticles can also be used as solid supports for homogeneous assays. A variety of microparticles that allow for 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.

[0170] A “chip” is a solid, non-porous material, such as metal, glass, or plastic. The material may optionally be completely or partially encapsulated. An array of points, visible or in coordinates, exists on the surface of the material. At each point, a defined polypeptide may be fixed, with or without connectors or spacers to the material surface. All documents mentioned above and below are incorporated herein by reference.

[0171] Preferably, the method of the present invention includes 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 to the target amino acid sequence, and wherein the method includes the following steps:

[0172] a. Contact the biological sample with a trapping agent immobilized to a solid support, wherein the trapping agent is selected from the following:

[0173] i. Proteins containing the target amino acid sequence,

[0174] ii. A peptide containing the target amino acid sequence or composed of the target amino acid sequence;

[0175] b. Separate biological samples from immobilized capture reagents;

[0176] c. Contacting the immobilized capture reagent-antibody complex with a detectable reagent, wherein the detectable reagent binds to an antibody of the reagent-antibody complex; and

[0177] d. The level of antibody bound to the capture reagent is measured using a detection method for the detection reagent, wherein the measurement step (D) preferably further includes a comparison with a standard curve to determine the level of antibody bound to the capture reagent.

[0178] Preferably, the detectable reagent that binds to the antibody in the reagent-antibody complex is a detectable antibody, more preferably a labeled second antibody.

[0179] The method of the present invention preferably further includes the step of determining the presence of one or more analytes selected from the following in a biological sample:

[0180] - For peptide-specific antibodies, the peptide comprises or is composed of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0181] In the context of the method of the present invention, the immunogenic composition is preferably the immunogenic composition described below.

[0182] As used herein, the term "biological sample" means any sample obtained 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, aspirates from the lungs and other organs, and lavage and enema solutions, as well as any other source available from a human or animal object. For animals, examples of "biological samples" include blood, cells, feces, diarrhea, milk, mucus, sputum, pus, saliva, semen, sweat, tears, urine, eye fluids, vaginal secretions, and vomit, if present in the animal.

[0183] As described herein, biological samples are preferably isolated from pigs and / or specifically selected from whole blood, plasma, serum, urine, and oral fluid. In this document, the term "serum" is intended to be equivalent to "blood serum".

[0184] As used herein, the term "oral fluid" specifically refers to one or more fluids found alone or in combination in the oral cavity. These include, but are not limited to, saliva and mucosal exudates. It should be understood in particular that oral fluids 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 alone or in combination. The term "saliva" refers to a combination of oral fluids typically found in the mouth, particularly after chewing. As used herein, the term "mucosal exudate" refers to fluids resulting from the passive diffusion of serum components from the oral mucosal interstitium into the oral cavity. Mucosal exudates typically form a component of saliva.

[0185] As described herein, the immobilized capture reagent is preferably coated on a microtiter plate, particularly a microtiter plate that can be read by an ELISA reader.

[0186] According to another aspect, the present invention provides a kit, particularly a test kit, for determining whether an individual has received an immunogenic composition containing the polypeptide of the present invention, 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 antibodies specific to a target amino acid sequence contained in the polypeptide of the present invention, and wherein the one or more capture agents are preferably selected from the following:

[0187] i. Proteins containing the target amino acid sequence; and

[0188] ii. A peptide containing or composed of the target amino acid sequence, especially a synthetic peptide.

[0189] Example

[0190] Example 1

[0191] Materials and procedures / design of mutants

[0192] Preparation of mutant PCV2b ORF2 baculovirus

[0193] The sequence encoding SEQ ID NO:1 (PCV2b ORF2-cmyc) was cloned into the baculovirus transfer vector pVL1393 and co-transfected with baculovirus DNA in Sf9 cells. The recombinant baculovirus was examined for PCV2b ORF2-cmyc expression by IFA. Amplification primary strains of the recombinant baculovirus were prepared in Sf+ cells and transfected via TCID45. 50 The method involves titration to determine the baculovirus titer.

[0194] Evaluation of PCV2b ORF2-cmyc baculovirus expression

[0195] Recombinant baculovirus was evaluated for expression of the PCV2b ORF2-cmyc coding sequence by infecting Sf+ cells with a target MOI of 0.1. Infection was allowed to proceed for 5–7 days, followed by harvesting by centrifugation at 20,000g for 20 minutes to remove cell debris and insoluble proteins. The harvest supernatant was filtered through a 0.2 μm filter, and PCV2b ORF2-cmyc expression was directly assessed using an α-PCV2 antibody via Western blotting. The presence of macromolecular structures in the harvest supernatant was also evaluated. Briefly, each sample of harvest supernatant was centrifuged at 100,000g for 2 hours. The resulting precipitates were resuspended in a small amount of TBS and separated by SDS-PAGE. The PCV2b ORF2-cmyc band was detected in the stained gel by comparison with the size of PCV2b ORF2. Figure 1 The resuspended particles were further separated along a 10%–60% discontinuous sucrose gradient by centrifugation at 100,000 g for 2 hours to partially purify PCV2b ORF2-cmyc protein for quantification and VLP confirmation by electron microscopy (EM). Figure 2 ).

[0196] After sucrose gradient separation, fractions containing PCV2b ORF2 were combined, and the PCV2b ORF2-cmyc concentration was determined by SDS-PAGE gel densitometer by comparison with a BSA standard curve. Additionally, the sucrose gradient purified material was further concentrated and submitted for VLP confirmation via EM, using phosphotungstic acid as negative staining (e.g., Figure 3 ).

[0197] Example 2

[0198] PCV2b ORF2-cmyc was identified by ELISA using (i) anti-PCV2b ORF2 antibody and (ii) anti-c-myc antibody.

[0199] Therefore, it can be seen that the PCV2 ORF2-c-myc VLP is recognized by both anti-PCV2b ORF2 antibody and anti-c-myc antibody. Figure 4 ).

[0200] Example 3

[0201] The purified PCV2 ORF2-c-myc VLP was evaluated in rabbits according to the following protocol:

[0202] Study 1-200 μg / dose

[0203] - Day 0 (French Complete)

[0204] - Day 7 (French incomplete)

[0205] - Day 14 (French incomplete)

[0206] Study 2-200 μg / dose

[0207] - Day 0 (French Complete)

[0208] Serum samples were assessed using ELISA to evaluate IgG responses to c-myc and PCV2 ORF2. Figure 5 ).

[0209] In the sequence list:

[0210] SEQ ID NO:1 corresponds to SEQ ID NO:2, which includes SEQ ID NO:5.

[0211] SEQ ID NO:2 corresponds to the sequence of the wild-type PCV2b ORF2 protein.

[0212] SEQ ID NO:3 corresponds to the sequence of the wild-type PCV2a ORF2 protein.

[0213] SEQ ID NO:4 corresponds to the polynucleotide sequence encoding SEQ ID NO:1.

[0214] SEQ ID NO:5 corresponds to the target amino acid sequence containing the c-myc tag peptide (corresponding to the C-terminal amino acids (410-419) of human c-myc protein).

[0215] SEQ ID NO:6 corresponds to the sequence of a peptide encoded by the ORF5 gene of the PRRS virus.

[0216] SEQ ID NO:7 corresponds to the sequence aa 1-11 of SEQ ID NO:6. sequence list <110> Boehringer Ingelheim Animal Health <120> PCV2 ORF2 carrier platform <130> P10-0170 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> corresponds to SEQ ID NO:2 with amino acid positions 58 to 64 being replaced by SEQ ID NO:6 <400> 1 Met Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Ile Gly Tyr Thr Val Glu Gln Lys Leu Ile Ser Glu 50 55 60 Glu Asp Leu Thr Thr Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn 65 70 75 80 Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val 85 90 95 Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro 100 105 110 Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val 115 120 125 Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp 130 135 140 Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser 145 150 155 160 Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile 165 170 175 Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu 180 185 190 Gln Thr Thr Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu 195 200 205 Asn Ser Ile Tyr Asp Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val 210 215 220 Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys 225 230 235 <210> 2 <211> 234 <212> PRT <213> Porcine circovirus <400> 2 Met Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Lys Thr Thr Val Arg Thr 50 55 60 Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140 Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr 145 150 155 160 Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro 165 170 175 Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn 180 185 190 Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp 195 200 205 Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe 210 215 220 Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys 225 230 <210> 3 <211> 233 <212> PRT <213> Porcine circovirus <400> 3 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Thr Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val 65 70 75 80 Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140 Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr 145 150 155 160 Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175 Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn 180 185 190 Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp 195 200 205 Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe 210 215 220 Asn Leu Lys Asp Pro Pro Leu Glu Pro 225 230 <210> 4 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> SEQ ID NO:1 encodes <400> 4 atgacgtatc caaggaggcg tttccgcaga cgaagacacc gcccccgcag ccatcttggc 60 cagatcctcc gccgccgccc ctggctcgtc cacccccgcc accgttaccg ctggagaagg 120 aaaaatggca tcttcaacac ccgcctctcc cgcaccatcg gttatactgt cgaacaaaag 180 ctcatctccg aggaagacct cacaacgccc tcctggaatg tggacatgat gagatttaat 240 attaatgatt ttcttccccc aggagggggc tcaaaccccc tcactgtgcc ctttgaatac 300 tacagaataa ggaaggttaa ggttgagttc tggccctgct ccccaatcac ccagggtgac 360 aggggagtgg gctccactgc tgttattcta gatgataact ttgtaacaaa ggccaatgcc 420 ctaacctatg acccctatgt aaactactcc tcccgccata ccataaccca gcccttctcc 480 taccactccc ggtactttac cccgaacct gtccttgata ggacaatcga ttactccaa 540 cccataaca aaagaaatca actctggctg agactacaaa ctactggaaa tgtagaccat 600 gtaggcctcg gcactgcgtt cgaaaacagt atatacgacc aggactacaa tatccgtata 660 accatgtag tacattcag agaatttaat cttaaagacc cccacttaa ccctaagtga 720 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> c‐myc epitope <400> 5 Leu Glu Gln Lys With Serving Leu Glu Asp Thr 1 5 10 <210> 6 <211> 16 <212> PRT <213> Swine reproductive and respiratory syndrome virus <400> 6 Ser Ser His Gln and Tyr Asn and Thr and Cys Glu Asn 1 5 10 15 <210> 7 <211> 11 <212> PRT <213> Swine reproductive and respiratory syndrome virus <400> 7 Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr 1 5 10

Claims

1. A recombinant PCV2 ORF2 protein, wherein the amino acid sequence of the recombinant PCV2 ORF2 protein is as shown in the amino acid sequence of SEQ ID NO:

1.

2. An immunogenic composition comprising the recombinant PCV2 ORF2 protein of claim 1.

3. A polynucleotide whose sequence encodes the recombinant PCV2 ORF2 protein of claim 1.

4. A plasmid comprising a polynucleotide whose sequence encodes the recombinant PCV2ORF2 protein of claim 1.

5. A cell comprising a plasmid, said plasmid comprising a polynucleotide whose sequence encodes the recombinant PCV2 ORF2 protein of claim 1.

6. A virus-like particle comprising a plurality of the recombinant PCV2 ORF2 protein of claim 1.

7. A baculovirus containing a polynucleotide whose sequence encodes the recombinant PCV2 ORF2 protein of claim 1.

8. An insect cell comprising a baculovirus containing a polynucleotide whose sequence encodes the recombinant PCV2 ORF2 protein of claim 1.

9. The following uses are permitted in the preparation of medicaments for the prevention of diseases caused by PCV2 infection: - The recombinant PCV2 ORF2 protein of claim 1, - The immunogenic composition of claim 2, - The polynucleotide of claim 3, - The virus-like particles of claim 6, - The baculovirus of claim 7, - The plasmid of claim 4, and / or - The insect cell of claim 8; The disease mentioned is post-weaning multisystem failure syndrome.

10. The use of claim 9, wherein the drug is a vaccine.

11. A method for producing the recombinant PCV2 ORF2 protein of claim 1, comprising transfecting cells with the plasmid of claim 4.

12. A method for producing the recombinant PCV2 ORF2 protein of claim 1, comprising infecting insect cells with the baculovirus of claim 7.

13. Use of the recombinant PCV2 ORF2 protein of claim 1 in the preparation of a kit for determining whether an individual has received an immunogenic composition containing the recombinant PCV2 ORF2 protein of claim 1 by the following methods. The method includes determining the presence or absence of one or more markers in a biological sample obtained from the individual, the presence of the one or more markers in the biological sample indicating that the individual has received the immunogenic composition.

14. The use of claim 13, wherein the one or more markers indicating that the individual has received the immunogenic composition are antibodies specific to a heterologous amino acid sequence, wherein the heterologous amino acid sequence is a c-myc-tagged peptide.

15. The use of claim 13 or 14, wherein the method comprises the following steps: - Contact the biological sample with a capture agent immobilized to a solid support, wherein the immobilized capture agent is capable of binding the one or more markers, and - Determine 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.

16. The use of claim 14, wherein the method comprises determining the presence or absence of one or more markers in the biological sample, wherein the marker is an antibody specific to the c-myc tag peptide, and wherein the method comprises the following steps: a. Contact the biological sample with a trapping agent immobilized to a solid support, wherein the trapping agent is selected from the following: i. Proteins containing the c-myc-tagged peptide sequence, ii. A peptide comprising the c-myc-tagged peptide sequence or composed of the c-myc-tagged peptide sequence; b. Separate the biological sample from the immobilized capture reagent; c. Contact the immobilized capture reagent-antibody complex with a detectable reagent, wherein the detectable reagent binds to the antibody of the reagent-antibody complex; and d. Measure the level of the antibody bound to the capture reagent using a detection method for the detectable reagent.

17. The use of claim 16, wherein the measurement step (D) further comprises a comparison with a standard curve to determine the level of antibody binding to the capture reagent.

18. The use of claim 16, wherein the detectable reagent that binds to the antibody in the reagent-antibody complex is a labeled second antibody.

19. The use of claim 13 or 14, wherein the individual is a pig or a rabbit.

20. The use of claim 13 or 14, wherein the biological sample has been isolated from a pig.

21. The use of claim 13 or 14, wherein the biological sample is selected from whole blood, plasma, serum, urine, and oral fluid.

22. The use of claim 15, wherein the fixed capture reagent package is placed on a microtiter plate.

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