Porcine circovirus type 3 immunogenic composition, method of making same, and method of using same

Abstract

To provide an immunogenic composition against a new species of circovirus, called porcine circovirus type 3 (PCV3), and methods of making and using the same.SOLUTION: A composition comprises: at least one porcine circovirus type 3 protein selected from the group consisting of ORF1, ORF2, and ORF3; and a veterinary-acceptable carrier selected from the group consisting of a solvent, a dispersion media, a coating agent, a stabilizing agent, a diluent, a preservative, an antimicrobrial agent, an antifungal agent, an isotonic agent, and an adsorption delaying agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 242,866, filed October 16, 2015, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing in written or computer readable form, the teachings and content of which are incorporated herein by reference. [Background technology]

[0003] Porcine circovirus type 2 (PCV2) was first identified sporadically in the mid-1990s in Canada in pigs suffering from postweaning multisystemic wasting syndrome (PMWS). Epidemics of severe systemic disease associated with PCV2 were subsequently identified in North America, Europe, and Asia. With the widespread use of commercial vaccines, PCV2-associated diseases (PCVAD), including PMWS, pneumonia, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive disorders, were effectively suppressed. Retrospective studies demonstrated that PCV2 circulated unnoticed among pigs for decades before widespread clinical disease. A shift in dominant genotype from PCV2a to PCV2b appears to correspond to the severity of PCVAD. Recently, a novel circovirus, designated porcine circovirus type 3 (PCV3), has been identified in sows and aborted fetuses with clinical symptoms typically associated with PCV2 infection. Summary of the Invention [Means for solving the problem]

[0004] Tissues were collected from four sows from a farm that had chronically reduced reproductive performance and died suddenly with clinical signs consistent with PDNS. Pathological examination of the sow tissues noted purple skin lesions due to acute necrosis of the dermis and epidermis associated with hemorrhage and perivascular accumulation of leukocytes. The kidneys showed tubular dilation with lymphocyte and macrophage clusters within the interstitium and glomeruli, and mild dysplasia in tubular epithelial cells. Despite the gross and microscopic lesions commonly seen with PCV2 infection, immunohistology and quantitative PCR (qPCR) were negative for PCV2 as well as porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). Concurrently, mummified fetuses were collected from an aborted sow with similar skin lesions from the same farm. Similarly, qPCR was negative for PCV2 as well as porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV).

[0005] Tissue homogenate pools were prepared from the three mummies and subjected to viral metagenomic sequencing as described above. Of the 989,478 collected reads, 926,380 reads were mapped against the reference wild boar (Sus scrofa) genome. De novo assembly of the unassembled reads yielded 27 contigs. The majority of reads (54%) that mapped to the 1246 bp contigs showed 98% similarity to a circovirus partial genome detected in commercial pork by metagenomic sequencing according to BLASTN analysis. The remaining reads showed no similarity to known eukaryotic viruses. Rolling circle amplification was then performed on the mummy pool DNA, followed by inverse PCR using primers overlapping the XhoI site. Agarose gel electrophoresis confirmed a single band of approximately 2 kb. The 2000 bp genome (SEQ ID NO:1) was determined by Sanger sequencing of overlapping amplicons spanning the entire genome.

[0006] Genetic analysis identified an open reading frame (ORF1: SEQ ID NO:3) encoding a predicted 296 amino acid protein (SEQ ID NO:4) that showed 96% identity to the partial replicase (rep) protein of the circovirus PorkNW2 / USA / 2009 (accession number ADU77001, 221, aa) and 54% identity to the 293 amino acid (aa) rep protein from a Chinese bat circovirus (accession number AIF76248) by BLASTN analysis. Conserved viral rep and helicase domains were identified from amino acids 9-93 and 162-251 of SEQ ID NO:1, respectively. As with PorkNW2 / USA / 2009, no proper start codon was identified, suggesting an alternative GTC start codon or splicing derived from the upstream ATG. The second ORF in the reverse orientation (ORF2: SEQ ID NO:5) encodes a predicted 214 amino acid protein (SEQ ID NO:6) that shows 87% identity to the partial capsid (cap) sequence of PorkNW2 / USA / 2009 (110 aa) and 36-37% identity to the capsids of PCV2 and duck circovirus (233 and 257 aa, respectively). Similar to other circovirus cap proteins, the N-terminus is composed of a highly arginine-rich region approximately 32 aa long. The third ORF (ORF3: SEQ ID NO:7), encoding a predicted 233 aa protein, has 94% identity to that identified in a partial circovirus genome detected in ground beef and 39% identity to a murine herpesvirus protein of unknown function. The predicted origin of replication contains a stem-loop structure with a loop nanomer identical to PCV1, TAGTATTAC (SEQ ID NO:15), located between rep and cap of the rep-encoding strand. Given its overall genetic and structural similarity to members of the Circovirus genus, and its less than 70% identity of the capsid aa to other species, this species is referred to herein as Porcine Circovirus 3 (PCV3).

[0007] Phylogenetic analysis was performed in MEGA using sequences aligned by ClustalW. Phylogeny was estimated using maximum likelihood method using optimal LG with gamma distribution model with tree shape confirmed by 500 bootstrap replicates (Figure 2). Scale representing number of amino acid changes is shown. GenBank accession numbers are shown in brackets. In Figure 2, CaCV is canary circovirus, GuCV is gull circovirus, FiCV is finch circovirus, StCV is starling circovirus, PiCV is pigeon circovirus, BFDV is beak feather disease virus, DuCV is duck circovirus, GoCV is goose circovirus, SwCV is swan circovirus, BtCV is bat circovirus, PCV1 is porcine circovirus type 1, and PCV2 is porcine circovirus type 2. As shown, PCV3 clusters with PorkNW2 / USA / 2009, which are most closely related to Chinese bat circoviruses. Interestingly, PCV1 and PCV2 are also evolutionarily related to another bat circovirus from Myanmar.

[0008] Tissue homogenates from three individual mummies and lung homogenates from four sows that died from PDNS were analyzed using a qPCR assay targeting the PCV3rep gene. All mummies were positive, with cycle threshold values ​​(Ct values) ranging from 16.7 to 21.3. Three of the four sows were also positive, with Ct values ​​ranging from 27.7 to 29.7.

[0009] A total of 271 porcine nasal swab, oral fluid, or lung homogenate samples submitted for diagnostic testing were screened using multiplex Taqman assays targeting the rep and cap genes to determine the occurrence of PCV3. Thirty-four (12.5%) samples were positive with similar Ct values ​​for both assays. Two samples were positive only for the assay targeting PCV3cap. Ct values ​​for positive samples ranged from 20.3 to 35.8.

[0010] A peptide encompassing amino acids 35-214 of SEQ ID NO:1 was expressed as an N-terminal 6x histidine fusion protein in E. coli and purified by affinity chromatography. Denaturing electrophoresis shows that the protein was expressed as a dimer, similar to that seen in PCV2. ELISA was performed as described above. Eighteen serum samples from 3-week-old pigs from specific pathogen-free herds were used as negative controls, with a mean optical density of 4.9. The cutoff for positivity was set at three standard deviations above the mean (OD>0.87). All serum samples from 10 ≥2-parous sows from the same farm were positive, with a mean OD of 1.2. Twenty-seven serum samples from sows from a gilt farm supplying replacement animals to the sow farm were also tested, of which 17 (63%) were positive. Of 83 serum samples (age of animals unknown) submitted for diagnostic testing from multiple states, 46 (55%) were positive. [Brief description of the drawings]

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the specific embodiments presented herein.

[0012] [Figure 1] Genomic organization of porcine circovirus type 3 [Diagram 2] Phylogenetic tree of circovirus replicase proteins

[0013] In the present specification, SEQ ID NO: 1 is the nucleotide sequence of the PCV3 genome, SEQ ID NO: 2 is the amino acid sequence of the PCV3 genome, SEQ ID NO: 3 is the nucleotide sequence of the PCV3 replicase (ORF1), SEQ ID NO: 4 is the amino acid sequence of the PCV3 replicase (ORF1), SEQ ID NO: 5 is the nucleotide sequence of the PCV3 capsid (ORF2), SEQ ID NO: 6 is the amino acid sequence of the PCV3 capsid (ORF2), SEQ ID NO: 7 is the nucleotide sequence of the PCV3 ORF3, SEQ ID NO: 8 is the amino acid sequence of the PCV3 ORF3, SEQ ID NO: 9 is the nucleotide sequence of the primer 1 PCV3 replicase, and SEQ ID NO: 10 is the nucleotide sequence of the primer 2 PCV3 replicase. SEQ ID NO:11 is the nucleotide sequence of the probe PCV3 replicase, SEQ ID NO:12 is the nucleotide sequence of the primer 1 PCV3 capsid, SEQ ID NO:13 is the nucleotide sequence of the primer 2 PCV3 capsid, SEQ ID NO:14 is the nucleotide sequence of the probe PCV3 capsid, SEQ ID NO:15 is a replication origin containing a stem-loop structure with a loop nanomer, SEQ ID NO:16 is an endogenous cap gene primer, SEQ ID NO:17 is an endogenous cap gene primer, SEQ ID NO:18 is a primer for a portion of the PCV3 gene that encodes amino acids (aa), and SEQ ID NO:19 is a primer for a portion of the PCV3 gene that encodes amino acids (aa). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The following detailed description and examples set forth preferred materials and procedures utilized in accordance with the present disclosure, however, it should be understood that these examples are presented for illustrative purposes only and should not be construed as limiting the overall scope of the present disclosure.

[0015] Immunogenic compositions, methods of making and using same

[0016] One aspect of the present disclosure provides a method for producing and / or recovering recombinant PCV3 ORF2 protein, the method comprising the steps of: (1) infecting a number of susceptible cells in a culture medium with a recombinant viral vector encoding a PCV3 protein; (2) expressing the PCV3 protein by the recombinant viral vector; (3) recovering the PCV3 protein; and (4) separating cellular debris from the expressed PCV3 protein.

[0017] In another embodiment of the present disclosure, it is preferable to include an inactivation step to inactivate the viral vector before recovering the PCV3 protein to be used as a vaccine in the immunogenic or immunological composition. The inactivation step can be carried out as step (5) in addition to steps (1)-(4) above.

[0018] In some forms, the inactivation step is performed either immediately before or after the filter or separation step. For the purposes of this disclosure, any conventional inactivation method can be used. Thus, inactivation can be performed by chemical and / or physical treatment. One representative inactivation method includes the addition of cyclized binary ethyleneimine (BEI).

[0019] Optionally, the method described above also includes a neutralization step after step (5) above. For example, when the inactivation agent is BEI, it is preferred to add sodium thiosulfate up to an equivalent amount. Preferably, sodium thiosulfate is added in an equivalent amount to the BEI added for inactivation.

[0020] An "immunogenic or immunological composition" refers to a composition comprising at least one antigen that induces an immunological response in a host, a cellular and / or antibody-mediated immune response to the subject composition or vaccine. Typically, an "immunological response" includes, but is not limited to, one or more of the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells, and / or ydT cells, which are specifically directed against the antigen contained in the subject composition or vaccine. Preferably, the host will exhibit either a therapeutic or prophylactic immunological response, which will result in enhanced resistance to new infections and / or reduced clinical severity of the disease. Such prevention will be indicated by a reduction in the severity or morbidity of symptoms or even the absence of symptoms, a rapid recovery period, and / or a reduction in viral titers that an infected host would normally exhibit.

[0021] In a preferred embodiment, particularly in the case of using recombinant PCV3 protein in an immunogenic composition such as a vaccine, each lot or only selected lots of harvested PCV3 protein are tested for inactivation. Thus, a further aspect of the present disclosure relates to an inactivation test method for determining the effectiveness of inactivation of a recombinant viral vector, comprising the steps of: (1) contacting at least a portion of a culture medium containing the recombinant viral vector with an inactivating agent; (2) adding a neutralizing agent to neutralize the inactivating agent; and (3) determining the remaining infectivity.

[0022] In a preferred embodiment, a recombinant viral vector containing PCV3 DNA and expressing PCV3 proteins used to infect cells is generated by transfecting a transfer vector carrying a cloned PCV3 gene into the viral vector. Preferably, only the portion of the transfer vector containing the desired PCV3 DNA (e.g., DNA encoding ORF1, ORF2, or ORF3) is transfected into the viral vector.

[0023] "Transfected into a viral vector" means "introducing" or "cloning" a heterologous DNA into a viral vector (e.g., a baculovirus vector) and is used synonymously. "Transfer vector" means a DNA molecule that contains at least one origin of replication, a heterologous gene (PCV3 DNA in the present invention) and a DNA sequence that allows the heterologous gene to be cloned into the viral vector. Preferably, the DNA sequences that allow the heterologous gene to be cloned into the viral vector flank the heterologous gene. More preferably, these flanking sequences have at least partial homology to the sequences of the viral vector. This sequence homology allows the recombination of both molecules (viral vector and transfer vector) to generate a recombinant viral vector containing the heterologous gene.

[0024] In a more preferred embodiment, the method of the present disclosure starts with the isolation of PCV3 DNA. Any PCV3 gene can be used for the purpose of the present disclosure. The PCV3 gene is preferably amplified using PCR. As a result, the DND is cloned into a transfer vector.

[0025] Therefore, in one aspect of the present disclosure, a method for constructing a recombinant viral vector containing PCV3 DNA is provided. The method generally includes the steps of (1) cloning at least one recombinant PCV3 gene into a transfer vector, and (2) transfecting the portion of the transfer vector containing the recombinant PCV3 gene into a viral vector to generate a recombinant viral vector. As described above, the PCV3 gene can code for ORF1, ORF2, or ORF3.

[0026] According to a further aspect, PCV3 DNA can be amplified in vitro and the flanking sequences of PCV3 DNA can be modified before the above step (1). The in vitro methods of amplifying PCV3 DNA, modifying the flanking sequences, and cloning the in vitro amplified PCV3 DNA into a transfer vector and a suitable transfer vector are described above or known to those skilled in the art. Thus, in a further aspect, the present disclosure relates to a method for constructing a recombinant viral vector containing PCV3 DNA and expressing a desired PCV3 protein, comprising the steps of: (1) amplifying PCV3 DNA in vitro and modifying the flanking sequences of PCV3 DNA; (2) cloning the amplified PCV3 ORF2 DNA into a transfer vector; and (3) transfecting the transfer vector or a portion of the vector containing the recombinant PCV3 DNA into a viral vector to generate a recombinant viral vector. In some embodiments, modification of the flanking sequences of PCV3 DNA is performed by introducing a 5' Kozak sequence and / or an EcoR1 site.

[0027] A further aspect of the present disclosure relates to a method for producing a composition comprising a PCV3 protein and an inactivated viral vector. This method generally includes the steps of (1) cloning the amplified PCV3 DNA into a transfer vector, (2) transfecting a portion of the transfer vector containing the recombinant PCV3 DNA into a virus, (3) infecting cells in a culture medium with the transfer vector, (4) allowing the transfected viral vector to express a recombinant protein derived from the PCV3 DNA, (5) separating the cells from the supernatant, (6) recovering the expressed PCV3 protein, and (7) inactivating the recombinant viral vector. In a preferred embodiment, as described above, after the above step (7), a neutralization step (8) is performed. Of course, as described above, before the above step (1), the PCV3 DNA can be amplified in vitro, preferably together with the flanking sequences of the PCV3 DNA.

[0028] In another aspect of the present disclosure, a method for producing an immunogenic composition, preferably a vaccine, for inducing an immune response against PCV3 is provided. In general, the method includes the steps of (1) transfecting a construct containing recombinant DNA derived from the ORF of PCV3 into a virus, (2) infecting cells in a growth medium with the transfected virus, (3) allowing the virus to express a recombinant ORF protein derived from PCV3, (4) recovering the expressed recombinant ORF protein, and (5) combining the recovered protein with a suitable adjuvant and / or other pharma- ceutically acceptable carrier to produce a composition. In some preferred forms, the composition also includes at least a portion of a viral vector expressing a PCV3 ORF protein, and / or a portion of a culture supernatant.

[0029] As used herein, "adjuvants" can include aluminum hydroxide, aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge, Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Alabama), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsions can be based in particular on light liquid paraffin oil (European Pharmacopoeia Form); isoprenoid oils, such as squalane or squalene obtained by oligomerization of alkenes, especially isobutene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearate. The emulsions are formed by using oils in combination with emulsifiers. The emulsifiers are preferably non-ionic surfactants, in particular esters of sorbitan, mannides (e.g. anhydromannitol oleate), glycols, polyglycerols, propylene glycol, oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, as well as polyoxypropylene-polyoxyethylene copolymer blocks, in particular the products of Pluronic, in particular L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, DES). JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0030] For example, 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 emulsion MF59 described on page 183 of the same book can be used.

[0031] Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid or copolymers of maleic anhydride and alkenyl derivatives. Suitable adjuvant compounds are polymers of acrylic or methacrylic acid crosslinked with polyalkenyl ethers, especially of sugars or polyalcohols. These compounds are called carbomers (Phameuropa Vol. 8, No. 2, June 1996). The skilled person may also refer to US Pat. No. 2,909,462, which describes acrylic polymers crosslinked with polyhydroxylated compounds having at least three, preferably up to eight, hydroxyl groups, in which the hydrogen atoms of at least three hydroxyl groups are replaced by unsaturated aliphatic radicals having at least two carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl, and other ethylenically unsaturated groups. The unsaturated radicals themselves may contain other substituents, such as methyl. Particularly suitable are the products sold under the name Carbopol (BF Goodrich, Ohio, USA). Carbopol is crosslinked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol 974P, 934P and 971P are suitable. Among the copolymers of maleic anhydride and alkenyl derivatives, the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene, is also suitable. Dissolving these polymers in water produces an acidic solution, which is preferably neutralized to physiological pH in order to incorporate the immunogenic, immunological or vaccine composition in the adjuvant solution.

[0032] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi), block copolymers (SytRx, Atlanta, GA), SAF-M (Chiron, Emeryville, CA), monophosphoryl lipid A, avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314, or muramyl dipeptide, among others.

[0033] Preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. 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.

[0034] In another aspect of the present disclosure, a method for producing an immunogenic composition, such as a vaccine, for inducing an immune response against PCV3 includes the steps of (1) expressing PCV3 ORF and recovering the expressed PCV3 ORF, and (2) mixing the recovered protein with a suitable adjuvant. Preferably, the above step (1) includes the steps described for producing and recovering PCV3 protein. Another optional step in this method includes cloning the amplified PCV3 ORF DNA into a first vector, excising the ORF DNA from the first vector, and using the excised PCV3 ORF DNA to clone into a transfer vector. Preferably, the above recovery step of this method also includes separating the medium from the cells and cell debris. This can be performed using any conventional method. A preferred method includes filtering the cells, cell debris, and growth medium using a filter with pores in the range of about 0.45-1.0 μM in size. In this embodiment, it is preferable to finally include a step of inactivating the virus before combining the recovered recombinant PCV3 ORF protein in a composition. If an inactivation step is included, it is preferable to further include a neutralization step, as described above.

[0035] In addition, the compositions of the present invention may include one or more pharma- ceutically acceptable carriers or veterinarily acceptable carriers. As used herein, a "pharma-ceutically acceptable carrier" or a "veterinarily acceptable carrier" includes any or all of a solvent, dispersion medium, coating, stabilizer, diluent, preservative, antibacterial agent, antifungal agent, isotonic agent, or absorption retardant. In a preferred embodiment, the compositions of the present invention include PCV3 ORF protein recovered from in vitro cultured cells infected with a recombinant viral vector containing PCV3 ORF DNA and expressing PCV3 ORF protein. The cell culture is treated to inactivate the viral vector. An equivalent concentration of a neutralizing agent is added to the cell culture, and an adjuvant and saline are also added. As in other embodiments, the PCV3 ORF protein may include ORF1, ORF2, or ORF3. When saline is included, the amount of saline is preferably about 50 to 90% (v / v), more preferably about 60 to 80% (v / v), and even more preferably about 70% (v / v). Optionally, the method can also further include a protectant. As used herein, "protectant" refers to an antimicrobial active agent, such as, for example, gentamicin or merthiolate. In particular, the addition of a protectant is most preferred in the preparation of a multi-dose composition. The antimicrobial active agent is added at a concentration effective to protect the composition from microbial contamination or inhibit the growth of microorganisms in the composition.

[0036] The methods of the present disclosure can also include the addition of any stabilizers, such as, for example, saccharides, trehalose, mannitol, sucrose, etc., to extend and / or maintain the shelf life and / or enhance the stability of the product.

[0037] In another aspect of the present disclosure, a product obtained by the above-mentioned method is provided. In particular, the present disclosure relates to a composition comprising recombinantly expressed PCV3 ORF protein. In some preferred forms, the composition also comprises a substance suitable for inactivating a viral vector. Such a product is useful as an immunogenic composition for inducing an immune response, more preferably conferring prophylactic immunity against clinical signs of PCV infection. The composition generally comprises, as an antigenic component, a polypeptide or fragment thereof expressed by ORF1, ORF2, ORF3 of PCV3, or any combination thereof. Of course, it should be understood that the PCV3 ORF polypeptide used in the immunogenic composition of the present disclosure can be derived by any method, including isolation and purification, standard protein synthesis, and recombinant methods.

[0038] Clinical signs of PCV3 infection occur both after weaning at approximately 6-8 weeks of age and during the growing and finishing stages at 12-14 weeks of age, and sporadically in other age groups. Clinical signs include the appearance of widespread, slightly elevated, reddish-purple spots of various sizes and shapes on the chest, abdomen, thighs, and front legs. Over time, the spots become covered with a dark crust and then fade, leaving scars. In addition, pigs may become listless, develop a high fever, be reluctant to move or eat, lose weight, have labored or difficult breathing, have swelling or fluid around the limbs or eyelids, have enlarged superficial lymph nodes, become hairy, have pale skin, develop jaundice, and may have diarrhea. Although overall mortality is high, most pigs with skin lesions die. Further evidence of PCV3 infection can be seen as gastric ulcers and bleeding present in the stomach and intestines, fluid present in the abdomen, and lesions seen in the lungs, tonsils, spleen, liver, and kidneys, which are swollen, pale, and mottled with multiple small hemorrhages visible through the skin.

[0039] Any PCV3 ORF is effective as a source of PCV3 ORF DNA and / or polypeptides used in the present disclosure. In a preferred form, the ORF DNA is ORF2. A preferred PCV3 ORF2 protein is that of SEQ ID NO:6, but it will be understood by those skilled in the art that this sequence, as well as the sequences of ORF1 and ORF3, can differ in sequence homology by up to 10% and still retain useful antigenicity in an immunogenic composition. The antigenicity of an immunogenic composition can be estimated, for example, by challenge experiments. Furthermore, the antigenicity of a modified antigen is still maintained if it confers at least 70%, preferably 80%, more preferably 90% protective immunity compared to the PCV3 ORF protein encoded by the polynucleotide sequence of SEQ ID NO:3, 5, or 7. In some forms, an immunogenic portion of a PCV3 ORF protein is used as the antigen component of the composition. As used herein, "immunogenic portion" refers to a truncated and / or substituted form, or fragment, of the PCV3 ORF2 protein and / or polynucleotide, respectively. Preferably, such truncated and / or substituted form, or fragment, comprises at least 6 consecutive amino acids from the full-length ORF polypeptide. More preferably, such truncated or substituted form, or fragment, comprises at least 10, preferably at least 15, more preferably at least 19 consecutive amino acids from the full-length ORF polypeptide. It is further understood that such sequences may be part of a larger fragment or truncated form. Preferably, such truncated or substituted form, or fragment, comprises at least 18 consecutive nucleotides from the full-length ORF nucleotide sequence (e.g., SEQ ID NO: 3, 5, or 7). More preferably, such truncated or substituted form, or fragment, comprises at least 30, preferably at least 45, more preferably at least 57 consecutive amino acids from the full-length OR nucleotide sequence.

[0040] "Sequence identity" as known in the art refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., between a reference sequence and a given sequence that is compared to the reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after optimally aligning the given sequence and the reference sequence to obtain the highest degree of sequence similarity (as determined by the match between strings of these sequences). Upon such alignment, sequence identity is ascertained position by position. For example, if the nucleotide or amino acid residue at a particular position is identical, the sequence is "identical" at that position. The total number of positions with such position identity is divided by the total number of nucleotides or residues in the reference sequence to determine the percentage of sequence identity. Sequence identity may be determined using techniques such as, but not limited to, 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, eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073-1076. (1988), the teachings of which are incorporated herein by reference.The preferred method of determining sequence identity is designed to obtain the maximum match between the sequences examined. The method of determining sequence identity is codified in publicly available computer programs that can determine the sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990)), the teachings of which are incorporated herein by reference. BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, 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 programs use default gap weighting to optimally align the given sequence with the reference sequence, thereby obtaining the highest level of sequence identity between the given sequence and the reference sequence. For example, if a given polynucleotide has a nucleotide sequence that has at least 85%, preferably 90%, more preferably 95% "sequence identity" with a reference nucleotide sequence, this means that the nucleotide sequence of the given polynucleotide is identical to the reference sequence, except that the given polynucleotide sequence may contain up to 15, preferably up to 10, more preferably up to 5 point mutations for every 100 nucleotides of the reference nucleotide sequence.In other words, in a polynucleotide having a nucleotide sequence with at least 85%, preferably 90%, more preferably 95% identity to a reference nucleotide sequence, up to 15%, preferably up to 10%, more preferably up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 15%, preferably up to 10%, more preferably up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may be made at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either interspersed individually between the nucleotides of the reference sequence or interspersed within one or more contiguous groups of the reference sequence. Similarly, when a given polypeptide has an amino acid sequence with at least 85%, preferably 90%, more preferably 95% sequence identity to a reference amino acid sequence, it means that the amino acid sequence of the given polypeptide is identical to the reference sequence, provided that the given polypeptide sequence may contain up to 15, preferably up to 10, more preferably up to 5 amino acid modifications per 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence with at least 85%, preferably 90%, more preferably 95% sequence identity to a reference amino acid sequence, up to 15%, preferably up to 10%, more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or up to 15%, preferably up to 10%, 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 modifications of the reference sequence may be made at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually interspersed among the residues of the reference sequence or interspersed within one or more consecutive groups of the reference sequence. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included in the match when determining sequence identity.

[0041] As used herein, "sequence homology" refers to a method for determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned and gaps are introduced if necessary. However, in contrast to "sequence identity", conservative 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%, more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or contain conservative substitutions with another amino acid or nucleotide. Alternatively, up to 15%, preferably up to 10%, more preferably up to 5% of the total number of amino acid residues or nucleotides (not including conservative substitutions) in the reference sequence may be inserted into the reference sequence. Preferably, the homologous sequence comprises at least 50, preferably 100, more preferably 250, even more preferably 500 consecutive nucleotides.

[0042] A "conservative substitution" refers to the replacement of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar properties or characteristics, including size, hydrophobicity, etc., such that the overall functionality is not significantly altered.

[0043] "Isolated" means altered "by the hand of man" from its natural state, i.e., altered or removed from its original environment, or both, if present in nature. For example, a polynucleotide or polypeptide that occurs naturally in an organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated" as that term is employed herein.

[0044] In a further embodiment of the present disclosure, an immunogenic composition effective for reducing the severity of clinical symptoms associated with PCV3 infection is provided, comprising a PCV3 ORF protein.Preferably, the PCV3 ORF2 protein is selected from the following group: 1) a polypeptide comprising the sequence of SEQ ID NO: 4, 6, 8 or any combination thereof; 2) any polypeptide that is at least 90% homologous to the polypeptide of 1); 3) any immunogenic portion of the polypeptides of 1) and / or 2); 4) an immunogenic portion of 3) comprising at least 10 consecutive amino acids contained in the sequence of SEQ ID NO: 4, 6 or 8; 5) A polypeptide corresponding to that encoded by DNA containing the sequence of SEQ ID NO: 3, 5 or 7 (due to the degeneracy of the genetic code); 6) Any polypeptide encoded by a polynucleotide that is at least 80% identical to the polynucleotide of 5); 7) any immunogenic portion of a polypeptide encoded by the polynucleotides of 5) and / or 6); or 8) The immunogenic portion of 7), wherein the polynucleotide encoding said immunogenic portion comprises at least 30 consecutive nucleotides contained in the sequence of SEQ ID NO: 3, 5 or 7.

[0045] In a preferred form, these immunogenic portions have the immunogenic characteristics of the PCV3 ORF proteins encoded by the sequences of SEQ ID NO:3, 5 or 7.

[0046] According to a further embodiment, the PCV3 ORF2 protein is provided in the immunological composition at an antigen loading level effective to induce a desired immune response, i.e., effective to reduce the incidence or severity of clinical symptoms resulting from PCV3 infection. Preferably, the PCV3 ORF2 protein loading level is at least 0.2 μg antigen / ml, more preferably about 0.2 to about 400 μg / ml of the final immunogenic composition.

[0047] The polypeptide is incorporated into a composition that can be administered to an animal susceptible to PCV3 infection. In a preferred form, the composition may also contain additional components known to those skilled in the art (see also Remington's Pharmaceutical Sciences, 18th ed. Mack Publ. Easton, 1990).

[0048] Those skilled in the art will understand that the compositions herein can incorporate known injectable and physiologically acceptable sterile solutions. To prepare a ready-to-use solution for parenteral injection or infusion, isotonic aqueous solutions, such as saline or corresponding plasma protein solutions, are readily available. In addition, the immunogenic and vaccine compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylenediaminetetraacetic acid, among others. Suitable adjuvants are as described above.

[0049] According to a further aspect, the immunogenic composition of the present disclosure further comprises a pharma- ceutically acceptable salt, preferably phosphate, at a physiologically acceptable concentration.Preferably, the pH of the immunogenic composition is adjusted to a physiological pH of about 6.5-7.5.

[0050] The immunogenic compositions described herein may include one or more other immunomodulatory agents, such as interleukins, interferons, or other cytokines. The immunogenic compositions may also include gentamicin and merthiolate. In another preferred embodiment, the present disclosure contemplates a vaccine composition comprising about 1 μg / ml-about 60 μg / ml of antibiotic, more preferably less than about 30 μg / ml of antibiotic.

[0051] The immunogenic compositions comprising the recombinant PCV3 ORF proteins provided herein are found to be highly effective in reducing the severity or incidence of clinical signs associated with PCV3 infection, including preventing such signs.

[0052] Another aspect of the present disclosure relates to a kit. Generally, the kit comprises a container containing at least one dose of the immunogenic composition of PCV3 ORF protein provided herein, with one dose containing at least 2 μg of PCV3 ORF protein. The container can contain 1-250 doses of the immunogenic composition. In some preferred forms, the container contains 1, 10, 25, 50, 100, 150, 200 or 250 doses of the immunogenic composition of PCV3 ORF protein. Preferably, each of the containers containing multiple doses of the immunogenic composition of PCV3 ORF protein further comprises an antimicrobial active agent. These agents are, for example, antibiotics of the gentamicin and merthiolate classes. Thus, one embodiment of the present disclosure relates to a container containing 1-250 doses of an immunogenic composition of PCV3 ORF protein, each dose containing at least 2 μg of PCV3 ORF protein and gentamicin and / or merthiolate, preferably about 1 μg / ml-about 60 μg / ml of antibiotic, more preferably less than about 30 μg / ml. In a preferred form, the kit also includes instructions for intramuscular administration of at least one dose of the immunogenic composition of PCV3 ORF protein to animals, preferably pigs and piglets, to reduce the incidence and / or severity of clinical symptoms associated with PCV3 infection. Moreover, according to a further embodiment, the instructions include information for administering a second or further dose of at least one dose of the immunogenic composition of PCV3 ORF2, the second or further dose being at least 14 days after the first or previous dose. In some preferred forms, the instructions also include information for administering an immunostimulant. Preferably, the immunostimulant is given at least twice. Preferably, an interval of at least 3 days, more preferably at least 5 days, and even more preferably at least 7 days is placed between the first and second or subsequent administrations of the immunostimulant. Preferably, the immunostimulant is given 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 of PCV3 ORF protein.It is understood that any immune stimulant known to those skilled in the art can be used. As used herein, "immunostimulant" refers to any agent or composition that can induce a general immune response, preferably without initiating or increasing a specific immune response, such as an immune response against a specific pathogen. It is further instructed to administer the immune stimulant in an appropriate dose. The kit can also include a second container that contains at least one dose of the immune stimulant.

[0053] Further aspects of the present disclosure include the immunogenic compositions and instructions for use of the PCV3 ORF provided herein, the instructions further comprising information for administering the PCV3 ORF immunogenic composition at the same time as an immunogenic composition comprising an additional antigen effective in reducing the severity or incidence of clinical signs associated with another swine pathogen. Preferably, the manual comprises information regarding the timing of administration of the PCV3 ORF-containing composition and the immunogenic composition comprising the additional antigen.

[0054] Further aspects relate to the use of any of the compositions provided herein as a medicine, preferably as a veterinary medicine, more preferably as a vaccine.Furthermore, the present disclosure also relates to the use of any of the compositions described herein for the preparation of a medicament for reducing the severity of clinical symptoms associated with PCV3 infection.Preferably, the medicament is for the prevention of PCV3 infection in pigs, more preferably piglets.

[0055] Further aspects include the following: (1) a method for preventing infection or reinfection by PCV3 in a subject, or (2) a method for reducing or eliminating the occurrence or severity of clinical symptoms caused by PCV3 in a subject, comprising administering an immunogenic composition to a subject in need thereof. Preferably, the subject is a pig. It is understood that this reduction is compared to a subject not receiving the composition of the present disclosure. Preferably, one or two doses of the immunogenic composition are administered, with one dose preferably containing at least about 2 μg of PCV3 ORF protein. Further aspects relate to the above treatment method, in which a second application of the immunogenic composition is administered. Preferably, the second administration is performed with the same immunogenic composition, preferably with the same amount of PCV3 ORF protein. Preferably, the second administration is performed at least 14 days after the first administration, and even more preferably at least 4 weeks after the first administration. In a preferred form, the method is effective after a single administration of the immunogenic composition, and does not require a second or subsequent administration to provide a protective benefit to the subject.

[0056] According to a further aspect, the present disclosure provides a multivalent combination vaccine comprising an immunological agent effective in reducing the incidence or severity of PCV3 infection and at least one immunologically active component against another pathogen in pigs.

[0057] In particular, the immunological agent effective for reducing the incidence or reducing the severity of PCV3 infection is PCV3 antigen.Preferably, said PCV3 antigen is PCV3 ORF protein provided herein, or any of the above immunogenic compositions comprising PCV3 ORF protein.PCV3 ORF protein can be ORF1, ORF2, ORF3, or any combination thereof.For example, ORF1 combined with ORF2, ORF2 combined with ORF3, ORF1 combined with ORF3, and ORF1, 2, and 3 are each possible combination of PCV3 ORF that can be combined in a single immunogenic composition composition.In a preferred form, ORF is a combination comprising ORF2 or ORF2.

[0058] The term "immunogenic protein", "immunogenic polypeptide" or "immunogenic amino acid sequence" as used herein refers to any amino acid sequence that elicits an immune response in a host against a pathogen that contains said immunogenic protein, immunogenic polypeptide or immunogenic amino acid sequence. As used herein, "immunogenic protein", "immunogenic polypeptide" or "immunogenic amino acid sequence" includes the full-length sequence of any protein, analogue thereof, or immunogenic fragment thereof. By "immunogenic fragment" is meant a fragment of a protein that contains one or more epitopes and thus elicits an immunological response against the relevant pathogen. Such fragments can be identified using any number of epitope mapping techniques well known in the art. See, for example, Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be measured by simultaneously synthesizing a large number of peptides on a solid support that correspond to, for example, portions of a protein molecule, and allowing the peptides to react with antibodies while still attached to the support. Such techniques are known in the art, see, for example, U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by methods that determine the spatial conformation of amino acids, such as, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, epitope mapping protocols, supra. Synthetic antigens, such as polyepitopes, contiguous epitopes, and other recombinant or synthetically derived antigens, are also included in the definition.See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol, and Cell Biol. 75:402-408; Gardner et al. (1998), 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998.

[0059] Preferably, other pathogens in pigs are Actinobacillus pleuropneumoniae, adenovirus, alphavirus (e.g. eastern equine encephalitis virus), Bordetella bronchiseptica, Brachyspira spp. (preferably Brachyspira hyodysenteriae, Brachyspira piosicoli), Brucella abortus (preferably biotypes 1, 2, and 3), classical hog fever virus, Clostridium spp. (preferably Clostridium difficile, Clostridium perfringens types A, B, and C, Clostridium , Clostridium septicum, Clostridium tetani), coronavirus (preferably porcine respiratory coronavirus), Erysipelothrix suis, Erysipelothrix rhusiopathiae, Escherichia coli, Haemophilus parasuis (preferably subtypes 1, 7, and 14), hemagglutinating encephalomyelitis virus, Japanese encephalitis virus, Lawsonia intracellularis, Leptospira spp. (preferably Leptospira australis, Leptospira canicola, Leptospira grippotyphos, Mycobacterium spp. (preferably Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis), Mycoplasma hyopneumoniae, Pasteurella multocida, Porcine cytomegalovirus, Porcine parvovirus, Porcine reproductive and respiratory syndrome (PRRS) virus, Pseudorabies virus, Rotavirus, Salmonella typhimurium ... The pathogen is selected from the group including the genus Staphylococcus (preferably Salmonella typhimurium and Salmonella cholerae suis), Staphylococcus hyicus, the genus Staphylococcus (preferably Streptococcus, preferably Streptococcus suis), swine herpes virus, swine influenza virus, swinepox virus, vesicular stomatitis virus, vesicular exanthema of swine virus, Leptospira hardjo, and Mycoplasma hyosinoeae, or at least any one of these.

[0060] As used herein, "immunologically active ingredient" refers to an ingredient that induces or stimulates an immune response in an animal to which said ingredient is administered. According to a preferred embodiment, said immune response is against said ingredient or a microorganism that contains said ingredient. According to a further preferred embodiment, the immunologically active ingredient is an attenuated microorganism, comprising a modified live virus (MLV), a killed microorganism or at least an immunologically active part of a microorganism.

[0061] As used herein, "immunologically active portion of a microorganism" means comprising proteins, sugars and / or glycoproteins of a microorganism comprising at least one antigen that induces or stimulates an immune response in an animal to which said component is administered. According to a preferred embodiment, said immune response is directed against said immunologically active portion of a microorganism or against a microorganism comprising said immunologically active portion.

[0062] In another aspect of the present disclosure, infectious chimeric DNA clones of porcine circovirus (PCV3) and live chimeric viruses derived from the DNA clones useful as vaccines are provided. The new live chimeric, genetically avirulent viruses are generated from non-pathogenic PCV1 genome structures in which the immunogenic genes of pathogenic PCV3 replace the genes of PCV1 that are typically in the corresponding same location. The present disclosure encompasses suitable host cells transfected with vectors containing DNA and immunogenic polypeptide expression products, such as biologically functional plasmids and viral vectors that contain the novel recombinant nucleic acid molecules described herein. Included within the scope of the present disclosure are novel methods and methods for providing protection or protection against viral infection or clinical signs of postweaning multisystemic wasting syndrome virus (PMWS), including pneumonia, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS) caused by PCV3, to pigs in need of such protection, including immunologically effective amounts of, for example, cloned chimeric DNA in a plasmid, chimeric viruses derived from the chimeric DNA clones, polypeptides expressed from the DNA described herein, or any combination thereof. Another aspect of the present disclosure aims to promote cell culture growth and ensure vaccine safety by introducing novel mutants of PCV3 immunogenic capsid genes and proteins, and mutations in chimeric clones. In yet another aspect, the present disclosure provides novel infectious PCV3 molecular DNA and reciprocal chimeric DNA clones of PCV, which are useful as experimental models for obtaining or identifying novel non-pathogenic virus vaccines.

[0063] According to one aspect of the present disclosure, there are provided infectious molecules and chimeric nucleic acid molecules of porcine circovirus (PCV), live chimeric viruses produced from the chimeric nucleic acid molecules and veterinary vaccines for protecting pigs from viral infection, pneumonia, reproductive failure, PMWS, and / or PDNS caused by PCV3. The present disclosure further provides immunogenic polypeptide expression products that can be used as vaccines. The novel non-pathogenic infectious chimeric DNA molecule PCV (PCV1-3) ​​comprises a nucleic acid molecule encoding infectious non-pathogenic PCV1, which contains an immunogenic open reading frame (ORF) gene of pathogenic PCV3 in place of the ORF gene in the PCV1 gene. The infectious chimeric PCV1-3 DNA clone preferably comprises an immunogenic capsid gene (ORF2) of PCV3 DNA cloned into the genomic backbone of infectious non-pathogenic PCV1 DNA clone. Generally, the capsid gene of PCV3 DNA replaces the ORF2 gene of PCV1 DNA in the non-pathogenic PCV1 genome structure, but it is believed that various positional substitutions can be constructed by genetic engineering to obtain other non-pathogenic or attenuated chimeric DNA clones.As an analytical control of the chimeric PCV1-3 clone of the present disclosure, a chimeric infectious PCV3-1 DNA clone with reciprocity between PCV1 and PCV3 is disclosed, which is constructed by replacing the capsid gene of PCV3 with that of PCV1 in the backbone of pathogenic PCV3.In addition to being an experimental model, the chimeric PCV3-1 DNA clone with reciprocity can be used in making customized vaccines.

[0064] In another embodiment, ORF3 of PCV3 can be substituted for ORF2 or ORF3 of the PCV1-3 or PCV3-1 infectious clone. In yet another preferred embodiment, ORF1 and ORF3 of PCV3 can be substituted for ORF2 of PCV3 of the PCV1-3 or PCV3-1 infectious clone.

[0065] The cloned genomic DNA of PCV3 described herein is shown to be infectious in vitro and in vivo when transfected into PK-15 cells and fed to pigs. The infectious PCV3 DNA clone produces pathological lesions characteristic of PMWS and / or PDNS in pigs, allowing improved identification of clinical pathology and understanding of viral distribution in tissue cells. This new, easily reproducible pathogen will aid in the development of appropriate vaccination programs to prevent PMWS and / or PDNS in pigs.

[0066] The novel chimeric PCV1-3 DNA clones are also infectious both by in vitro transfection of PK-15 cells and by in vivo administration to pigs. In transfected PK-15 cells, one preferred chimeric PCV1-3 DNA clone will express PCV3 ORF2 capsid antigen (immunogenic capsid protein of PCV3), while the chimeric PCV3-1 DNA clone will express PCV1 capsid antigen, which can be demonstrated by immunofluorescence assay (IFA) using antibodies specific for PCV1 or PCV3 capsid antigen. Seroconversion to PCV3-specific antibodies will be detectable in pigs inoculated with chimeric PCV1-3 clones as well as infectious PCV3 clones. Detection of seroconversion to PCV3-specific antibodies will establish that the chimeric PCV1-3 DNA clones induce PCV3-specific antibodies in infected pigs, and thus act to protect the inoculated pigs from PCV3 infection.

[0067] Surprisingly and advantageously, the chimeric PCV1-3 infectious DNA clone with the immunogenic capsid gene (ORF2) of pathogenic PCV3 cloned into the non-pathogenic PCV1 genomic backbone induces a specific antibody response against the pathogenic PCV3 capsid antigen while preserving the non-pathogenic nature of PCV1 in pigs. Animals inoculated with the chimeric PCV1-3 infectious DNA clone develop a mild infection similar to PCV1-inoculated animals while seroconverting to antibodies against the ORF2 capsid protein of pathogenic PCV3. The average length of viremia observed in PCV1 and chimeric PCV1-3-inoculated animals will be shorter than that in pathogenic PCV3-inoculated animals. The lack of detectable chimeric PCV1-3 viremia in some inoculated animals does not affect seroconversion to antibodies against the PCV3 ORF2 capsid protein in PCV1-3-inoculated pigs. This result would indicate that even if chimeric PCV1-3 viremia is short or undetectable in some inoculated animals, the chimeric PCV1-3 virus would be able to induce antibody responses against PCV3 ORF2 capsid protein. The special ability of the chimeric PCV1-3 infectious DNA clone to induce specific immune responses against the pathogenic PCV3 immunogenic ORF2 capsid protein, but remain non-pathogenic to pigs, makes the chimeric PCV1-3 clone particularly useful for genetically engineered live attenuated vaccines and other types of vaccines. Appropriate cells containing the chimeric nucleic acid molecule will produce live infectious chimeric porcine circovirus on their own. In some preferred forms, live infectious chimeric viruses will be generated from the chimeric DNA clone by transfecting PK-15 cells via in vitro and in vivo transfection. The present disclosure further contemplates that the chimeric viruses are derived from a nucleotide sequence having a complementary or high degree of homology to the chimeric nucleotide sequence, such as at least 90%, more preferably 91, 92, 93, 94, 95, 96, 97, 98 or 99%.

[0068] The scope of the present disclosure includes biologically functional plasmids, viral vectors, and the like, including vectors containing the novel recombinant nucleic acid molecules, chimeric and molecular DNA clones described herein, and suitable host cells transfected with vectors containing immunogenic polypeptide expression products. Some particularly preferred immunogenic proteins will have the amino acid sequences set forth in SEQ ID NOs: 4, 6, or 8. Biologically active variants thereof are further encompassed by the present disclosure. Those skilled in the art know how to modify, substitute, delete, etc., amino acids from a polypeptide sequence and readily generate biologically active variants that retain the same or substantially the same activity as the parent sequence.

[0069] To produce an immunogenic polypeptide product of this disclosure, the process includes the steps of: rendering prokaryotic or eukaryotic host cells transfected with the novel recombinant nucleic acid molecules described herein under appropriate nutrient conditions capable of expressing said nucleic acid molecules by standard methods known in the art, and isolating the desired polypeptide product. It is contemplated that immunogenic proteins may also be provided by other techniques, such as, for example, biochemical synthesis.

[0070] Another embodiment of the present disclosure relates to novel mutations in the nucleotide and amino acid sequences of PCV3 immunogenic capsid gene and protein. PCV3 is the causative agent of PMWS, PDNS, pneumonia and reproductive failure, but PK-15 cell culture-derived porcine circovirus type I (PCV1) is non-pathogenic to pigs. PCV1-3 vaccines containing mutations can be further tested in cell culture to promote cell culture growth by routine procedures under cell culture or to select combinations that ensure improved safety measures when followed during vaccination of pigs for further attenuation of PCV3 virulence. While the advantage of PCV1-3 chimera is its natural non-toxic properties, the use of mutant PCV3 ORF2 instead to create PCV1-3 chimera provides another embodiment of the present disclosure, thereby ensuring that safer natural live chimeric vaccines will be available in the future.

[0071] Chimeric virus and molecular DNA clone vaccines and methods of using them are also included within the scope of the present disclosure. Inoculated pigs are protected from serious viral infections, pneumonia, reproductive failure, PDNS and PMWS caused by PCV3. A novel method of protecting pigs in need of protection against viral infections, pneumonia, reproductive failure, PDNS or PMWS is by administering to the pig an immunologically effective amount of a vaccine according to the present disclosure, such as a vaccine containing an immunogenic amount of chimeric PCV1-3 DNA, cloned chimeric virus, plasmid or viral vector containing chimeric DNA of PCV1-3, polypeptide expression product, recombinant PCV3 DNA, etc. Other antigens and immune stimulants as described above can be administered to the pig simultaneously to provide broad spectrum protection against infection. Such co-administration can be provided in separate administrations or can be a single-administered multi-effect combination vaccine.

[0072] The vaccine comprises, for example, infectious chimeric PCV1-3 DNA, cloned PCV chimeric DNA genome in a suitable plasmid or vector, such as pSK vector, non-pathogenic live chimeric virus, inactivated chimeric virus, etc., in combination with a physiologically acceptable carrier and optionally one or more adjuvants, as described herein. The vaccine may also comprise an infectious PCV3 molecular DNA clone as described herein. The non-pathogenic live virus vaccine of the present disclosure offers advantages over traditional viral vaccines that use either attenuated live viruses that have the risk of reverting to a virulent state or killed cell culture grown whole viruses that do not induce sufficient antibody immune responses.

[0073] Although live virus vaccines have some advantages, other types of vaccines can be used to inoculate pigs with the new chimeric viruses and other antigens described herein. To provide an inactivated virus vaccine, for example, virus propagation from an infectious DNA clone is performed by methods known in the art or described herein. Successive virus inactivation is then optimized by protocols commonly known to those skilled in the art.

[0074] Inactivated virus vaccines can be provided by treating isolated viruses or chimeric viruses derived from cloned PCV DNA with inactivating agents such as formalin or hydrophobic solvents, acids, UV or X-ray irradiation, or heat. Inactivation is accomplished by methods understood in the art. A virus is considered inactivated if it is unable to infect susceptible cells.

[0075] To provide attenuated vaccines from pathogenic clones, tissue culture is applied and produced by methods known in the art, typically by serial passage through cell culture, live pathogenic PCV3 is first attenuated (non-pathogenic or harmless). Attenuation of pathogenic clones can also be achieved by gene deletion or virus-producing gene mutation. The attenuated PCV3 virus can then be used to construct further chimeric PCV1-3 viruses that retain the non-pathogenic phenotype of PCV1, but the strength of selected immunogenic traits from the PCV3 genome can be altered by recombinant techniques.

[0076] The present disclosure also relates to a vaccine comprising the genomic nucleotide sequence of PCV3, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or a homolog or fragment thereof, and an acceptable pharmaceutical or veterinary carrier. In one embodiment of the present disclosure, the nucleotide sequence is selected from a homolog or a fragment thereof. In another embodiment of the present disclosure, the homolog has at least 90% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. In yet another embodiment, the vaccine further comprises an adjuvant.

[0077] A further aspect of the present disclosure relates to a vaccine comprising a vector and an acceptable pharmaceutical or veterinary carrier, wherein the vector comprises a nucleotide sequence of the genome of PCV3, such as SEQ ID NO: 2, 4, 6, 8, or any combination thereof, or a homolog or fragment thereof.

[0078] The present disclosure also relates to a vaccine immunogenic composition comprising an acceptable pharmaceutical or veterinary carrier, wherein the cell is transformed with a nucleotide sequence of the genome of PCV3, a homologue or a fragment thereof.

[0079] The present disclosure further relates to a vaccine or immunogenic composition comprising a pharma- ceutically acceptable carrier and a single polypeptide, wherein the single polypeptide consists of SEQ ID NO: 2, 4, 6 or 8.

[0080] Furthermore, the present disclosure relates to a method of immunizing a mammal against PCV3, comprising administering to the mammal an effective amount of the above-described vaccine or immunogenic composition.

[0081] The present disclosure relates to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 of the genome of PCV3 or one of their fragments.

[0082] The present disclosure also relates to a nucleotide sequence selected from the following a) to e): a) The nucleotide sequence of a specific fragment of the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or one of its fragments. b) a nucleotide sequence which is homologous to a nucleotide sequence as defined in a). c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b) and the corresponding nucleotide sequence of RNA. d) A nucleotide sequence capable of hybridizing under stringent conditions to a sequence as defined in a), b) or c). e) A nucleotide sequence comprising a sequence as defined in a), b), c) or d). f) A nucleotide sequence modified by a nucleotide sequence as defined in a), b), c), d) or e).

[0083] Nucleotide, polynucleotide or nucleic acid sequence are understood in accordance with the present disclosure to mean both double-stranded or single-stranded DNA in monomeric and dimeric (so-called tandem) form and the transcription products of said DNA.

[0084] It must be understood that the present disclosure does not relate to genomic nucleotide sequences taken in their natural environment, i.e. in a natural state, but rather to sequences that have been isolated, purified or partially purified, starting from separation methods such as ion exchange chromatography, exclusion based on molecular size, or fractionation techniques based on affinity or solubility in different solvents, or starting from genetic engineering methods such as amplification, cloning and subcloning, which allow the sequences of the present disclosure to be carried by vectors.

[0085] A complementary nucleotide sequence of a sequence of the present disclosure is understood to mean any DNA whose nucleotides are complementary to the nucleotides of a sequence of the present disclosure but in reverse orientation (antiparallel sequence).

[0086] Hybridization under stringent conditions with a nucleotide sequence according to the present disclosure is understood to mean hybridization under conditions of temperature and ionic strength selected to allow the maintenance of hybridization between two fragments of complementary DNA.

[0087] Among the nucleotide sequences according to the present disclosure, those that can be used as primers or probes in methods for obtaining homologous sequences according to the present disclosure, such as, for example, polymerase chain reaction (PCR), nucleic acid cloning and sequencing, are also preferred, which are well known to those skilled in the art.

[0088] Among the nucleotide sequences according to the present disclosure, those that can be used as primers or probes in methods that allow for the presence of PCV3 or one of its variants as defined below to be diagnosed are also preferred.

[0089] Also preferred are nucleotide sequences of the present disclosure that can regulate, inhibit or induce the expression of PCV3 genes and can regulate the replication cycle of PCV3 in host cells and / or organisms. The replication cycle is understood to designate the spread from host cell to host cell and the proliferation and invasion of PCV3 within the host organism.

[0090] According to the present disclosure, among the nucleotide sequences, those corresponding to open reading frames encoding polypeptides, such as, for example, SEQ ID NO: 4 (ORF1), SEQ ID NO: 6 (ORF2) and SEQ ID NO: 8 (ORF3), respectively, are called ORF sequences. The nucleotide sequence fragments according to the present disclosure can be obtained, for example, by specific amplification, such as PCR, or after digestion of the nucleotide sequence according to the present disclosure with appropriate restriction enzymes, which methods are described in particular in the work of Sambrook et al. in 1989. Said representative fragments can also be obtained by chemical synthesis, provided that their size is not too large and according to methods well known to those skilled in the art.

[0091] Modified nucleotide sequences shall be understood to mean all nucleotide sequences obtained by mutagenesis by techniques well known to those skilled in the art and include modifications of normal sequences according to the present disclosure, such as regulatory sequences and / or promoter sequences for the expression of a polypeptide, and in particular sequences for the modification of the rate of expression of said polypeptide or sequences for the regulation of the replication cycle.

[0092] A modified nucleotide sequence is likewise understood to mean any nucleotide sequence which codes for a modified polypeptide as defined below.

[0093] The present disclosure relates to a nucleotide sequence of PCV3 according to the present disclosure, characterized in that it is selected from a sequence comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or one of their fragments.

[0094] The present disclosure also relates to a nucleotide sequence characterized in that it meets any of the following nucleotide sequence conditions selected by a) to e): a) The sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or one of their fragments. b) The nucleotide sequence of a specific fragment of a sequence as defined in a). c) a homologous nucleotide sequence having at least 80% identity with a sequence as defined in a) or b). d) The complementary nucleotide sequence or sequences of RNA corresponding to a sequence as defined in a), b) or c). e) A nucleotide sequence modified by a sequence as defined in a), b), c) or d).

[0095] As far as the homology with the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or one of their fragments is concerned, the homologous, particularly specific sequence is preferably an amino acid sequence having at least 80%, preferably 90% or 95% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or one of their fragments. The specific homologous sequence may, for example, include sequences corresponding to the sequences of ORF1, ORF2, ORF3 of PCV3. Similarly, these specific homologous sequences may correspond to variants related to mutations within strains of PCV3, in particular corresponding to truncations, substitutions, deletions and / or additions of at least one nucleotide.

[0096] The present disclosure includes polypeptides encoded by a nucleotide sequence according to the present disclosure, preferably polypeptides whose sequences are represented by fragments, particularly specific fragments, whose six amino acid sequences correspond to polypeptides that can be encoded according to one of three possible reading frames of the sequence of SEQ ID NO:1 or the sequence of SEQ ID NO:2.

[0097] The disclosure also relates to polypeptides, characterized in that they comprise a polypeptide selected from the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or one of their fragments.

[0098] The present disclosure also relates to a polypeptide comprising a polypeptide selected from: a) A specific fragment of at least 5 amino acids of a polypeptide having an amino acid sequence according to the present disclosure. b) A polypeptide which is homologous to a polypeptide as defined in a). c) specific biologically active fragments of a polypeptide as defined in a) or b). d) A polypeptide modified by a polypeptide as defined in a), b) or c).

[0099] Among the polypeptides according to the present disclosure, the polypeptides of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 are also preferred, and these polypeptides are particularly effective against antibodies produced during infection with PCV3. Thus, these polypeptides have epitopes specific to PCV3 and can therefore be used in particular in the diagnostic field or as immunogenic agents to protect pigs against infection with PCV3.

[0100] As used herein, the terms polypeptide, peptide and protein are interchangeable.

[0101] It should be understood that the present disclosure does not relate to naturally occurring forms of polypeptides, i.e., in other words, they are not obtained in a natural environment, but may be isolated or obtained by purification from a natural source, or alternatively, may be obtained recombinantly or produced by chemical synthesis, and therefore, as described below, they may contain unnatural amino acids.

[0102] A polypeptide fragment according to the present disclosure is understood to designate a polypeptide comprising at least 5 consecutive amino acids, preferably 10 consecutive amino acids or 15 consecutive amino acids.

[0103] In the present disclosure, a particular polypeptide fragment is understood to designate a contiguous polypeptide fragment encoded by a particular fragment nucleotide sequence according to the present disclosure.

[0104] A homologous polypeptide is understood as designating, with respect to a naturally occurring polypeptide, in particular a polypeptide having a given modification to any amino acid, including a deletion, addition or substitution, truncation, extension, chimeric fusion, and mutation of at least one amino acid or at least any one of these. Among the homologous polypeptides, those whose amino acid sequence has at least 80%, preferably 90%, homology with the amino acid sequence of the polypeptide according to the present disclosure are preferred.

[0105] A particular homologous polypeptide is understood to designate a homologous polypeptide as defined above having a particular fragment of the polypeptide described herein.

[0106] In the case of substitution, one or more consecutive or non-consecutive amino acids are replaced by "equivalent" amino acids. The term "equivalent" amino acids as used herein refers to any amino acid that can be replaced by one of the amino acids of the basic structure without essentially modifying the biological activity of the corresponding peptide, as described below. These equivalent amino acids can be determined depending on their structural homology with the amino acid they replace, or by the results of comparative tests of biological activity between different polypeptides that can be carried out. By way of example, possible substitutions that can be carried out without extensive modification of the biological activity of the corresponding modified polypeptide are, for example, the replacement of leucine with valine or isoleucine, aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, etc., with the reverse substitutions being of course conceivable under the same conditions.

[0107] Particular homologous polypeptides similarly correspond to the polypeptides encoded by particular homologous nucleotide sequences as defined above and therefore, within the definition of the present invention, may be present in PCV3 and include in particular truncations, deletions and / or additions of at least one amino acid residue.

[0108] A specific biologically active fragment of a polypeptide according to the present disclosure is understood to specify a specific polypeptide fragment as defined above, having at least one of the characteristics of a polypeptide according to the present disclosure, in particular a polypeptide fragment having at least any one of the following characteristics: an ability to induce an immunogenic response against PCV3, a polypeptide that can be recognized by a specific antibody of the polypeptide according to the present disclosure and a polypeptide that can be linked to a nucleotide sequence of PCV3, an ability to exert a physiological activity, even if only partially, such as, for example, a disseminated or structural (capsid) activity, an ability to modify, induce or inhibit the expression of a PCV3 gene or one of its mutants, and an ability to regulate the replication cycle of PCV3 in a cell and / or a host organism.

[0109] A polypeptide fragment according to the present disclosure may correspond to an isolated or purified fragment naturally occurring in PCV3, or may correspond to a fragment that can be obtained by splitting said polypeptide with proteolytic enzymes such as trypsin or chymotrypsin or collagenase, or with chemical reagents such as cyanogen bromide (CNBr), or alternatively by placing said polypeptide in a very acidic environment, such as pH 2.5. Such a polypeptide fragment can also be easily produced by chemical synthesis, from a host transformed with an expression vector according to the present disclosure that contains a nucleic acid under the control of suitable control and / or expression elements, allowing the expression of said fragment.

[0110] A "modified polypeptide" of a polypeptide according to the present disclosure is understood to refer to a polypeptide obtained by genetic recombination or chemical synthesis as described below, which has at least one modification with respect to the normal sequence. These modifications may concern, in particular, amino acids involved in the origin of specificity, pathogenicity and / or toxicity, or in the origin of the conformation of the structure and the membrane insertion ability of the polypeptide according to the present disclosure. It will therefore be possible to create polypeptides of equal, increased or decreased activity, and equal, narrower or broader specificity. Among the modified polypeptides, it is necessary to mention polypeptides in which up to 5 amino acids can be modified, truncated at the N-terminus or C-terminus, or even deleted or added.

[0111] As indicated, modification of the polypeptide includes, inter alia, that it can regulate, inhibit or induce the expression of PCV3 genes and / or regulate the replication cycle of PCV3 in cells and / or the host organism, allowing its incorporation into a combination vaccine and / or modifying its bioavailability as a compound for therapeutic use.

[0112] The methods to be disclosed as enabling said regulation for eukaryotic or prokaryotic cells are well known to those skilled in the art. It is likewise understood that it is possible to use the nucleotide sequence encoding said modified polypeptide for said modification, for example via the vectors disclosed and described below, for preventing or treating pathologies associated with, for example, infections.

[0113] Prior modified polypeptides may be obtained, for example, by the use of combinatorial chemistry, which allows for systematic variation of portions of a polypeptide before testing them in models, cell cultures or microorganisms to select compounds with the most active or desired properties.

[0114] Similarly, chemical synthesis has the advantage that it can use non-natural amino acids or non-peptide bonds. Therefore, to improve the longevity of the polypeptides according to the present disclosure, it may be important to use non-natural amino acids, such as D-forms, or amino acid analogs, especially sulfur-containing forms.

[0115] Finally, the structure of the polypeptides according to the present disclosure, their specific or modified homologous forms, can be incorporated into polypeptide types or other chemical structures, and therefore it can be important to provide compounds at the N- and C-termini that are not recognized by proteases.

[0116] Nucleotide sequences encoding the polypeptides according to the present disclosure are also part of the present disclosure.

[0117] The present disclosure also relates to nucleotide sequences that can be used as primers or probes, characterized in that said sequences are selected from the nucleotide sequences according to the present disclosure.

[0118] Cloning and sequencing of PCV3 confirmed that the sequences of these nucleic acid fragments are strictly specific for PCV3 after comparative analysis with the nucleotide sequences of other porcine circoviruses, but they correspond to consensus sequences of porcine circoviruses other than PCV3. Similarly, there is a strong need for nucleotide sequences that can be used as primers or probes specific to all other known and non-pathogenic porcine circoviruses.

[0119] The present invention also relates to specific polypeptides of known porcine circoviruses other than PCV3, which are encoded by consensus nucleotide sequences and can be obtained by purification from natural polypeptides, by genetic recombination, or by chemical synthesis according to well-known methods, as specifically described below, by procedures known to those skilled in the art. Likewise, the labeled or unlabeled mono- or polyclonal antibodies directed against the specific polypeptides encoded by the consensus nucleotide sequences are also part of the present disclosure.

[0120] The consensus nucleotide sequences, the corresponding polypeptides and the antibodies against the polypeptides may be used in place of or in addition to the nucleotide sequences, polypeptides or antibodies as described above in procedures or sets for detection and identification or either one of the above, as described below, specific for PCV3 types A and / or B.

[0121] These protocols have been refined for the differential detection of circular monomeric forms of replicating virions or specific replicative forms of DNA, and for the differential detection of dimeric forms found in so-called tandem molecular constructs.

[0122] The present disclosure relates to the use of nucleotide sequences as well as primers or probes for the detection and / or amplification of nucleic acid sequences.

[0123] The nucleotide sequences according to the present disclosure may therefore be used to amplify nucleotide sequences, in particular by the PCR technique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; Rolfs et al., 1991 and White et al., 1997). These oligodeoxyribonucleotide or oligoribonucleotide primers advantageously have a length of at least 8 nucleotides, preferably at least 12 nucleotides, and even more preferably at least 20 nucleotides. Other target nucleic acid amplification techniques can be advantageously used as alternatives to PCR.

[0124] The nucleotide sequences of the present disclosure, and in particular the primers according to the present disclosure, can be used in other procedures for amplification of target nucleic acids as well, such as TAS technology (Transcription-Based Amplification System) Kwoh et al. (1989), 3SR technology (Self-Sustained Sequence Replication) Guatelli et al. (1990), NASBA technology (Nucleic Acid Sequence Based Amplification) Kievitis et al. (1991), SDA technology (Strand Displacement Amplification) Walker et al. (1992), TMA technology (Transcription-Mediated Amplification), and the like.

[0125] The polynucleotides of the present disclosure can also be used in techniques for amplifying or modifying nucleic acids to act as probes, such as the LCR technique (Ligase Chain Reaction) Landegren et al. (1988) and its improved technique (using a thermostable ligase) Barany et al. (1991), the RCR technique (Repair Chain Reaction) Segev (1992), the CPR technique (Cycling Probe Reaction) Duck et al. (1990), the Q-beta replicase amplification technique Miele et al. (1983) and its special improvements Chu et al. (1986), Lizardi et al. (1988), Burg et al., and Stone et al. (1996).

[0126] If the target polynucleotide to be detected is possibly RNA, for example MRNA, it will be possible to use an enzyme of the reverse transcriptase type to obtain cDNA from the RNA contained in the biological sample before employing an amplification reaction with the aid of at least one primer according to the present disclosure, or a detection procedure with the aid of at least one probe according to the present disclosure, and the cDNA thus obtained will serve as a target for the primers or probes used in the amplification or detection procedure according to the present disclosure.

[0127] The detection probe is selected such that it hybridizes with the target sequence or with an amplicon generated from the target sequence. By way of sequence, such a probe advantageously has a sequence of at least 12 nucleotides, in particular at least 20 nucleotides, preferably at least 100 nucleotides.

[0128] The present disclosure also includes nucleotide sequences that can be used as probes or primers according to the present disclosure, characterized in that they are labeled with radioactive or non-radioactive compounds. The sequences are generally labeled with radioactive elements (32P, 35S, 3H, 125I) or with non-radioactive molecules (biotin, acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein), but non-labeled nucleotide sequences can be used directly as probes or primers, obtaining probes that can be used for many applications. Examples of non-radioactive labeling of nucleotide sequences are, for example, French Patent No. 78.10975 or Urdea et al. or Sanchez-Pescador et al. (1988). In the latter case, it would also be possible to use one of the labeling methods listed in patents FR-2,422,956 and FR-2,518.755.

[0129] Hybridization techniques can be carried out in various ways (Matthews et al., 1988). The most common methods consist in immobilizing a nucleic acid extract of cells on a support (e.g., nitrocellulose, nylon, polystyrene) and incubating the immobilized target nucleic acid with a probe under well-defined conditions. After hybridization, the excess probe is removed and the hybrid molecules formed are detected by an appropriate method (fluorescence or radioactivity measurement of the enzyme activity bound to the probe).

[0130] The present disclosure also includes nucleotide sequences according to the present disclosure, characterized in that they are covalently or non-covalently immobilized on a support.

[0131] According to another advantageous embodiment of the present disclosure, the nucleotide sequence can be used immobilized on a support, and thus can capture the target nucleic acid obtained from the biological sample to be tested by specific hybridization.Optionally, the solid support is separated from the sample, and then the hybridization complex formed between said capture probe and the target nucleic acid is detected by using a second probe, so-called detection probe, which is labeled with an easily detectable element.

[0132] Another subject of the present disclosure is a vector for the cloning and / or expression of a sequence, characterized in that it comprises a nucleotide sequence according to the present disclosure.

[0133] Vectors according to the present disclosure, characterized in that they contain elements that allow the expression and / or secretion of said nucleotide sequence in a determined host cell, are likewise part of the present disclosure. The vector must then contain a promoter, signals for the initiation and termination of translation, as well as appropriate regions of regulation of transcription. It must be able to be stably maintained in the host cell and, depending on the choice, can have specific signals that specify the secretion of the translated protein. These different elements are selected as a function of the host cell used. For this purpose, the nucleotide sequence according to the present disclosure can be inserted into a self-replicating vector in the selected host, or into an integrating vector of the selected host. Such vectors are prepared by methods currently available to those skilled in the art, and it is possible to introduce the clones obtained from said vectors into a suitable host by standard methods, such as lipofection, electroporation and heat shock. The vectors according to the present disclosure are, for example, vectors of plasmid or viral origin. A preferred vector for the expression of the polypeptides of the present disclosure is the baculovirus.

[0134] These vectors are useful for transforming host cells to clone or express the nucleotide sequences of the present disclosure. The present disclosure also includes host cells transformed by vectors according to the present disclosure. These cells can be obtained by introducing the nucleotide sequence inserted in the vector as described above into a host cell and then culturing said cell under conditions that allow the replication and / or expression of the transfected nucleotide sequence. The host cells can be selected from prokaryotic or eukaryotic systems, such as bacterial cells (Olins and Lee, 1993), as well as yeast cells (Buckholz, 1993), and animal cells, particularly mammalian cultures (Edwards and Aruffo, 1993), in particular Chinese Hamster Ovary (CHO) cells, but also insect cells, for example, which can also use baculovirus procedures (Luckow, 1993). A preferred host cell for the expression of the proteins of the present disclosure is constituted by sf9 insect cells.

[0135] The present disclosure also relates to animals comprising one of the transformed cells according to the present disclosure. The transgenic animals according to the present disclosure that overexpress one or more genes of PCV3 or parts thereof are preferably obtained in rats, mice or rabbits by methods well known to those skilled in the art, such as, for example, viral or non-viral transfection. It is possible to obtain transgenic animals that overexpress one or more genes by transfecting multiple copies of said genes under the control of promoters that are highly ubiquitous or selective for one type of tissue. Similarly, it is possible to obtain transgenic animals by homologous recombination in embryonic cell lines, transferring these cell lines into embryos, selecting affected chimeras at the level of the germ line, and growing said chimeras. The transformed cells and transgenic animals according to the present disclosure can be used in the procedure of preparing recombinant polypeptides.

[0136] It is now possible to produce relatively large amounts of recombinant polypeptides by genetic recombination using cells transformed with an expression vector according to the present disclosure or by using transgenic animals according to the present disclosure. The present invention also includes a procedure for preparing a polypeptide of the present disclosure in recombinant form, characterized in that a vector and / or a cell transformed with a vector according to the present disclosure and / or a transgenic animal comprising one of said transformed cells according to the present disclosure is used. Among said procedures for preparing a recombinant polypeptide of the present disclosure, the preparation procedure using a vector and / or a cell transformed with said vector and / or a transgenic animal comprising one of said transformed cells is preferred, which contains a nucleotide sequence according to the present invention that codes for a PCV3 polypeptide. The recombinant polypeptide obtained as shown above can exist in glycosylated as well as non-glycosylated form, and can have or not have a native tertiary structure.

[0137] Preferred variants are those which produce recombinant polypeptides for use as "carrier" proteins (chimeric proteins). The advantage of this system is that it allows stabilization and reduction of proteolysis of the recombinant product, increased solubility during in vitro renaturation and / or simplified purification when the fusion partner has affinity for a specific ligand.

[0138] More particularly, the present disclosure relates to a process for preparing a polypeptide of the present disclosure, comprising the steps of (a) culturing a transformed cell under conditions allowing expression of a recombinant polypeptide of a nucleotide sequence according to the present disclosure, and (b) optionally, recovering said recombinant polypeptide.

[0139] When the method for preparing a polypeptide of the present disclosure uses a transgenic animal according to the present disclosure, the recombinant polypeptide is then extracted from said animal.

[0140] The present disclosure also relates to polypeptides obtainable by the disclosed procedures as described above.

[0141] The present disclosure also includes a method for the preparation of a synthetic polypeptide, characterized in that it uses an amino acid sequence of a polypeptide according to the present disclosure. The present disclosure also relates to a synthetic polypeptide obtained by a procedure according to the present disclosure.

[0142] The polypeptides according to the present disclosure can likewise be prepared by conventional techniques in the field of peptide synthesis. The synthesis can be carried out in homogeneous solution or in solid phase. For example, reference can be made to the synthesis technique in homogeneous solution described in Houben-Weyl 1974. This synthesis method consists in the condensation of consecutive amino acids two by two in the required order, or of previously formed amino acids and fragments which already contain some amino acids in the appropriate order, or of condensing some of the fragments thus previously prepared, and as will be understood, all reactive functional groups which these amino acids or fragments possess must be protected beforehand, except in the case of an amine function on one side and a carboxyl on the other side or vice versa, which must usually be included in the formation of the peptide bond in the known methods of peptide synthesis, especially after activation of the carboxyl function. According to another preferred technique of the present disclosure, the technique described in Merrifield is used. To prepare the peptide chain in the Merrifield method, a highly porous polymeric resin is used on which the first C-terminal amino acid of the chain is immobilized. This amino acid is immobilized on the resin via its carboxyl group and its amine function is protected. In this way, the amino acids forming the peptide chain are immobilized one after the other on the previously deprotected amino groups of each part of the already formed peptide chain that is bound to the resin. When the entire desired peptide chain has been formed, the protecting groups of the different amino acids forming the peptide chain are removed and the peptide is cleaved from the resin with an acid.

[0143] The present disclosure further relates to hybrid polypeptides comprising at least one polypeptide according to the present disclosure and having the sequence of a polypeptide capable of inducing an immune response in humans or animals.

[0144] Advantageously, the antigenic determinants are such that they are capable of inducing a humoral and / or cellular response. Such antigenic determinants may comprise the polypeptide according to the present disclosure in glycosylated form, used to obtain an immunogenic composition capable of inducing the synthesis of antibodies against multiple epitopes. Said polypeptide or its glycosylated fragments are likewise part of the present disclosure. These hybrid molecules may be formed in part from a polypeptide carrier molecule according to the present disclosure or a fragment thereof, optionally with an immunogenic moiety, such as, in particular, an epitope of diphtheria toxin or tetanus toxin, the surface antigen of the hepatitis B virus (French patent FR79 21811), the VP1 antigen of the poliovirus or any other viral or bacterial toxin or antigen. The procedures for the synthesis of the hybrid molecules encompass the methods used for genetic modification to construct a hybrid nucleotide sequence encoding the desired polypeptide sequence. For example, it may be convenient to refer to the techniques for obtaining genes encoding fusion proteins described in Minton 1984. Said hybrid nucleotide sequence encoding a hybrid polypeptide, as well as a hybrid polypeptide according to the present disclosure, characterized in that it is a recombinant polypeptide obtained by expression of said hybrid nucleotide sequence, are likewise part of the present disclosure.

[0145] The present disclosure also includes vectors, characterized in that they contain one of said hybrid nucleotide sequences. Host cells transformed by said vectors, transgenic animals containing one of said transformed cells, as well as methods for the preparation of recombinant polypeptides using said vectors, said transformed cells and / or said transgenic animals are of course likewise part of the present disclosure.

[0146] The polypeptides according to the present disclosure, the antibodies according to the present disclosure described below and the nucleotide sequences according to the present disclosure can be advantageously used in procedures for the detection and / or identification of PCV3 or porcine circoviruses other than PCV3 in biological samples (biological tissues or fluids) that may contain them. These procedures, according to the specificity of the polypeptides, antibodies and nucleotide sequences to be used, can in particular detect and / or identify PCV3 or porcine circoviruses other than PCV3 or other than PCV3.

[0147] The polypeptides according to the present disclosure can be advantageously used in a procedure for the detection and / or identification of PCV3 in a biological sample (biological tissue or liquid) that may contain them, characterized in that the procedure comprises the steps of (a) contacting this biological sample with a polypeptide according to the present disclosure or one of its fragments (under conditions that allow an immunological reaction between said polypeptide and antibodies possibly present in the biological sample) and (b) demonstrating any antigen-antibody complexes that may be formed. Preferably, the biological sample is a fluid, for example porcine serum or whole blood, or formed by biopsy. Any conventional procedure can be used to carry out the detection of such antigen-antibody complexes that may be formed. By way of example, preferred methods involve the ELISA technique, immunofluorescence, or an immunoenzymatic process by radioimmunoassay (RIA) or equivalent methods.

[0148] Thus, the present disclosure also relates to a polypeptide according to the present disclosure, labeled with the aid of a suitable label, such as of enzymatic, fluorescent or radioactive type. Such a method may, for example, comprise the following steps: (1) depositing a predetermined amount of a polypeptide composition according to the present disclosure in a well of a microtiter plate, (2) introducing increasing dilutions of serum or a biological sample other than the one previously defined into said wells for analysis, (3) incubating the microtiter plate, and (4) introducing labeled antibodies against porcine immunoglobulins into the wells of the microtiter plate, the labeling of which is carried out by detecting the amount of substrate hydrolyzed with the aid of an enzyme selected from a group of enzymes capable of hydrolyzing the substrate, at least at a predetermined wavelength, for example 550 nm, by detecting the change in radiation absorption of the substrate, in comparison with a control test.

[0149] The present disclosure also relates to a kit or set for detecting and / or identifying PCV3, characterized in that it comprises the following elements: (1) a polypeptide according to the present disclosure; (2) if necessary, a reagent for forming a medium favorable for an immunological or specific reaction; (3) if necessary, a reagent allowing the detection of an antigen-antibody complex generated by an immunological reaction between the polypeptide according to the present disclosure and an antibody that may be present in a biological sample, which may also have a label or be recognized by a labeled reagent (but more particularly, when the polypeptide according to the present disclosure is not labeled); (4) if necessary, a biological reference sample lacking an antibody recognized by the polypeptide according to the present disclosure (negative control); and (5) if necessary, a biological reference sample containing a predetermined amount of an antibody recognized by the polypeptide according to the present disclosure (positive control).

[0150] The polypeptide according to the present disclosure allows the preparation of monoclonal or polyclonal antibodies characterized by specific recognition of the polypeptide according to the present disclosure. Conveniently, monoclonal antibodies can be prepared from hybridomas according to the technique described in Kohler and Milstein 1975. Polyclonal antibodies can be prepared, for example, by immunizing animals, particularly mice, with the polypeptide or DNA according to the present disclosure together with an adjuvant for immune response, and then purifying the antigen-specific antibodies contained in the serum of the immunized animal on an affinity column on which the polypeptide that was the antigen has been immobilized. Polyclonal antibodies according to the present disclosure can also be prepared by purifying the antibodies contained in the serum of pigs infected with PCV3 on an affinity column on which the polypeptide according to the present disclosure has been immobilized.

[0151] The present disclosure also relates to monoclonal or polyclonal antibodies or fragments thereof, or chimeric antibodies, characterized in that they are capable of specifically recognizing the polypeptides according to the present disclosure. Similarly, the antibodies of the present disclosure can be labeled in the same manner as described above for the nucleic acid probes of the present disclosure, such as with enzymatic, fluorescent or radioactive type labels.

[0152] The present disclosure further relates to a procedure for detection and / or identification of PCV3 in a biological sample, characterized in that it comprises the following steps: (a) contacting a biological sample (biological tissue or fluid) with a monoclonal or polyclonal antibody according to the present disclosure under conditions allowing an immune reaction between said antibody and polypeptides of PCV3 comprising ORF1, ORF2, and / or ORF3 that may be present in the biological sample; and (b) demonstrating any antigen-antibody complexes that may be formed.

[0153] Within the scope of the present invention is a kit or set for detecting and / or identifying PCV3, characterized in that it comprises: (a) a polyclonal or monoclonal antibody according to the present disclosure, which may be labeled if necessary; (b) if necessary, a reagent for forming a medium favorable for carrying out an immunological reaction; (c) if necessary, a reagent allowing the detection of an antigen-antibody complex produced by the immune reaction, which may also have a label or, in particular if the monoclonal or polyclonal antibody is not labeled, which in turn may be recognized by a labeled reagent; and (d) if necessary, a reagent for carrying out lysis of cells of the tested sample.

[0154] The present disclosure also relates to a procedure for the detection and / or identification of PCV3 in a biological sample, characterized by using a nucleotide sequence according to the present disclosure. The present disclosure particularly includes a procedure for the detection and / or identification of PCV3 in a biological sample, characterized by the following steps: (a) isolation of DNA from the biological sample to be analyzed, if necessary; (b) specific amplification of the DNA of the sample with the aid of at least one primer or pair of primers according to the present disclosure; and (c) demonstration of the amplification products. These can be detected, for example, by molecular hybridization techniques utilizing a nucleic acid probe according to the present disclosure. The probe is advantageously labeled with a non-radioactive (low temperature probe) or radioactive element.

[0155] In this specification, the expressions "DNA of a biological sample" or "DNA contained in a biological sample" are to be understood to mean either the DNA present in the biological sample considered, or, as the case may be, the cDNA obtained after the action of reverse transcriptase on the RNA present in the biological sample.

[0156] Another object of the present disclosure is to provide a method according to the present disclosure, characterized in that it comprises the following steps: (a) contacting a nucleotide probe according to the present disclosure with a biological sample, whereby, if necessary, the DNA contained in the biological sample has previously been made accessible for hybridization under conditions allowing hybridization of the probe with the DNA of the sample, and (b) verifying a hybrid formed between the nucleotide probe and the DNA of the biological sample.

[0157] The present disclosure also relates to a procedure according to the present disclosure, characterized in that it comprises the following steps: (a) contacting a nucleotide probe immobilized on a support according to the present disclosure with a biological sample, whereby, if necessary, the DNA contained in the biological sample has been made accessible for hybridization beforehand under conditions that allow hybridization of the probe with the DNA of the sample, (b) contacting the hybrid formed between the nucleotide probe immobilized on the support and the DNA contained in the biological sample with a labeled nucleotide probe according to the present disclosure, whereby, if necessary, after removing the DNA of the biological sample that has not hybridized to the probe, and (c) verification of the new hybrid formed in step (b). According to an advantageous embodiment of the procedure for detection and / or identification defined above, it is characterized in that, prior to step (a), the DNA of the biological sample is first amplified with the aid of at least one primer according to the present disclosure.

[0158] The present disclosure further relates to a kit or set for detecting and / or identifying PCV3, characterized in that it comprises the following elements: (a) a nucleotide probe according to the present disclosure, (b) if necessary, reagents necessary for performing a hybridization reaction, and (c) if necessary, at least one primer according to the present disclosure, as well as reagents necessary for a DNA amplification reaction.

[0159] The present disclosure similarly relates to a kit or set for detecting and / or identifying PCV3 or a porcine circovirus other than PCV3, characterized in that it comprises the following components: (a) a nucleotide probe, called a capture probe, according to the present disclosure, (b) an oligonucleotide probe, called an exposed probe, according to the present disclosure, and (c) if necessary, at least one primer, according to the present disclosure, as well as reagents necessary for a DNA amplification reaction.

[0160] The present disclosure also relates to a kit or set for detecting and / or identifying PCV3, characterized in that it comprises the following elements: (a) at least one primer according to the present disclosure, (b) if necessary, reagents necessary for performing a DNA amplification reaction, and (c) if necessary, components for verifying the sequence of the amplified fragment, more specifically an oligonucleotide probe according to the present disclosure.

[0161] The present disclosure further relates to the use of nucleotide sequences according to the present disclosure, polypeptides according to the present disclosure, antibodies according to the present disclosure, cells according to the present disclosure, and / or transformed animals according to the present disclosure for the selection of organic or inorganic compounds capable of regulating, inducing or inhibiting expression of genes and / or capable of regulating cellular replication of PCV3 or inducing or suppressing pathology, including reducing the incidence and severity of clinical signs associated with infection with PCV3.

[0162] The present disclosure also includes a method for selecting a compound capable of binding to a polypeptide or one of its fragments according to the present disclosure, binding to a nucleotide sequence according to the present disclosure, or recognizing an antibody according to the present disclosure, and / or regulating, inducing or inhibiting expression of a gene, and / or modifying cellular replication of PCV3, or inducing or inhibiting a pathology (including reducing the incidence and severity of clinical symptoms associated with infection with PCV3), comprising the steps of: (a) contacting the compound with the polypeptide, the nucleotide sequence, or administering the compound to a transformed cell according to the present disclosure and / or to a transformed animal according to the present disclosure; and (b) determining the ability of the compound to bind to the polypeptide or the nucleotide sequence, or to regulate, induce or inhibit a gene, or to regulate the growth or replication of PCV3, or to induce or inhibit a pathology associated with infection with PCV3 in the transformed animal (activity of the compound). The compound that can be selected can be an organic compound such as a polypeptide or a carbohydrate, any other organic compound known, an inorganic compound, or a new organic compound that is refined by molecular modeling techniques and obtained by chemical or biochemical synthesis (these techniques are known to those skilled in the art).The selected compound can be used to regulate the cell replication of PCV3 and thus control the infection by this virus, and this method allows the determination of said regulation, which is well known to those skilled in the art.This regulation can be carried out, for example, by an agent that can bind to a protein and thus inhibit or enhance its biological activity, or that can bind to the envelope protein of the outer surface of the virus and block the penetration of the virus into host cells, or that can assist the action of the immune system of the infected organism directed against the virus.This regulation can likewise be achieved by an agent capable of binding to a nucleotide sequence in the DNA of the virus and blocking, for example, expression of a polypeptide having a biological or structural activity necessary for the replication or propagation of the virus from a host cell into a host animal.

[0163] The present disclosure relates to compounds that can be selected by the selection method according to the present disclosure.

[0164] The present disclosure also relates to a pharmaceutical composition comprising a compound selected from the following compounds: (a) a nucleotide having a nucleotide sequence according to the present disclosure, (b) a polypeptide according to the present disclosure, (c) a vector, a viral particle or a transformed cell according to the present disclosure, (d) an antibody according to the present disclosure, and (e) a compound that can be selected by a selection method according to the present disclosure, optionally in combination with a pharma- ceutically acceptable carrier, and if necessary, one or more suitable immunological adjuvants.

[0165] The present disclosure also relates to an immunogenic and / or vaccine composition, characterized in that it comprises a compound selected from the following compounds: (a) a nucleotide sequence according to the present disclosure, (b) a polypeptide according to the present disclosure, (c) a vector or viral particle according to the present disclosure, and (d) a cell according to the present disclosure.

[0166] In one embodiment, the vaccine composition according to the present disclosure comprises a mixture of at least two of said compounds (a), (b), (c) and (d), wherein one of said two compounds is related to PCV3.

[0167] In another embodiment of the present disclosure, the vaccine composition comprises at least one of the above compounds (a), (b), (c), or (d) associated with PCV3. In yet another embodiment, the vaccine composition comprises at least one of the above compounds (a), (b), (c), or (d) associated with ORF2 of PCV3.

[0168] A compound related to PCV3 is understood herein to denote a compound obtained from the genomic sequence of PCV3 and / or ORF1, ORF2 and ORF2 of PCV3, respectively.

[0169] The present disclosure is further directed to an immunogenic and / or vaccine composition, characterized in that it comprises at least one of the following compounds: (1) a nucleotide of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or one of their fragments or homologs; (2) a polypeptide of the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or one of their fragments, or a variant thereof; (3) a vector or viral particle comprising a nucleotide of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or one of their fragments or homologs; (4) a transformed cell capable of expressing a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or one of their fragments, or a variant thereof; or (5) a mixture of at least two of the aforementioned compounds.

[0170] The present disclosure also includes immunogenic and / or vaccine compositions according to the present disclosure comprising said mixture of at least two of said compounds as a combination product for simultaneous, separate or chronic use for the prevention or treatment of infection by PCV3.

[0171] In a preferred embodiment, the vaccine composition according to the present disclosure comprises the following compounds: (1) a pcDNA3 plasmid comprising a nucleic acid having the sequence of SEQ ID NO:1; (2) a pcDNA3 plasmid comprising a nucleic acid having the sequence of SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; (3) a pcDNA3 plasmid containing a nucleic acid encoding a GM-CSF protein; (4) a recombinant baculovirus comprising a nucleic acid having the sequence of SEQ ID NO:1; (5) a recombinant baculovirus comprising a nucleic acid having the sequence of SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; and (6) optionally, a suitable immune adjuvant, in particular the adjuvant AIFTM.

[0172] The present disclosure also relates to a pharmaceutical composition according to the present disclosure for the prevention or treatment of infection with PCV3.

[0173] "Prevention" as used in this disclosure includes complete prevention of infection by PCV3, but also includes reducing the severity or incidence of clinical signs associated with or caused by PCV3 infection. Such prevention is also referred to herein as a protective effect.

[0174] The present disclosure also relates to the use of a composition according to the present disclosure for the preparation of a medicament for the prevention or treatment of infection with PCV3.

[0175] In another aspect, the present disclosure relates to a vector, a viral particle or a cell according to the present disclosure for the treatment and / or prevention of a disease by gene therapy.

[0176] Finally, the present disclosure includes the use of a vector, a viral particle or a cell according to the present disclosure for the preparation of a medicament for the treatment and / or prevention of a disease by gene therapy.

[0177] Polypeptides of the present disclosure to be included in an immunogenic or vaccine composition according to the present disclosure can be selected by techniques known to those skilled in the art, for example, depending on the ability of said polypeptide to stimulate T cells (e.g., their proliferation or their ability to be translated by secretion of interleukins, resulting in the production of antibodies against said polypeptide).

[0178] In pigs, as in mice, weight doses of the vaccine composition comparable to those used in humans are administered, and the antibody response is tested according to conventional techniques by obtaining serum and then examining the formation of complexes between the antibodies present in the serum and the antigens of the vaccine composition.

[0179] The pharmaceutical composition according to the present disclosure may be present in said compound in an effective amount, i.e., in an amount sufficient to obtain the desired effect, such as, for example, the regulation of PCV3 cell replication.Those skilled in the art know how to determine this amount, for example, as a function of the age and weight of the individual to be treated, the progression of the pathology, possible side effects, and evaluation tests of the effect obtained in the population range, which tests are known in the art.

[0180] In accordance with the present disclosure, the vaccine combination is preferably combined with a pharma- ceutically or veterinarily acceptable carrier and, if necessary, with one or more suitable immunological adjuvants.

[0181] Today, various types of vaccines are available to protect animals or humans against infectious diseases, including live attenuated microorganisms (M. bovis-BCG for tuberculosis), inactivated microorganisms (influenza virus), cell extracts (B. pertussis for whooping cough), recombinant proteins (surface antigen of Hepatitis B virus), polysaccharides (Streptococcus pneumoniae), etc. Vaccines prepared from synthetic peptides or genetically engineered microorganisms expressing heterologous antigens are in the process of experimentation. More recently, recombinant plasmid DNA carrying genes encoding prophylactic antigens has been proposed as an alternative vaccine strategy. This type of vaccination is performed with specific plasmids derived from Escherichia coli (E. coli) plasmids that do not replicate in vivo and that uniquely code for the vaccinating protein. Animals are immunized by simply injecting naked plasmid DNA into the muscle. This technique leads to the expression of the vaccine protein in situ and to an immune response of both cellular (CTL) and humoral (antibody) types. This dual induction of immune response is one of the main advantages of the naked DNA vaccination technique.

[0182] The constitutive nucleotide sequences of the vaccine composition according to the present disclosure can be injected into a host after being conjugated to a compound that facilitates the penetration of the polynucleotide into the cell interior or its transport to the cell nucleus. The resulting complex can be encapsulated in polymeric microparticles as described in International Publication WO 94 / 027238 (Medisorb Technologies International).

[0183] According to another embodiment of the vaccine composition according to the present disclosure, the nucleotide of the sequence of the present invention, preferably DNA, is complexed with DEAE-dextran (Pagano et al., 1967), with nucleoproteins (Kaneda et al., 1989), or with lipids (Felgner et al., 1987), or is encapsulated in liposomes (Fraley et al., 1980), or is introduced in the form of a gel that facilitates its transfection into cells (Midoux et al., 1993, Pastore et al., 1994). The polynucleotide or vector according to the present disclosure may also be suspended in a buffer solution or combined with liposomes.

[0184] Conveniently, such vaccines are prepared by the techniques described in Tacson et al., or Huygen et al. 1996, or alternatively in Davis et al., International Publication No. WO 95 / 011307.

[0185] Such vaccines can be prepared in the form of a composition containing a vector according to the present disclosure, which is under the control of regulatory elements that allow its expression in humans or animals. For example, it is possible to use the in vivo expression vectors of the polypeptide antigen of interest, the plasmid pcDNA3 or the plasmid pcDNA1 / neo, both of which are marketed by Invitrogen (R&D Systems, Abingdon, UK). It is also possible to use the plasmid V1Jns.tPA (described in Shiver et al. 1995). Such vaccines advantageously contain, apart from the recombinant vector, a saline solution, for example a sodium chloride solution.

[0186] As far as vaccine formulations are concerned, these may contain suitable immunological adjuvants known to those skilled in the art, for example as described above.

[0187] These compounds can be administered by the systemic route (systemic administration), in particular by the intravenous, intramuscular, intradermal or subcutaneous route, or by oral route. In a more preferred embodiment, the vaccine composition comprising the polypeptide according to the present disclosure is administered by the intramuscular route, through food, or by spraying several times at staggered times.

[0188] Their administration methods, dosages and optimal pharmaceutical forms can be determined according to criteria generally considered in establishing a treatment suitable for a certain animal, such as age or weight, the severity of its general pathology, resistance to treatment, and factors such as indicated secondary effects. Preferably, the vaccine of the present disclosure is administered in an amount that provides a protective or protective effect against PCV3 infection.

[0189] For example, in the case of a vaccine according to the present disclosure comprising a polypeptide encoded by a nucleotide sequence of the genome of PCV3 or a homologue or fragment thereof, the polypeptide is administered in a single dose or multiple doses at a dose of about 0.1 μg-10 μg per kg of animal body weight, preferably at a dose of about 0.2 μg / kg-about 5 μg / kg, more preferably about 0.5 μg / kg-about 2 μg / kg, and spread directly or by transformed cells capable of expressing the polypeptide.

[0190] The present disclosure also relates to the construction of self-replicating retroviral vectors of the nucleotide sequence of PCV3 according to the present disclosure and their use in therapeutic applications, in particular in the field of in vivo gene therapy.

[0191] The feasibility of gene therapy applied to humans no longer needs to be demonstrated anew, which is relevant for many therapeutic applications such as genetic diseases, infectious diseases and cancer. Many documents in the prior art describe the means of using gene therapy, especially via viral vectors. In general, vectors are obtained by deletion of at least some of the viral genes, which are replaced by genes of therapeutic interest. Such vectors are defective for replication, but can be propagated in complementation systems that provide the missing viral functions in trans to generate viral vector particles capable of infecting host cells. To date, retroviral vectors are the most widely used, and their mode of infection is widely described in the literature available to the skilled artisan.

[0192] The principle of gene therapy is to deliver a functional gene, called a gene of interest, whose RNA or corresponding protein produces the desired biochemical effect in targeted cells or tissues. On the one hand, gene insertion allows for long-term expression of complex and unstable molecules such as RNA or proteins that may be extremely difficult or even impossible to obtain or administer directly. On the other hand, the controlled insertion of the desired gene into specific targeted cells allows the expression product to be regulated in a given tissue. For this purpose, it is necessary to have an insertion method available that allows the desired therapeutic gene to be inserted into selected cells, thus allowing specific targeting of selected cells or tissues. Some preferred genes of interest for the present disclosure are those that code for ORF1, ORF2, or ORF3.

[0193] The use of viral particles, such as retroviruses, in gene insertion methods such as microinjection, especially injection of naked plasmid DNA, electroporation, homologous recombination, etc., is widespread. However, when applied in vivo, recombinant retrovirus-type gene transfer systems suffer from low infectivity (insufficient concentration of viral particles) and at the same time lack specificity with respect to the selected target cells.

[0194] The production of cell-specific viral vectors with tissue-specific tropism and capable of being properly translated by target cells can be achieved, for example, by fusing a specific ligand of the target host cell to the N-terminus of the surface protein of the envelope of PCV3. For example, retroviral particles with CD4 molecules on the envelope surface to target human cells infected with HIV virus (viral particles with peptide hormones fused to the envelope protein specifically infect cells expressing the corresponding receptors), or alternatively, viral particles with fusion polypeptides that can be immobilized to the receptor of epidermal growth factor (EGF) are also envisioned. In another approach, single-chain fragments of antibodies against surface antigens of target cells are inserted by fusion with the N-terminus portion of the envelope protein.

[0195] For the purposes of this disclosure, the gene of interest used in this disclosure can be obtained from eukaryotes or prokaryotes, or from viruses by any conventional technique. It can preferably produce an expression that has a therapeutic effect, and the expression can be a product that is homologous to the cellular host or a heterologous product. Within the scope of this disclosure, the gene of interest can code for (1) an intracellular product that is present on the surface of the host cell, or (2) a membrane product, or (3) a membrane product that is secreted outside the host cell. Therefore, it can include appropriate additional elements, such as a sequence that codes for a secretion signal. These signals are known to those skilled in the art.

[0196] According to the objectives pursued by the present disclosure, the gene of interest can code for a protein corresponding to all or part of a natural protein as found in nature. It can also be a chimeric protein resulting, for example, from the fusion of polypeptides of various origins, or from a variant with improved and / or modified biological properties. Such mutants can be obtained by conventional biological techniques, by substitution, deletion and / or addition of one or more amino acid residues.

[0197] Thus, the present disclosure relates to vectors comprising the nucleotides of the nucleotide sequence of PCV3 according to the present disclosure and characterized in that they additionally comprise a gene of interest.

[0198] The present disclosure also relates to a viral particle produced from said vector according to the present disclosure.The present invention further relates to a method for preparing a viral particle according to the present disclosure, characterized in that a vector according to the present disclosure containing a viral pseudoparticle (VLP, virus-like particle) is used.

[0199] The present disclosure also relates to animal cells transfected with a vector according to the present disclosure. Similarly, the present disclosure includes animal cells, particularly mammalian cells, infected with a viral particle included in the present disclosure.

[0200] One preferred vaccine uses live chimeric virus DNA clones, specifically those containing the immunogenic genes of PCV3 cloned into the backbone of non-pathogenic PCV1. Advantageously, when constructed by genetic engineering, the live chimeric virus is naturally avirulent, and does not require time-consuming attenuation procedures. Uniquely, this virus functions as a live but non-pathogenic replicating virus that produces immunogenic proteins against PCV3 during viral replication, thereby inducing a full range of immune responses against pathogenic PCV3.

[0201] As an additional advantage, the live chimeric virus of the present disclosure provides a genetically stable vaccine that is easier to make, store and deliver than other types of attenuated vaccines.Avirulent or attenuated vaccines based on chimeric viruses are generally considered safer, if not safer, than conventionally modified live vaccines.For example, the ChimeriVax-JE vaccine against Japanese encephalitis virus (JEV), a genetically engineered derivative of the yellow fever virus vaccine YFV17D, in which the genes encoding the structural proteins prM and E of YFV17D are replaced with the corresponding genes of attenuated JEV SA14-14-2 strain, has been shown to be genetically stable after longer passages both in vitro and in vivo. Another chimeric virus vaccine against dengue virus type 2, ChimeriVax-D2, an attenuated chimeric yellow fever (YF)-dengue virus type 2 (dengue 2) virus, has also been found to be genetically stable, and its sequence was reported to remain unchanged after 18 passages in Vero cells.

[0202] Another preferred vaccine of the present disclosure utilizes a plasmid suitable for delivering the non-pathogenic chimeric DNA clone to pigs. Unlike conventional vaccines that use live or killed cell culture grown whole viruses, the present disclosure provides for direct inoculation of pigs with plasmid DNA containing the infectious chimeric viral genome.

[0203] The further genetically engineered vaccines desirable in the present disclosure are produced by techniques known in the art. Such techniques include, but are not limited to, further manipulation of recombinant DNA, modification or substitution of amino acid sequences of recombinant proteins, etc. Genetically engineered vaccines based on recombinant DNA technology are made, for example, by identifying alternative portions of viral genes that code for proteins responsible for inducing stronger immune or protective responses in pigs (e.g., proteins from ORF1, ORF2, ORF3). Such identified genes, i.e., immunodominant fragments, can be cloned into standard protein expression vectors, such as baculovirus vectors, and used to infect suitable host cells. The host cells can be cultured and the desired vaccine proteins can be expressed, purified to the desired extent, and formulated into suitable vaccine products.

[0204] If the clone retains the natural ability to cause any undesirable disease, it is also possible to identify the nucleotide sequence in the viral genome that is responsible for the residual toxicity and genetically engineer the avirulent virus, for example, via site-directed mutagenesis. Site-directed mutagenesis can add, delete or change one or more nucleotides. An oligonucleotide containing the desired mutation is synthesized and annealed to a portion of the single-stranded viral DNA. A hybrid molecule is obtained from the procedure and used to transform bacteria. If the isolated double-stranded DNA containing the appropriate mutation is then used to generate a full-length DNA, it is generated by ligation to a restriction enzyme fragment of the full-length DNA that is then transfected into an appropriate cell culture. Ligation of the genome into a suitable vector can be accomplished by any standard technique known to those skilled in the art. Transfection of the vector into a host cell for the production of viral progeny can be performed using any of the conventional methods, such as calcium phosphate or DEAE-dextran mediated transfection, electroporation, protoplast fusion and other well-known techniques. The cloned virus then exhibits the desired mutation. Alternatively, two oligonucleotides containing the appropriate mutation can be synthesized. These can be annealed to form a double stranded DNA that can be inserted into the viral DNA to produce a full length DNA.

[0205] The genetically engineered proteins useful for vaccines can be expressed, for example, in insect cells, yeast cells or mammalian cells. The genetically engineered proteins can be purified or isolated by conventional methods and directly inoculated into pigs to provide protection against postweaning multisystemic wasting syndrome (PMWS) caused by viral infection or PCV3. Insect cell lines (such as HI-FIVE) can be transformed with a transfer vector containing a nucleic acid molecule obtained from a virus or copied from a viral genome encoding one or more of its immunodominant proteins. Transfer vectors include, for example, linearized baculovirus DNA and a plasmid containing the desired polynucleotide. To generate a recombinant baculovirus, a host cell line can be co-transfected with linearized baculovirus DNA and a plasmid.

[0206] Alternatively, DNA from pigs harboring PMWS, infectious PCV3 molecular DNA clones, or cloned PCV chimeric DNA genomes encoding pneumonia, reproductive failure, PDNS, and / or one or more capsid proteins can be inserted into a live vector (e.g., poxvirus or adenovirus) and used as a vaccine.

[0207] An immunologically effective amount of the vaccine or immunogenic composition of the present disclosure is administered to pigs in need of protection against viral infection, pneumonia, reproductive failure, PDNS and / or PMWS. The immunologically effective or immunogenic amount to inoculate the pig can be easily determined or easily titrated by routine testing. An effective amount is an amount that achieves a sufficient immune response to the vaccine to protect pigs exposed to the virus that causes PMWS. Preferably, the vaccinated pig is protected to the extent that one or all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or completely prevented.

[0208] The vaccine can be administered in a single dose or multiple doses, with a single dose being preferred. A single dose vaccine provides protection after a single dose without the need for any booster or subsequent doses. Protection includes complete prevention of clinical signs of infection, or a reduction in the severity, duration, or likelihood of the onset of one or more clinical signs of infection. The range of dosage is, for example, about 1 μg to about 1,000 μg of plasmid DNA containing an infectious chimeric DNA genome, preferably 100 μg to 200 μg of chimeric PCV1-3 DNA clone, but should not contain a sufficient amount of virus-based antigen to produce adverse reactions or physiological symptoms of viral infection. Methods are known in the art for determining or titrating the appropriate dosage of an effective antigenic agent to find the minimum effective dosage based on pig weight, antigen concentration, and other typical factors. Preferably, an infectious chimeric virus DNA clone is used as the vaccine, or a live infectious chimeric virus is generated in vitro and then the live chimeric virus is used as the vaccine. In this case, the 50% tissue culture infectivity dose (TCID ) of the live chimeric virus is, for example, about 50 to about 10,000. 50 ) can be fed to pigs.

[0209] Desirably, the vaccine is administered to pigs that have not been exposed to the PCV virus. Vaccines containing chimeric PCV1-3 infectious DNA clones, or other antigen forms, can be administered intranasally, transdermally (i.e., applied on or to the skin surface for systemic absorption), parenterally, and the like. Parenteral routes of administration include, but are not limited to, intramuscular, intravenous, intraperitoneal, intradermal (i.e., injected or otherwise placed under the skin) routes, and the like. In other studies with viral infectious DNA clones, injection via intramuscular and intradermal routes have been successful, so these routes are most preferred in addition to the actual intranasal route of administration. Although less convenient, it is also conceivable to vaccinate pigs via injection via lymphoblastic route. One unique and preferred method of administration involves injecting plasmid DNA containing PCV1-3 chimeras directly into pigs via intramuscular, intradermal, intralymphatic, and other routes.

[0210] When administered as a liquid, the vaccine may be prepared in the form of an aqueous solution, syrup, elixir, tincture, or the like. Such formulations are known in the art and are generally prepared by dissolving the antigen and other typical additives in a suitable carrier or solvent system. Suitable carriers or solvent systems include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerol, and the like. Typical additives are, for example, approved dyes, flavors, sweeteners, and antimicrobial preservatives such as thimerosal (sodium ethylmercurithiosalicylate). Such solutions may be stabilized, for example, by the addition of partially hydrolyzed gelatin, sorbitol, or culture cell media, and may be buffered in a conventional manner by using agents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, mixtures thereof, and the like.

[0211] Liquid formulations may also include suspensions and emulsions, which include suspending agents or emulsifying agents in combination with other standard formulations.These types of liquid formulations can be prepared by conventional methods.Suspensions can be prepared, for example, using a colloid mill.Emulsifying agents can be prepared, for example, using a homogenizer.

[0212] Parenteral formulations designed for injection into the body fluid system require appropriate isotonicity and pH buffering for the corresponding levels of porcine body fluids. Isotonicity can be appropriately adjusted with sodium chloride and other salts as necessary. Appropriate solvents such as ethanol or propylene glycol can be used to increase the solubility of the components in the formulation and the stability of the liquid preparation. Additional additives that can be used in the vaccine include, but are not limited to, dextrose, conventional antioxidants, and conventional chelating agents such as ethylenediaminetetraacetic acid (EDTA). Parenteral dosage forms must also be sterilized prior to use.

[0213] In another embodiment of the present disclosure, a protein is provided that is encoded by a chimeric DNA molecule. The protein may be administered separately or in addition to other compositions described herein. In a preferred form, the protein is administered in an amount ranging from about 0.2 to about 400 μg / ml. The chimeric DNA molecule is preferably as described herein.

[0214] As described herein, by constructing infectious DNA clone of PCV3, biologically pure and homogenous infectious virus stock can be generated for pathogenicity study and development of non-pathogenic chimeric vaccine.By using this molecular DNA clone and the biologically pure and homogenous infectious PCV3 virus stock derived from molecular DNA clone, the clinical disease, virus distribution and pathological lesion course associated with PCV3 infection can be more clearly characterized than observed in the past, which is useful for the development of the desired vaccine product of the present disclosure.

[0215] PCV3 molecular clones are generated by tandemly linking two copies of the complete PCV3 genome to a pSK vector. In sharp contrast to the single copy genomes disclosed in the art, the infectious DNA PCV3 clones generated by the methods described herein contain two complete copies of the PCV3 genome linked together in direct repeats. Linking two copies of the genome in tandem provides a similar circular genome that mimics the normal circular genome of PCV3. The advantage of having two copies of the genome in tandem in an infectious DNA PCV3 clone is that it can maximize replication when the infectious DNA clone is transfected in vitro and in vivo. Thus, the clones of the present disclosure work more effectively and efficiently than traditional single copy genomes.

[0216] Infection of animals with molecular viral clones is extremely useful to study viral replication in the host and genetic determinants of virulence. PCV3 has been identified as the causative agent of pneumonia, reproductive disorders, PMWS, and PDNS. PMWS and PDNS are complex disease syndromes in pigs, and multiple factors may contribute to the clinical manifestations of PMWS and / or PDNS. However, the difficulty of producing biologically pure forms of PCV3 due to the presence of other common pigs in tissue homogenates of diseased pigs has prevented conclusive characterization of clinical disease and pathological lesions attributable solely to PCV3 infection. This is the first time that an infectious molecular DNA clone of PCV3 has been constructed and used to characterize disease and pathological lesions associated with PCV3 infection by direct in vivo transfection of pigs with the molecular clone.

[0217] Homologous PCV3 live virus stocks derived from molecular clones are shown to be infectious in vitro when transfected into PK-15 cells. Cloned PCV3 genomic DNA also infects specific pathogen-free (SPF) pigs when injected directly into the liver and superficial iliac lymph nodes. Animals injected with cloned PCV3 plasmid DNA develop infections and disease similar to those induced by intranasal inoculation with homologous, infectious PCV3 live virus stocks. Seroconversion to PCV3-specific antibodies is detectable in the majority of pigs in the inoculated group 35 days post-inoculation (35 DPI).

[0218] The onset and duration of viremia in pigs inoculated with chimeric PCV1-3 DNA clones is similar to that of pigs inoculated with non-pathogenic PCV1 DNA clones, whereas viremia in pigs inoculated with PCV3 clones develops earlier and for longer. Beginning at 14 DPI and lasting approximately 2-6 weeks, viremia is detectable in the majority of PCV3-inoculated animals. Similarly, the majority of inoculated pigs necropsied at 35 DPI will seroconvert to PCV3 antibodies. PCV3 antigen is detected in various tissues and organs of inoculated pigs. Gross lesions are restricted to lungs and lymph nodes and are characterized by histologically enlarged tan lymph nodes, failed to collapse lungs, and mild multifocal tan consolidation lesions. In both non-pathogenic PCV1-vaccinated and chimeric PCV1-3-vaccinated pigs, gross lesions affecting lymph nodes are mild and limited to a few animals, whereas pathogenic PCV3-vaccinated pigs have a higher incidence of moderate to severe swelling and discoloration of lymphoid tissues. Statistical analysis reveals that the scores of gross lesions in lymph nodes of chimeric PCV1-3-vaccinated animals are similar to those in lymph nodes of non-pathogenic PCV1-vaccinated pigs. At 21 DPI, PCV3-vaccinated pigs have statistically more severe gross lesions than PCV1-vaccinated and chimeric PCV1-3-vaccinated pigs. Histopathological lesions and PCV3-specific antigens are detected in numerous tissues and organs of inoculated (infected) pigs, including brain, lung, heart, kidney, tonsils, lymph nodes, spleen, ileum, and liver. Histopathological lesions in multiple tissues and organs similar to those of PMWS are reproduced by PCV3 molecular DNA clones and infectious viruses prepared in vitro from the molecular DNA clones. Microscopically, chimeric PCV1-3-inoculated animals have statistically fewer microscopic lesions than PCV3-inoculated animals at both 21 and 49 DPI. Microscopic lesion scores in lymph nodes of chimeric PCV1-3-inoculated pigs are similar to nonpathogenic PCV1-inoculated pigs, reciprocal chimeric PCV3-1-inoculated pigs, and uninoculated control animals. Moderate to severe microscopic lesions are found in multiple tissues of pathogenic PCV3-inoculated animals, including lung, liver, lymphocytes, spleen, brain, heart, kidney, and tonsil tissues.However, in chimeric PCV1-3 inoculated animals, mild to moderate microscopic lesions were restricted to liver, lymph node and kidney tissues.

[0219] The availability of an infectious DNA clone of PCV3 described herein makes feasible the development of a genetically engineered attenuated vaccine to prevent PCV3 infection in pigs, as well as PDNS and PMWS.

[0220] Because of the availability of the PCV3, PCV1, chimeric PCV1-3, and reciprocal chimeric PCV3-1 infectious DNA clones described herein, the structural and functional relationships of the PCV genes are better understood.

[0221] The construction of infectious PCV3 molecular DNA clones and the demonstration of infection by direct injection of cloned PCV3 plasmid DNA into pig liver and lymph nodes in the context of this disclosure are advantageous for PCV3 research. This in vivo transfection system tests different regions or genes of PCV3 for their role in viral replication and pathogenicity in the host by enhancing the study of the structural and functional relationships of PCV3 genes with recombinant plasmids constructed in vitro. PCV3 replication and pathogenicity can be studied in vivo without producing infectious virus stocks by propagating PCV3 in cultured cells. This is advantageous because continuous cell culture passaging can select for viral variants. Another advantage of using cloned PCV3 genomic DNA instead of live virus for animal studies is the relative ease of quantifying the inoculation dose. The amount of cloned PCV3 DNA used for animal inoculation can be easily determined by spectrophotometer, whereas the amount of live PCV3 virus requires infectivity titration in cell culture and confirmation of infection by IFA. Direct injection of animals with cloned PCV3 plasmid DNA eliminates problems associated with the presence of other indigenous porcine agents in tissue homogenate inocula in animal studies.

[0222] In one embodiment of the present disclosure, the unique structure of chimeric PCV1-3 infectious DNA clone is generated by switching the immunogenic ORF2 capsid gene between pathogenic PCV3 and non-pathogenic PCV1. Surprisingly and advantageously, the chimeric PCV1-3 infectious clone replicates, expresses the immunogenic ORF2 capsid antigen in vitro and in vivo, and induces specific antibody response against PCV3 ORF2, but retains the non-pathogenicity of PCV1. The chimeric PCV1-3 infectious DNA clone has the ability to induce a strong immune response against PCV3, but only induces limited infection with mild pathological lesions similar to non-pathogenic PCV1. For vaccine development, the relatively easy storage and stability of cloned DNA, and the economy of large-scale recombinant PCV3 plasmid DNA and chimeric PCV1-3 DNA clone production provide an attractive means to deliver live infectious virus DNA vaccines or genetically engineered attenuated virus vaccines to pigs. Therefore, the chimeric PCV1-3 infectious DNA clone taught in this disclosure is a useful vaccine candidate against PCV3 infection and PMWS.

[0223] It is to be understood that all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purposes of this disclosure, the term "infectious" means that the virus replicates in the pig, regardless of whether it causes any disease. "SPF" refers to specific pathogen-free pigs.

[0224] By "gnotobiotic" pigs it is intended that the pigs are germ-free. The terms "PCV3 plasmid DNA," "PCV3 genomic DNA," and "PCV3 molecular DNA" are used interchangeably as they refer to the same cloned nucleotide sequence.

[0225] The following examples demonstrate certain aspects of the present disclosure. However, it should be understood that these examples are illustrative and do not claim to be completely definitive with respect to the conditions and scope of the present disclosure. It should be understood that when typical reaction conditions (e.g., temperature, reaction time, etc.) are given, conditions above and below the specified ranges can both be used, but are generally less favorable. The examples are carried out at room temperature (about 23°C-about 28°C) and atmospheric pressure. All parts and percentages referred to herein are by weight, and all temperatures are expressed in degrees Celsius, unless otherwise indicated. Furthermore, unless otherwise indicated, all components of the present disclosure are understood to be disclosed as encompassing the claims' "comprising," "consisting essentially of," and "consisting of," as commonly used terms in the claims.

[0226] Another aspect of the present disclosure is the preparation of a combination vaccine or immunological composition. Such combination can be any of the different vaccine components described herein. For example, the vaccine of the present disclosure can include both the protein portion and the DNA portion of PCV3 as described herein, which are administered simultaneously or separately. Furthermore, the combination can be any of the PCV3 vaccine components described herein and antigens of other pathogens as described above.

[0227] In a further embodiment, the vaccine or immunological composition is first dehydrated. If the composition is first lyophilized or dehydrated by other methods, it is rehydrated in an aqueous solution (e.g., saline, PBS (phosphate buffered saline)), or a non-aqueous solution (e.g., oil emulsion (mineral oil, or vegetable / metabolizable oil based / single or double emulsion based), aluminum-based, carbomer-based adjuvants) prior to vaccination.

[0228] According to the present disclosure, an effective amount of the combination vaccine administered to a pig provides an effective immune or protective effect against microbial infection caused by PCV3 and at least one additional pathogen. Preferred combinations of antigens for the treatment and prevention of microbiological diseases in pigs are listed above.

[0229] In a further embodiment, the combination vaccine is administered to pigs in one or two doses at an interval of about 2-4 weeks. For example, the first administration is administered when the animals are about 2-3 weeks to about 8 weeks old. The second administration is administered about 1 to about 4 weeks after the first administration of the primary vaccination. In a further embodiment, revaccination is performed at an interval of 3-12 months after administration of the second dose. Administration of subsequent vaccine doses is preferably performed every 6 months. In another preferred embodiment, animals vaccinated before reaching about 2-3 weeks of age should be revaccinated. Administration of subsequent vaccine doses is preferably performed annually. If one of the components of the combination vaccine is effective after a single administration, such component needs to be administered only once with the other component(s) administered according to the preferred regimen.

[0230] The amount of effective combination vaccine depends on the vaccine components and the schedule of administration. Typically, when inactivated or modified live virus preparations are used in combination vaccines, about 10 per dose is used. 2 -about 10 9 TCID 50 A vaccine amount containing about 10 3 -about 10 8 TCID 50 More preferably, the amount of vaccine containing about 10 4 -about 10 8 TCID 50 In general, inactivated antigens are usually used in larger amounts than live modified viruses. Typically, when bacterial antigens are used in combination vaccines, the vaccine contains about 10 3 -about 10 9 Colony forming units (CFU), preferably about 10 per dose4 -about 10 8 (CFU), more preferably about 10 per dose 5 -about 10 6 Subunit vaccines are typically administered at an antigen-containing level of at least 0.2 μg antigen per dose, preferably from about 0.2 to about 400 μg / dose, even more preferably from about 0.3 to about 200 μg / dose, even more preferably from about 0.35 to about 100 μg / dose, even more preferably from about 0.5 to about 50 μg / dose, even more preferably from about 0.45 to about 30 μg / dose, even more preferably from about 0.6 to about 15 μg / dose, even more preferably from about 0.75 to about 8 μg / dose, even more preferably from about 1.0 to about 6 μg / dose, and even more preferably from about 1.3 to about 3 μg / dose. For example, the antigen content level of the PCV3 ORF2 antigen, preferably the PCV3 ORF2 protein provided herein, contains about 2 μg to about 150 μg, preferably about 2 μg to about 60 μg, even more preferably about 2 μg to about 50 μg, even more preferably about 2 μg to about 40 μg, even more preferably about 2 μg to about 30 μg, even more preferably about 2 μg to about 25 μg, even more preferably about 2 μg to about 20 μg, even more preferably about 4 μg to about 20 μg, and even more preferably about 4 μg to about 16 μg.

[0231] The composition according to the present disclosure may be applied intradermally, intratracheally, or intravaginally. The composition may preferably be applied intramuscularly or intranasally. In the animal body, it proves advantageous to apply such pharmaceutical compositions via intravenous injection or direct injection into the target tissue. For systemic application, intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral or intrathecal routes are preferred. More localized application may be performed subcutaneously, intradermally, intradermally, intracardially, intralobarly, intramedullary, intrapulmonary, or directly in or near the tissue to be treated (connective tissue, bone tissue, muscle tissue, nerve tissue, epithelial tissue). Depending on the desired duration and effectiveness of the treatment, the composition according to the present disclosure may be administered once or several times, and also intermittently, for example daily for several days, weeks, or months, in various doses.

[0232] Example 1

[0233] This example develops a qPCR for the specific detection of the PCV3 capsid gene.

[0234] Viral DNA was isolated from clinical specimens using the MagMax-96 Total Nucleic Acid Isolation Kit according to the manufacturer's protocol. Nucleic acid from formalin-fixed paraffin-embedded tissues was extracted with the QIAamp DNA FFPE Tissue Kit as instructed by the manufacturer (Qiagen, Valencia, CA, USA). A 5' nuclease assay was designed to target a 112 bp region of PCV3 cap gene nucleic acid in the samples: probe, 5'-FAM-ACC CCA TGG-Zen-CTC AAC ACA TAT GAC C-Iowa Black-3' (SEQ ID NO: 14), forward, 5'-AGT GCT CCC CAT TGA ACG-3' (SEQ ID NO: 13), reverse, 5'-ACA CAG CCG TTA CTT CAC-3' (SEQ ID NO: 12). Quantitative PCR was performed using the Qiagen Quantitect PCR kit under the following conditions: 95°C, 15 min; 45 cycles of 94°C, 15 s and 60°C, 60 s. The sensitivity and specificity of the assay were determined using a dilution series of a plasmid containing the entire PCV3 cap gene cloned into pSF-CMV-AMP (Oxford Genetics, UK) and PCV2 nucleic acid extracted from cultured cells (pSF-CMV-cap). The cycle threshold values ​​(Ct values) determined from the plasmid dilution series were used to generate a standard curve for calculation to determine genome copies / ml (gc / ml).

[0235] The complete genome of PCV3 was determined from fetal tissue homogenate pools from PDNS outbreak farms and samples from prevalence studies using Sanger sequencing of four overlapping amplicons generated with primers shown in Table 1. PCR was performed using TaKaRa Taq® as follows: 94°C, 4 min; followed by 40 cycles of 94°C, 20 s; 50°C, 30 s; 72°C, 1 min; and 72°C, 5 min. Sequencing to confirm selected PCV3 positive samples was performed using a 330 bp internal cap gene primer set: 5'-CCA CAG AAG GCG CTA TGT C-3' (SEQ ID NO: 16) and 5'-CCG CAT AAGGGT CGT CTT G-3' (SEQ ID NO: 17). The cap gene PCR reaction was performed using TaKaRa Taq® as outlined above. PCR products were Sanger sequenced for validation.

[0236] Example 2

[0237] In this example, a virus was isolated.

[0238] Virus isolation was attempted on porcine testis (ST) and porcine kidney (PK-15) cells maintained in minimum essential medium (MEM) supplemented with L-glutamine and 5% fetal bovine serum. Cells were seeded onto 6-well plates (60-80% confluent) and 100 μl samples were placed into 1 ml of virus exchange medium consisting of MEM and penicillin-streptomycin solution. Cells were observed daily for cytopathic effects and PCV3 growth was monitored by quantitative PCR and immunofluorescence.

[0239] Example 3

[0240] This example demonstrates the cloning, expression, and purification of the PCV3 capsid protein.

[0241] To generate the recombinant PCV3 cap construct, primers were designed to amplify a portion of the PCV3 gene encoding amino acids (aa) 35-214: forward, 5'-AAA AAA GCT AGC GCT GGA ACA TAC TAC ACA-3' (SEQ ID NO: 18); reverse, 5'-AAA AAA GAA TTC TTA GAG AAC GGA CTT GTA ACG-3' (SEQ ID NO: 19). The 5' ends of the forward and reverse primers contained an NheI restriction site (GCT AGC) and an EcoRI restriction site (GAA TTC), respectively. The PCR product was cloned into the pET28a (Novagen, Madison, WI, USA) vector to allow expression of the N-terminally truncated PCV3 cap as an N-terminal 6x His fusion protein in E. coli. The pET28a cap plasmid was transformed into E. coli BL21(DE3) cells and expressed as previously described. After expression, bacteria were harvested by centrifugation and lysed using B-PER reagent (Pierce, Rockford, IL, USA) followed by purification on Ni-NTA agarose (Qiagen, Valencia, CA, USA) as per the manufacturer's instructions. Purity and identity of the recombinant proteins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions and by Western blot detecting the His tag at the N-terminus of the recombinant proteins.

[0242] Example 4

[0243] This example demonstrates the production and in vitro characterization of anti-PCV3 capsid monoclonal antibodies.

[0244] BALB / c mice were immunized with purified truncated capsid protein (35-214aa). Mice were inoculated with 50 μg of antigen mixed with Freund's incomplete adjuvant once every 2 weeks for a total of 8 weeks. Subsequently, mouse splenocytes were fused with NS-1 myeloma cells. Monoclonal antibodies (MAbs) specific for PCV3 were identified by immunofluorescence antibody assay (IFA) using HEK293 cells expressing native PCV3 cap. HEK293 cells on 6-well plates maintained in MEM containing 5% fetal bovine serum (FBS) and antibiotics (ciprofloxacin, penicillin, streptomycin, and gentamicin) were transfected with pSF-CMV-AMP or pSF-CMV-AMP-derived plasmids containing the complete PCV3 cap gene cloned into pSF-XhoI restriction site (pSF-CMV-cap). On 6-well plates, 10 6 Cells were transfected with Lipofectamine® 2000 (Invitrogen) according to the manufacturer's instructions and 10 μg of DNA. After 48 h, plates were fixed with 80% acetone for 10 min at room temperature (RT) and dried. Transfected cells were incubated with undiluted anti-PCV3 cap MAb for 1 h at 37°C, followed by three washes with phosphate-buffered saline (PBS) and incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA) diluted 1:100 in PBS for 1 h at 37°C. After the final wash, cells were visualized using an Eclipse TE2000-U inverted fluorescence microscope (Nikon). PCV2-infected ST cells were used to assess the specificity of PCV3 MAb.

[0245] Example 5

[0246] This example demonstrates PCV3 detection in PDV cases and immunohistochemistry.

[0247] Tissues were fixed in 10% neutral buffered formalin at room temperature and tissue sections were embedded in paraffin until sectioning. Immunohistochemistry was performed via standard protocols and slides were visualized with an LSM700 confocal scanning microscope (Zeiss).

[0248] Example 6

[0249] This example demonstrates the development of a recombinant PCV3 capsid ELISA.

[0250] ELISA was performed similarly to the assay described above using 2 μg / ml of purified recombinant PCV3 capsid protein per well to coat Corning EIA / RIA high binding plates (33-35).

[0251] Results of Examples 1-6

[0252] The complete genome sequences of PCV3 used for phylogenetic analysis were submitted to the gene bank under accession numbers KT869077 (PCV3 strain 29160) and KX458235 (PCV3 strain 2164). Already published sequences used for phylogenetic analysis include: Barbel Circovirus (BarCV) GU799606, Bat Circovirus-1 (BtCV-1) JX863737, Bat Circovirus-2 (BtCV-2) KC339249, Bat Circovirus-3 (BtCV-3) JQ814849, Beak and Feather Disease Virus (BFDV) AF080560, Canary Circovirus (CaCV) AJ301633, Canine Circovirus (CanineCV) KC241983, Chimpanzee Fecal Associated Circovirus (CHFACV) GQ404851, Duck Circovirus (DuCV) AY228555, European catfish circovirus (EcatfishCV) JQ011377, Finch circovirus (FiCV) DQ845075, Goose circovirus (GoCV) AJ304456, Gull circovirus (GuCV) DQ845074, Human fecal-associated circovirus (HufaCV) GQ404856, Mink circovirus (MiCV) KJ020099, Pigeon circovirus (PiCV) AF252610, Porcine circovirus 1 (PCV1) Y09921, Porcine circovirus 2 (PCV2) AF027217, PorkNW2 / USA / 2009 HQ738638, Raven Circovirus (RaCV) DQ146997, Starling Circovirus (StCV) DQ172906, Swan Circovirus (SwCV) EU056309, and Zebra Finch Circovirus (ZfiCV) KP793918.

[0253] Clinical and histological findings of an outbreak of PDNS-like disease in a commercial sow farm

[0254] Impact on commercial sows in June 2015 resulted in a 10.2% increase in mortality and a 0.6% decrease in conception rate compared to the historical farm average due to PDNS outbreaks. Clinically, affected sows were anorexic and presented with multifocal papular, macular and superficial dermatitis. Tissue samples were submitted to ISUVDL for diagnostic testing. Histologically, skin lesions were characterized by acute necrotizing dermatitis and epidermitis associated with lymphoplasmacytic perivascular cell infiltrates. Kidneys showed dilated cortical ducts, attenuation and regeneration of tubular epithelium, and large clusters of lymphocytes and macrophages diffusely infiltrating the cortical interstitium and glomeruli. The farm experienced an increase in mummified aborted fetuses per liter above the historical average abortion rate, at 1.19. Aborted fetuses included mummified fetuses of various gestational ages consistent with previously described PCV2-associated abortions. Although the gross and histological lesions and the presence of abortions observed in the sows were consistent with PCVAD, all sow tissues, including kidneys, lymph nodes, lungs and skin, were negative for PCV2, PRRSV and IAV when tested by IHC and qPCR, and fetal tissues were negative for PCV2, PRRSV and PPV when tested by qPCR.

[0255] Metagenome sequencing

[0256] Tissue homogenate pools prepared from three fetuses were analyzed by viral metagenomic sequencing. The Miseq run generated 989,478 total reads, with 926,380 mapping to the host genome, i.e., boar. The remaining reads were de novo assembled, yielding 27 contigs. When analyzed by BLASTN analysis, approximately 54% of the reads mapped to a 1,246 bp contig, which was 98% similar to PorkNW2 / USA / 2009 (accession no. HQ738638), a partial circovirus genome identified in commercial ground pork. The remaining reads did not show similarity to any known eukaryotic viruses.

[0257] Metagenomic sequencing was also performed on tissue homogenates from a sow with PDNS-like lesions. De novo assembly of sequences not mapping to the boar reference sequence yielded 735 contigs that were analyzed by BLASTN analysis. Two contigs had approximately 97% identity to Torque Teno Virus 1 (TTV-1). Assembly with the PCV3 reference sequence (KT869077) identified four reads that mapped to the genome. The remaining reads showed no homology to known eukaryotic viruses.

[0258] Genetic analysis

[0259] After rolling circle amplification, PCR and Sanger sequencing of the resulting amplification products (amplicons) allowed the assembly of a 2,000 nucleotide (nt) circular genome from fetal tissue homogenates. ORF analysis identified three ORFs encoding proteins of more than 200 amino acids (aa), with two ORFs showing homology to circovirus rep and cap proteins oriented in opposite directions by BLASTP (Figure 1). Within the 235 nt 5'-intergenic region between the rep and cap ORFs on the rep gene strand, there was a predicted stem-loop structure with a 9 nt stem and loop nonamer identical to PCV1 (TAGTATTAC) (SEQ ID NO: 15). By convention, the "A" residue at position 8 in the loop nonamer was defined as position "1" in the genome.

[0260] The largest ORF, encoding a 297aa protein predicted by BLASTP, was 69.4% identical to the partial replicase protein of Circoviridae PorkNW2 / USA / 2009 (accession no. ADU77001, 221aa) and 54% identical to a bat circovirus from China (accession no. AIF76248, 293aa). The PorkNW2 / USA / 2009 genome was obtained from a commercial pork product and encodes a complete replicase and partial capsid gene that is most similar in organization and sequence to circoviruses. Conserved circovirus replicase and helicase domains were identified by BLASTP from aa 9-93 and 162-251 in the rep ORF, respectively. Further examination of the rep ORF protein sequence revealed a conserved rolling circle replication (RCR) motif and a P-loop motif similar to GoCV and PiCV. Of the three RCR motifs conserved among circoviruses, the FTLNN (SEQ ID NO:20) motif contained a single mutation present as FTINN (SEQ ID NO:21) in PCV3. This mutation is found in other circoviruses such as GoCV. Two other RCR motifs, HLQG (SEQ ID NO:22) and YCKK (SEQ ID NO:23), are also present in PCV3. In addition, three motifs conserved but with unknown function were identified in PCV3 and among circovirus replicase proteins, including WWDGY (amino acids 196-200) (SEQ ID NO:24), DDFYGWVP (amino acids 209-216) (SEQ ID NO:25), and DRYP (amino acids 225-228) (SEQ ID NO:26). Interestingly, no canonical start codon was identified. At the 5' end of the ORF with the nearest in-frame ATG (SEQ ID NO:28) present approximately 400 bp downstream, there is a GTC (SEQ ID NO:27) codon (encoding valine). This alternative start codon was also found in PorkNW2 / USA / 2009. Alternative start codons have been proposed for several avian circoviruses, including goose circovirus, pigeon circovirus, and beak and feather disease virus.

[0261] The putative cap ORF, in the opposite orientation of rep, encodes a 214 aa protein that is 87% identical to the partial capsid sequence of PorkNW2 / USA / 2009 (110 aa) and 36-37% identical to PCV2 and duck circovirus (233 and 257 aa, respectively) by BLASTP (Figure 1). Similar to other circovirus capsid proteins, the N-terminus was highly basic, containing many arginine residues. A conserved circovirus capsid domain was identified by BLASTP from aa 26-173. Furthermore, the PCV3 cap protein had no predicted N-linked glycosylation sites and two predicted O-linked glycosylation sites, at aa 146 and 150 (S and T, respectively). This is in contrast to PCV2, which has two experimentally verified N-linked glycosylation sites.

[0262] A third ORF, oriented on the same strand as the predicted rep, encodes a 231 aa protein, a protein of unknown function, 94% identical to an ORF identified as PorkNW2 / USA / 2009 and 39% identical to Murid herpesvirus M169. As with rep, the start codon for ORF3 is unknown. The 5' terminal codon is TCG (encoding serine). A methionine at ORF3 aa55 is another possible start site that would give rise to a 177 aa protein.

[0263] Due to genetic and structural similarities to the genus Circovirus and less than 70% capsid aa identity to other species, the novel species has been proposed as porcine circovirus 3 (PCV3). The PCV3 genome sequence has been submitted to the gene bank under accession numbers KT869077 and KX458235.

[0264] Phylogenetic analysis

[0265] To investigate the evolutionary relationship of PCV3 to other members of the Circoviridae family, we analyzed genome sequences from 23 members of the Circoviridae family and two PCV3 genomes. Analysis of the complete circovirus genome grouped both PCV3 sequences in a separate clade with all other members of the genus PorkNW2 / USA / 2009 (Figure 2). The phylogeny showed that PCV3 is most closely related to canine circovirus (KC241983), but this relationship lacked strong bootstrap support. The phylogeny also suggests that PCV3 and canineCV share a common ancestor with a clade that includes PCV1, PCV2, and BatCV-2 (KC339249). With the exception of human circovirus (HufaCV, GQ404856), mammalian and avian circoviruses belonged to separate, well-supported clades.

[0266] Detection of PCV3 by PCR

[0267] To confirm the presence of PCV3 in porcine samples, a 5'-nuclease assay was designed to determine the presence of the PCV3 cap gene. Fetal tissue homogenate samples from the development farm were strongly positive for PCV3 with Ct values ​​of 16.7-21.3, approximately 1.88 × 10 8 and 7.55×10 6 Tissues from three sows with PDNS-like lesions corresponded to high levels of PCV3 (2.13 × 10 4 -8.62×10 4 The serum samples were positive for PCV3 in 5.63 × 10 2 -2.28×10 4All samples were positive for PCV3 at 1000 mg / ml. The serum sample with the highest PCV3 titer was amplified by PCR to generate overlap amplicons and obtain a second complete PCV3 genome, which was 99.0% identical to the PCV3 from the outbreak farm. This second PCV3 genome was submitted to the gene bank under the accession number KX458235.

[0268] Furthermore, to investigate the prevalence of PCV3, a total of 271 samples submitted to ISUVDL for respiratory disease diagnostic testing were analyzed by qPCR. Thirty-four of the samples (12.5%) had a PCV3 prevalence of 3.00 × 10 2 -1.52×10 7 The gc / ml titer was positive.

[0269] Characterization of PCV3capMAb14

[0270] HEK293 cells transfected with pSF-CMV-cap were separately incubated with cell supernatants from the four different MAb clones and screened by IFA. Nuclear-localized fluorescence was observed for clone 14 (MAb14), as expected due to the predicted strongly basic nuclear localization signal. No fluorescence was observed for cells transfected with pSF-CMV-Amp. Furthermore, PCV2-infected ST cells had no detectable fluorescence.

[0271] Virus isolation

[0272] Virus isolation was attempted in ST and PK-15 cells. Cells were inoculated with filtered fetal tissue homogenates and passaged three times. No cytopathic effect was evident, and Ct values ​​increased with each successive passage. No fluorescence was observed by IFA using MAb14.

[0273] Histopathological lesions associated with the presence of PCV3 antigen in PDNS cases

[0274] Tissue samples from sows with PDNS-like lesions and archived PDNS cases were examined by H&E staining and IHC with PCV3 MAb14. Lungs showed variable degrees of bronchointerstitial pneumonia occasionally complicated by secondary suppurative bronchopneumonia. Small and medium-sized airways and small vessels were cuffed by peribronchial and perivascular aggregates of lymphocytes and plasma cells. Adjacent alveolar septa were infiltrated with lymphocytes and plasma cells. Within the alveolar spaces, there was abundant intraluminal edema admixed with modest numbers of foamy macrophages, rare multinucleated giant cells, and small clusters of neutrophils. Occasionally, lymphocytes and scattered macrophages showed moderate intracytoplasmic immunostaining for PCV3. In skin sections, the dermis and subcutaneous tissues had characteristic necrotizing vasculitis with fibrinoid degeneration and transmural neutrophilic infiltrates, hemorrhage, and fibrin exudation. The inflammatory infiltrate often extended into the surrounding dermis and subcutis. There were also scattered lymphoplasmocytic aggregates with occasional cuffing around blood vessels and dermal adnexa. In some cases, the epidermis was hyperplastic with mildly normokerotic hyperkeratotic changes. The dermal lymphocytic infiltrate showed prominent intracytoplasmic immunostaining for PCV3. Minimal background staining was evident when PCV3 MAb14 was replaced with PBS.

[0275] Lymph nodes showed diffuse granulomatous lymphadenitis. Cortical follicles had moderate lymphoid depletion and were infiltrated with histiocytes and numerous multinucleated giant cells. Subdermal and perifollicular lymphocyte populations were expanded with moderate amounts of edema and hemorrhage and admixed with numerous macrophages and plasma cells. Follicular and perifollicular lymphocyte populations showed diffuse dense intracytoplasmic staining for PCV3 compared to background staining when PCV3 MAb14 was substituted with PBS or lymph node tissue from PCV3 qPCR-negative pigs.

[0276] Renal sections were characterized by the presence of diffuse membranoproliferative glomerulonephritis. Severe glomerulosclerosis was present, with Bowman's capsule often thickened, cortical tubules attenuated, and associated with variable interstitial fibrosis. The glomerular mesangium was hypercellular and thickened by amorphous eosinophilic material. The tubules were sometimes dilated, lined by attenuated epithelium, and sometimes exhibited prominent proteinosis. Scattered throughout the sections were small- to medium-sized clusters of lymphocytes and plasma cells in the interstitium. The tubular epithelium showed random areas of positive staining for PCV3. Minimal background fluorescence was observed for slides mock-stained with PBS and goat anti-mouse FITC or kidney tissue from PCV3 qPCR-negative pigs.

[0277] Detection of PCV3 nucleic acid in tissues with PDNS lesions

[0278] To further investigate the pathogenic role of PCV3 in PDNS, we evaluated 48 cases with histologic lesions consistent with PDNS that were already negative for PCV2 by IHC. Tissue scrolls from paraffin-embedded tissue blocks were assayed for PCV3 by qPCR. Viral titers of 1.60-3.47 × 10 4 There were 45 (93.8%) PCV3 positive cases with a titer of 0.01 gc / ml. To confirm these results, the five samples with the highest viral titers were analyzed by PCR targeting a 330 bp fragment of the cap gene. The target amplicon was amplified in all samples evaluated, and the amplicon product sequence showed 100% identity with the PCV3 genome sequence. Tissues from the five PCV3 PCR positive cases were tested by PCV3-IHC. Three of the five were positive.

[0279] PCV3 seroprevalence

[0280] The prevalence of anti-PCV3cap antibodies in swine sera was investigated by ELISA using rPCV3cap antigen. Eighteen serum samples from 3-week-old pigs from specific pathogen-free herds with negative PCV3 qPCR test results were used as negative controls. The mean absorbance was 0.49. The cutoff value for distinguishing positive and negative sera was determined to be 3 standard deviations above the mean of the negative controls (0.87). All sera from 10 sows from the outbreak farm collected during the 3 months after the PDNS outbreak were positive, with a mean absorbance of 1.27. In addition, 27 sera from sows from farms supplying mating animals to the sow farm were also tested, with 17 animals (63%) having absorbances between 0.88 and 1.37. Anti-PCV3cap antibodies were detected in 47 (56.6%) of 83 samples submitted for unrelated diagnostic testing from multiple states. Of the positive samples, 13 were from Iowa, 1 from Indiana, 5 from Mexico, 4 from North Carolina, 5 from Nebraska, 1 from Oklahoma, and 18 from unknown origin.

[0281] Discussion

[0282] First described in 1993 in Europe, PDNS has been reported in many countries worldwide. Although the prevalence of disease within herds is typically low (<1%), mortality in affected pigs can be high. The incidence of PDNS may exceed that of PMWS in Europe and the UK. The etiology of PDNS is unknown, but PCV2 nucleic acid is commonly detected in affected pigs using qPCR, whereas PCV2 antigen is not consistently detected. This has led to speculation regarding the role of PCV2 in PDNS. Here, a highly distinct novel porcine circovirus, designated PCV3, was identified in a mummified fetus following abortion from a sow with PDNS-like lesions, and in a sow that died suddenly with clinical signs consistent with PDNS. PCV3 was the only virus identified by sequencing of pooled fetal tissue and confirmed by qPCR. A cycle threshold of 16.7-21.3 in pooled fetal tissues was detected in 7.55 × 10 samples. 6-1.8×10 8 They show high viral titers of 100 gc / ml. There is a correlation between PCV2 titers in the fetus and reproductive disease, with PCV2 levels of 107 PCV2 DNA copies / 500 ng fetal tissue, or PCV2 levels more associated with PCV2-associated reproductive failure, including mummification. Based on the amount and tissue distribution of PCV3 nucleic acid detected during this outbreak, a similar correlation exists for PCV3 and reproductive failure.

[0283] PCV3 was also detected by PCR and IHC in the skin, lungs, kidneys, and lymph nodes of sows with PDNS-like lesions. Both PCR, IHC, and metagenomic sequencing failed to confirm PCV2 in samples recovered from outbreak farms. These results indicate that PCV3 infection contributes to PDNS-like lesions and that abortion and the presence of PCV3 in fetuses are the result of vertical transmission. In support of this etiologic role for PCV3 in PDNS lesions, screening of archived PCV2 IHC-negative PDNS cases found PCV3 nucleic acid to be highly prevalent (93.8%), with 3 of 5 cases investigated for PCV3 by IHC being positive. It is noted that the relatively low titers of PCV3 in tissues may limit consistent detection.

[0284] Although attempts to experimentally reproduce PDNS with PCV2 have been unsuccessful, PDNS was experimentally reproduced in the absence of PCV2 using tissue homogenates containing PRRSV and TTV. Little is known about the clinical significance of TTV infection. TTV is ubiquitous in pigs worldwide. Although TTV is commonly detected in healthy pigs, some studies suggest that TTV infection mitigates disease severity during coinfection. For example, inoculation of pigs with tissue homogenates containing TTV followed by PCV2 resulted in PMWS, whereas monoinfection did not result in PMWS. Sows from a PDNS-affected farm were infected with both PCV3 and TTV1. The effect of TTV1 coinfection is unknown. It is unknown whether coinfection with genetically diverse small circular DNA viruses such as TTV1 (Anelloviridae), PCV2 and PCV3 affects the development of PDNS. For PMWS, an additive effect of coinfection with PCV2 on disease severity has been demonstrated. Co-infection with PPV and PCV2 has been shown to exacerbate disease. Co-infection with PCV2 and PRRSV is a key component of the swine respiratory disease complex. Infection of herds with these agents causes severe respiratory disease and economically impactful sow abortions and deaths.

[0285] The pathogenesis of PDNS, including the characteristic necrotizing vasculitis, is thought to be a manifestation of immune complex-mediated disease involving PCV2. In a case-control study, all pigs with clinical signs of PDNS were PCV2-PCR positive and had significantly higher PCV2 antibody titers than clinically normal pigs. Examination of the kidneys of PDNS-positive pigs found increased fibrinoid deposits in the glomeruli consisting of accumulations of IgG1, IgG2, IgM, and complement factors C1q and C3 compared with clinically normal pigs. PCV2 antigen was identified in the lung tissue of these PDNS pigs by IHC, but PCV2 antigen was not identified in immune complexes. This result is similar to the inconsistent detection of PCV2 in kidney tissue of PDNS pigs reported by other studies. It is known that viral infection contributes to an immunological disease with a similar pathogenesis to PDNS, Aleutian mink disease (AMD), caused by the Aleutian mink disease virus (AMDV). The pathogenesis of AMD is associated with the overproduction of AMDV-specific IgG antibodies. The role of PCV3 in immune complex-mediated diseases that may result in the pathogenesis of PDNS requires further investigation.

[0286] PCV2 is one of the most economically important swine viral pathogens worldwide. The most common genotypes associated with PCVAD are PCV2a and PCV2b. Prior to 2003, PCV2a was the predominant genotype identified in the United States and Canada, but both PCV2a and PCV2b have been found internationally. Around 2003, a dramatic shift in the frequency of PCV2 genotypes from PCV2a to PCV2b occurred worldwide, concomitant with severe systemic disease associated with PCV2b. The development of commercial vaccines containing PCV2a antigens, which have been shown to be cross-protective, has led to successful containment of the global epidemic. More recently, a new genotype, PCV2d, was first detected in Switzerland in 1999 and spread to China and then to the United States. Similar to that seen with PCV2b, epidemiological studies suggest that genotypic shifts are ongoing, resulting in reported PCV2 vaccine failures and increased clinical disease. PCV2d has been implicated in more severe clinical signs and lesions.

[0287] Circoviruses are genetically diverse, infect a wide range of hosts, and cross-species transmission has been reported. Phylogenetic analysis suggests the closest evolutionary relationship between PCV3 and CanineCV. Interestingly, CanineCV was identified in the liver of dogs exhibiting necrotizing vasculitis and granulomatous lymphadenitis, both of which were observed in sows infected with PCV3 and also reported in PCV2-infected pigs. It is unclear whether PCV3 has been evolving in pigs since it was detected some time ago, or whether it arose via cross-species transmission or recombination between unidentified parent circoviruses. PCV2 can cross species barriers and cause fatal disease in species other than pigs. A recent report identified PCV2 in six mink that died of diarrhea in China.

[0288] The discovery of a novel porcine circovirus that may be the etiological agent in PDNS and reproductive failure is surprising. Retrospective studies suggest that PCV2 sporadically caused systemic disease as early as 1985, before becoming epidemic in the late 1990s. The possibility that PCV3 is on a similar trajectory merits further study and production of the immunological composition of the present disclosure. Importantly, given the approximately 30% identity between PCV2 and PCV3 capsid proteins, interference is unlikely to occur.

[0289] conclusion

[0290] Porcine circovirus 2 is one of the most important swine pathogens worldwide. Here, a highly distinct new species of porcine circovirus, PCV3, was identified in sows undergoing abortion and exhibiting PDNS, the latter being a clinical symptom typically associated with PCV2 infection. The PCV3 genome contains ORF1, ORF2, and ORF3. No other viruses were detected by metagenomic sequencing, and PCR was negative for PCV2, PPV, PRRSV, and IAV. This, coupled with the high viral load in mummified tissues, suggests that PCV3 may cause clinical disease similar to PCV2. Molecular and serological assays for PCV3 also suggest that the virus is commonly circulating among American swine producers. Further study of PCV3 is warranted, as PCV2 infection was initially asymptomatic and only caused sporadically disease before becoming a global epidemic.

[0291] The teachings and contents of the following references are incorporated herein by reference:

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Claims

1. A method for producing a PCV3 protein, comprising: Transforming a host cell with an expression vector enabling expression of a PCV3 protein, The vector comprises a heterologous nucleotide sequence encoding a protein, The method, wherein the protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

6.

2. The method described in claim 1, wherein the protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

6.

3. A method for producing a PCV3 protein, comprising: Transforming a host cell with an expression vector enabling expression of a PCV3 protein, The method of claim 1, wherein the vector comprises a heterologous nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

5.

4. The method described in claim 3, wherein the heterologous nucleotide sequence has at least 95% sequence identity to SEQ ID NO:

5.

5. A method for producing a PCV3 protein, comprising: Transforming a host cell with an expression vector enabling expression of a PCV3 protein, The method of claim 1, wherein the vector comprises a heterologous nucleotide sequence comprising a heterologous RNA sequence corresponding to a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

5.

6. The method described in claim 1, 3 or 5, wherein the vector is a viral vector or a plasmid.

7. The method described in claim 6, wherein the vector is a viral vector.

8. The method described in claim 1 or 3, wherein the vector is a baculovirus vector.

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