Fusion peptides and vaccines

Abstract

The present invention relates to novel immunogenic polypeptides and their use in vaccine compositions. The present invention also relates to nucleic acids, vectors and cells expressing said polypeptides and their use. More specifically, the polypeptides of the present invention comprise an immunogenic domain and a cell membrane addressing domain derived from the B5R gene. The present invention is particularly suitable for producing vaccines for non-human animals, in particular for vaccinating pigs against PCV2 infection.

Description

Technical Field

[0001] The present invention relates to novel immunogenic polypeptides and their use in vaccine compositions. The present invention also relates to nucleic acids, vectors and cells expressing the polypeptides and their use. More specifically, the polypeptides of the present invention comprise an immunogenic domain and a cell membrane addressing domain derived from the B5R gene. The present invention is particularly suitable for producing vaccines for non-human animals, in particular for vaccinating pigs against PCV2 infection. Background Art

[0002] Porcine circovirus (PCV) was initially identified as a contaminant of porcine kidney cell culture (PK15ATCC CCL-33). PCV virions have been characterized as icosahedral non-enveloped viruses with single-stranded circular DNA of about 1.76 kb. PCV is classified in the genus Circovirus of the Circoviridae family, which is also composed of other animal circoviruses, such as psittacine beak-feather disease virus, goose circovirus, canary circovirus and pigeon circovirus. Two PCV genotypes have been identified. The PCV of PK15 cell origin has been considered to be non-pathogenic for pigs and has been designated as PCV type 1 (PCV1). On the other hand, it is recognized that PCV type 2 (PCV2) is the main infectious agent involved in several pig diseases. PCV2-related diseases have caused significant economic losses to pig producers around the world. PCV2-related diseases are described in WO2007 / 076520 and include, for example, Postweaning Multisystemic Wasting Syndrome (PMWS), Porcine Dermatitis and Nephropathy Syndrome (PDNS), Porcine Respiratory Disease Syndrome (PRDC), reproductive failure, granulomatous enteritis, exudative epidermitis, necrotizing lymphadenitis, and congenital tremor. The emergence of PCV2 subtype A (PCV2A) and PCV2 subtype B (PCV2B) was specifically reported in Western Europe in 2000 and in Central Europe in 2003. Similar changes were recently reported in wild boars in 2008.

[0003] Currently developed PCV2 vaccines (such as (Merial), CircoFLEX (Boehringer lngelheim Vetmedica) or ) are either inactivated PCV2 vaccines or subunit vaccines. Regarding inactivated PCV2 vaccines, current PCV2 strains of subtype A or subtype B have some disadvantages. In particular, PCV2 viruses can only be produced at low titers, usually less than 10 5 TCID50 virus particles / ml. In addition, these viruses cannot be maintained in tissue culture and permanently infected cell lines. Regarding PCV2 subunit vaccines, they usually use purified recombinant PCV2 capsid protein, which is produced by expressing the ORF2 gene of PCV2 in a baculovirus system. In this regard, the protein encoded by ORF2 of PCV2 isolate Imp1011 has been reported in EP1741785. The protein encoded by ORF2 of PCV2 isolate PCV2Rm has been reported in WO2010 / 061000. The protein encoded by ORF2 of PCV2 isolate 412 has been reported in EP1816200. Another protein encoded by ORF2 of another PCV2 isolate has been reported in EP1036180 or EP2225367.

[0004] However, these natural capsid proteins have poor expression efficiency and immunogenicity and do not always provide the desired level of immune protection in vaccinated animals. In particular, the ORF2 protein contains a nuclear localization sequence, resulting in protein expression in the nucleus of cells. Such intracellular localization is not convenient for the extraction or purification of the protein and may also prevent or reduce the effectiveness of DNA vaccines or vector vaccines expressing the ORF2 protein in animals.

[0005] WO2010 / 068969 proposes to improve the expression profile of ORF2 by using an external secretion signal peptide sequence to express the ORF2 antigen in a soluble form. In this application, it is proposed to fuse ORF2 with a secretion signal or with a cell membrane signal, and to include a cleavage site in the construct so that the soluble ORF2 can be released in a soluble form. The application proposes a long prophetic list of possible candidate secretory peptides. However, the application does not contain any experimental data indicating that effective or improved expression / immunogenicity can be obtained by improving the expression profile of ORF2. There is no disclosure of a construct that allows effective immunization.

[0006] The present invention proposes new and improved constructs for expressing antigenic polypeptides. The present invention discloses fusion products specifically suitable for improved expression of antigenic polypeptides on the cell surface, especially improved expression on the cell surface using viral expression vectors (such as swinepox virus).

[0007] The present invention shows that by expressing antigenic polypeptides on the surface of infected / transduced cells using B5R-derived addressing signals, an improved immune response is achieved, resulting in effective protection. By presenting antigens on the cell surface in an animal, the vaccine of the present invention most effectively delivers and exposes antigens to the immune system, particularly to immune cells (such as lymphocytes, dendritic cells and macrophages). By presenting antigens on the cell surface, the present invention provides immunogens in an active conformation, thereby eliciting a strong protective immune response. The present invention can be applied to any antigenic polypeptide, particularly viral antigens. Summary of the invention

[0008] The present application provides a polypeptide comprising a signal peptide operably linked to a heterologous antigenic polypeptide, wherein the signal peptide is derived from the B5R gene of vaccinia virus.

[0009] In a specific embodiment, the signal peptide comprises SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1. The antigenic polypeptide may be a viral antigen, a bacterial antigen or a parasite antigen. The antigenic polypeptide is preferably a viral antigen, more particularly, the antigenic polypeptide is a capsid protein or an immunogenic domain thereof.

[0010] In a specific embodiment, the antigenic polypeptide is ORF2 of PCV2 virus or its antigenic domain. A preferred embodiment uses an antigenic polypeptide containing the sequence of SEQ ID NO: 3 or a sequence having at least 80% identity with SEQ ID NO: 3.

[0011] The polypeptides of the invention may be synthetic or recombinant and may contain post-transcriptional modifications (eg, glycosylation), added chemical groups, etc. Most preferably, the polypeptides of the invention have no cleavage site between the addressing peptide and the antigenic peptide.

[0012] Another object of the invention is a cell expressing on its surface a polypeptide as defined above.

[0013] Another object of the invention relates to a nucleic acid encoding a polypeptide as defined above.

[0014] Another object of the present invention is a vector containing the nucleic acid of the present invention. The vector is preferably a viral vector, for example, most preferably pseudorabies virus (PRV) or swine pox virus (SPV). The use of swine pox is particularly advantageous because the B5R signal peptide derived from vaccinia virus has improved compatibility with swine pox.

[0015] Another object of the present invention is a composition comprising a polypeptide, a cell, a nucleic acid or a vector as defined above.

[0016] Another object of the invention is a vaccine comprising a polypeptide, cell, nucleic acid or vector as defined above and optionally an adjuvant.

[0017] The present invention also relates to a method of immunizing or inducing an immune response in a non-human animal (eg, a pig), the method comprising administering to the animal a polypeptide, nucleic acid, cell, vector or vaccine as described above.

[0018] The present invention also relates to a method for treating and / or preventing a PCV2-related disease in a non-human animal (eg, a pig), the method comprising administering to the animal a polypeptide, nucleic acid, cell, vector or vaccine as described above.

[0019] The present invention also relates to the use of a signal peptide derived from the B5R gene of vaccinia virus for expressing a heterologous antigenic polypeptide in a cell.

[0020] The present invention can be used to induce an immune response and / or vaccinate any non-human animal. It is particularly useful for vaccinating pigs against PCV2 infection or PCV2 disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :Construction scheme of homology plasmid pSP72-Ess_ORF2.

[0022] Figure 2 : Black plaque analysis of rSPV (SVR3, SVR7) using monoclonal antibody 36F1. ESK-4 cells were infected with parental SPV or recombinants (SVR3 or SVR7). Six days later, cells were analyzed for black plaques using monoclonal antibody 36F1 (1:500), biotin-conjugated rabbit anti-mouse IgG and ABC-ALP (Vecterstain).

[0023] Figure 3 : IFA of SPV-infected cells. ESK-4 cells were infected with rSPV (SVR3 or SVR7) or parental SPV. Five days later, the infected cells were treated with acetone / methanol (a) or without (b) and reacted with primary antibodies [rabbit anti-β-galactosidase (α-βGal) and mouse anti-PCV2-ORF2 (α-ORF2) (1:1000)] and secondary antibodies [goat anti-rabbit IgG Alexa Fluor 488 and anti-rat IgG Alexa Fluor 546 (1:1000)].

[0024] Figure 4: Serological IF titers against PCV2. RPL-2 cells infected with Rm40 were plated on each well of a 96-well plate. The cells were fixed with acetone / methanol and 2-fold dilutions of the sera were incubated for 1 hr after blocking (0.5% skim milk powder in PBS). Anti-swine IgG-FITC antibody produced in rabbit (SIGMA catalog number: F1638, 1:1000) was used as a secondary antibody. The IF titer of the standard positive porcine serum PAB-PCV2 (VMRD) was 2560. DETAILED DESCRIPTION

[0025] The present invention relates to novel immunogenic polypeptides and their uses, in particular in vaccine compositions. The present invention also relates to nucleic acids, vectors and cells expressing the polypeptides and their uses. More specifically, the polypeptides of the present invention comprise an operably linked immunogenic domain and a cell membrane addressing domain, wherein the cell membrane addressing domain is derived from the B5R gene. The present invention is particularly suitable for producing vaccines for non-human animals, in particular for vaccinating pigs against PCV2 infection.

[0026] Cell membrane-addressed peptide derived from B5R

[0027] The term "derived from" means that the sequence of the addressing peptide is identical or substantially similar to the signal peptide sequence of the B5R gene of vaccinia virus. In a preferred embodiment, the addressing peptide comprises a sequence having at least 90% identity, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the signal peptide sequence of the B5R gene of vaccinia virus.

[0028] The most preferred example of the signal peptide sequence of the B5R gene is SEQ ID NO: 1, or a sequence having at least 90% identity to SEQ ID NO: 1, more preferably a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. Therefore, in a specific embodiment, the polypeptide of the present invention comprises a signal peptide sequence, and the signal peptide comprises SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1.

[0029] The signal peptide may contain other amino acids derived from the B5R gene, as long as these amino acids do not change the membrane addressing properties of the peptide. In this regard, in a specific embodiment, the signal peptide comprises SEQ ID NO: 2. Amino acids 23-36 of SEQ ID NO: 2 are not substantially involved in the membrane addressing function. However, the results shown indicate that these amino acids contribute to the stability and correct conformation of the fusion polypeptide.

[0030] Alternatively or additionally, the signal peptide may comprise other amino acids not derived from the B5R gene, as long as these amino acids do not alter the membrane addressing properties of the peptide. These amino acids may have cloning utility (eg, restriction sites), or may be involved in the stability of the polypeptide.

[0031] The signal peptide preferably comprises no more than 100 amino acids, even more preferably no more than 50 amino acids.

[0032] As explained above, the present invention includes a signal peptide comprising a sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2. The degree of homology between two amino acid sequences or nucleic acid sequences can be determined with the aid of computer programs known per se in the art, for example, GAP (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) provided in the GCG program package (Needleman, SB and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453). DNA sequence comparisons were performed using GAP with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3. Nucleic acid molecules / amino acid molecules can be aligned to each other using the Pileup alignment software (available as part of the GCG program package) using default settings, for example, a gap creation penalty of 5 and a gap width penalty of 0.3.

[0033] Suitable experimental conditions for determining whether a given nucleic acid molecule hybridizes to a specified nucleic acid may include pre-soaking a filter containing a sample of the nucleic acid of interest to be examined in 5×SSC for 10 minutes, pre-hybridizing the filter in a solution of 5×SSC, 5×Denhardt's solution, 0.5% SDS, and 100 μg / ml of ultrasonically denatured salmon sperm DNA, followed by hybridization at about 45° C. for 12 hours in the same solution containing a probe labeled with P-dCTP at a concentration of 10 ng / ml, according to the hybridization method described by Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbour, New York). The filter is then washed twice (30 minutes) in 2×SSC (0.5% SDS) at at least 55° C. (low stringency), at least 60° C. (medium stringency), at least 65° C. (medium / high stringency), at least 70° C. (high stringency), or at least 75° C. (very high stringency). Hybridization can be detected by exposing the filter to x-ray film.

[0034] In a preferred embodiment, the signal peptide comprises SEQ ID NO:1 or SEQ ID NO:2.

[0035] In a specific embodiment, the signal peptide consists of SEQ ID NO:1 or SEQ ID NO:2.

[0036] Antigenic Peptide

[0037] The present invention can be used with any antigenic polypeptide, i.e., any polypeptide containing one or more epitopes capable of causing an immune response. The polypeptide can be a complete protein, a fragment of a protein, or a small peptide (e.g., a small peptide of 10 amino acids). Preferably, the antigenic polypeptide comprises less than about 500 amino acids.

[0038] The antigenic polypeptide is "heterologous" relative to the signal peptide, which means that the antigenic polypeptide and the signal peptide are not naturally associated in nature. Typically, the antigenic polypeptide is not a sequence of the B5R protein. The term "heterologous" indicates that the antigenic polypeptide is, for example, from a virus other than vaccinia virus, or, for example, from a protein other than the B5R protein.

[0039] The antigenic polypeptide may be of viral origin, cellular (eg, bacterial) origin, or parasitic origin.

[0040] In this regard, as explained above, in a preferred embodiment, the antigenic polypeptide is the ORF2 protein of the PCV2 virus or an antigenic domain thereof.

[0041] The ORF2 protein of PCV2 isolate Imp1011 has been reported in EP1741785. The ORF2 protein of PCV2 isolate PCV2Rm has been reported in WO2010 / 061000. The ORF2 protein of PCV2 isolate 412 has been reported in EP1816200. Another ORF2 protein of another PCV2 isolate has been reported in EP1036180 or EP2225367. All of these ORF2 proteins are contemplated for use in the present invention.

[0042] In a preferred embodiment, the ORF2 protein used in the present invention comprises SEQ ID NO:3, or any sequence having at least 80% identity to SEQ ID NO:3, even more preferably at least 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3.

[0043] Typically, the PCV2 ORF2 protein contains about 234 amino acids. This sequence contains a nuclear localization sequence, which typically corresponds to amino acids 1-42 of this sequence.

[0044] For the present invention, it is preferred to use a portion of the ORF2 protein lacking the native nuclear localization sequence (i.e., the sequence located at residues 1-42 of native ORF2), and preferably replace this sequence with a membrane addressing peptide. SEQ ID NO: 3 is the amino acid sequence of the ORF2 protein lacking the nuclear localization sequence.

[0045] Sequences identical to SEQ ID NO:3 and retaining PCV2 immunogenic activity can be artificially derived, or obtained from the PCV2 serotypes listed above or from another different serotype. Typically, the first 42 amino acids of the ORF2 protein are removed to inhibit the nuclear localization function.

[0046] As described, the present invention can be used with immunogenic polypeptides of other viral antigens or pathogenic antigens, for example, any protein (e.g., glycoprotein, capsid protein, or antigenic fragments thereof) of a virus or pathogen selected from, for example, Actinobacillus pleuropneunomia; adenovirus; Alphavirus, such as Eastern equine encephalomyelitisviruses; Balantidium coli; Bordetellabronchiseptica; Brachyspira spp., preferably B. hyodyentheriae, B. pilosicoli, B. innocens, Brucella suis, suis) (preferably biovars 1, 2 and 3); classical swine fever virus, African swine fever virus; Chlamydia / Chlamydophila sp., preferably C. pecorum and C. abortus; Clostridium spp., preferably Clostridium difficile, Clostridium perfringens types A, B and C, Cl. novyi, Cl. septicum, Cl. tetani; digestive and respiratory coronaviruses; Cryptosporidium parvum; Eimeria spp.; Eperythrozoonis suis, currently named Mycoplasma suis haemosuis; Erysipelothrixrhusiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtype 1, subtype 7 and subtype 14; Hemagglutinating encephalomyelitis virus; Isospora suis; Japanese encephalitis virus; Lawsonia intracellulars; Leptospira spp.), preferably Leptospira australis, Leptospira canicola, Leptospira typhoid, Leptospira icterohaemorrhagicae, Leptospira interrogans, Leptospira Pomona and Leptospira tarassovi; Mannheimia haemolytica; Mycobacterium spp., preferably Mycobacterium avium, Mycobacterium intracellular and Mycobacterium bovis; Mycoplasma hyponeumoniae; Parovirus; Pasteurella multocida multocida; porcine cytomegolovirus; porcine parvovirus, porcine reproductive and respiratory syndrome virus; pseudorabies virus; rotavirus; Sagiyama virus; Salmonella spp., preferably S. thyhimurium and S. choleraesuis; Staphylococcus spp., preferably S. hyicus; Streptococcus spp., preferably Streptococcus suis; porcine cytomegalovirus; porcine herpesvirus; swine influenza virus; swine poxvirus; Toxoplasma gondii; porcine vesicular stomatitis virus or virus of exanthema; or other isolates and subtypes of porcine circovirus.

[0047] Peptide assembly and production

[0048] The polypeptide of the present invention generally comprises a cell membrane addressing peptide operably linked to an antigenic polypeptide. The term "operably linked" indicates that two domains are fused to each other directly or indirectly in a manner that allows the polypeptide to be addressed to the cell membrane and to express the antigenic polypeptide outside the cell.

[0049] Most preferably, the cell membrane addressing peptide is located at the N-terminus and the immunogenic polypeptide is located at the C-terminus. The two domains are covalently linked, preferably via an amino bond. Therefore, the polypeptide of the present invention preferably comprises from the N-terminus to the C-terminus:

[0050] .Cell membrane addressing peptide,

[0051] .Immunogenic polypeptides.

[0052] The polypeptide may further comprise additional domains or sequences. For example, the polypeptide may comprise a linker sequence between the cell membrane addressing peptide and the immunogenic polypeptide. However, the two domains are preferably directly connected without a linker sequence. In addition, in a preferred embodiment, the polypeptide has no cleavage site, so that the immunogenic polypeptide is exposed on the cell surface and is substantially not released in a soluble form.

[0053] Preferred and specific examples of the polypeptide of the present invention comprise the amino acid sequence of SEQ ID NO:4.

[0054] The polypeptide of the present invention may further comprise an N-terminal methionine residue.

[0055] The polypeptides of the invention may be glycosylated.

[0056] The polypeptides of the present invention can be produced by synthetic methods or by recombinant means. Preferably, the polypeptides of the present invention are engineered to be directly produced / expressed in cells or whole organisms by expressing encoding nucleic acid molecules. In fact, the new expression profiles of the polypeptides of the present invention are particularly suitable for the design of DNA vaccines or vector vaccines, which contain nucleic acid molecules encoding the polypeptides of the present invention. After introduction into the organism, the nucleic acid enters the cell and expresses the polypeptide, which is exposed on the cell surface and exhibits improved immunogenicity. As will be discussed below, the nucleic acid can be naked nucleic acid, or formulated with any suitable carrier. Alternatively, the present invention can use a cell vaccine, wherein the polypeptide is expressed on the cell surface in culture, and the resulting cells are used as a vaccine composition.

[0057] Therefore, the present invention also encompasses and utilizes nucleic acid molecules encoding the polypeptides as defined above.

[0058] Nucleic Acids

[0059] Another object of the present invention relates to a nucleic acid molecule encoding a polypeptide as defined above. The nucleic acid can be used to produce the polypeptide in vitro, or to produce a cell expressing the polypeptide on its surface, or to produce a vaccine in which the active agent is the nucleic acid or a vector containing the nucleic acid.

[0060] The nucleic acid of the present invention may be single-stranded or double-stranded DNA or RNA. The nucleic acid is usually cDNA or RNA. The nucleic acid can be produced by techniques known in the art, such as synthesizing, cloning, or amplifying sequences encoding immunogenic polypeptides; synthesizing, cloning, or amplifying sequences encoding cell membrane addressing sequences; connecting the sequences and cloning / amplifying them in suitable vectors and cells.

[0061] In a specific embodiment, the present invention relates to a nucleic acid molecule comprising SEQ ID NO:5.

[0062] Carrier

[0063] The nucleic acid molecules according to the present invention can be provided in the following forms: nucleic acid molecules themselves, such as naked nucleic acid molecules; vectors; viruses or host cells, etc., whether from prokaryotic or eukaryotic sources. Vectors include expression vectors containing nucleic acid molecules of the present invention. For example, the vectors of the present invention may contain a transcription promoter and / or a transcription terminator, wherein the promoter is operably linked to the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked to the transcription terminator.

[0064] In this regard, a particular object of the present invention is a viral vector comprising a nucleic acid as defined above. The viral vector may be derived from different types of viruses, for example, preferably swine pox virus, fowlpox virus, pseudorabies virus, Aujesky virus, Salmonella, vaccinia virus, BHV (bovine herpes virus), HVT (turkey herpes virus), adenovirus, TGEV (Transmissible Gastroenteritidis Coronavirus), Erythrovirus and SIV (Simian Immunodeficiency Virus).

[0065] In a preferred embodiment, the vector is a recombinant swine pox virus. In pigs, swine pox virus (SPV) is only mildly pathogenic and induces a protective immune response. Therefore, SPV is an excellent candidate for a viral vector in pigs. The general procedure for generating recombinant SPV is described in several references (Vet Rec. 1994 Jan 1; 134(1): 13-8). In a typical method, the first step in constructing a recombinant SPV is to generate a homologous plasmid that can direct the insertion of the transcription unit of the antigenic polypeptide gene of the present invention into the SPV genome. The insertion occurs by homologous recombination, which requires that the inserted DNA be flanked by contiguous SPV genomic regions. In the case of SPV recombinants, the thymidine kinase (TK) gene is preferred as an insertion site, which has been shown to be a non-essential region for viral replication. Because the transcription machinery of poxviruses cannot recognize host cell promoters, the antigenic polypeptide gene of the present invention is preferably linked to a poxvirus promoter. For expression in recombinant SPV, a vaccinia virus promoter (e.g., P11 or P7.5) is preferred, but other poxvirus promoters may also be used. Once the homologous plasmid is generated, it is transfected into competent cells (such as embryonic porcine kidney (ESK-4) cells or porcine kidney (PK-15) cells) that have been previously infected with the SPV. Recombinants are generated by homologous recombination between the replicated SPV genome and the transfected plasmid. Recombinant SPVs can be selected / screened using conventional techniques. One identification method utilizes the E. coli lacZ gene as a marker. In this case, the chromogenic substrate 5-bromo-4-chloro-3-indole-β-D-galactoside (X-gal), which is converted to a blue compound by the action of the expressed enzyme (β-galactosidase), is then used to identify viral plaques produced by the recombinant virus in the progeny compared to a background of colorless plaques produced by non-recombinant virus. In the absence of a marker gene, the production of the antigenic polypeptide expressed by the recombinant SPV can be verified in an immunofluorescence assay by using specific antibodies against the antigenic polypeptide. Using the above protocol, a recombinant SPV expressing the antigenic polypeptide of the present invention can be generated.

[0066] Other expression systems and vectors, such as plasmids that replicate and / or integrate in yeast cells, may also be used.

[0067] The present invention also relates to a method for preparing a polypeptide of the present invention, the method comprising culturing a host cell containing a nucleic acid or a vector as defined above under conditions suitable for nucleic acid expression, and recovering the polypeptide. As described above, proteins and peptides can be purified according to techniques known per se in the art. The present invention also provides an expression kit comprising (a) a host cell (preferably an insect cell or a yeast cell); (b) a means for expressing a polypeptide of the present invention (e.g., a means containing a vector system capable of replicating in the cell); and (c) a means for recovering a protein or peptide of the present invention.

[0068] Vaccine composition

[0069] As used herein, the term "vaccine" includes agents that can be used to induce, stimulate or enhance the immune system of an animal (eg, a pig) against a pathogen. The vaccine of the present invention is capable of inducing or stimulating or enhancing immunity against the PCV2 virus.

[0070] The term "immunization" includes methods of delivering an immunogen to a subject. For example, immunization can achieve sustained high levels of antibodies and / or cellular responses, wherein T lymphocytes are able to kill or inhibit pathogens in the immunized non-human animal (e.g., pig), directed against pathogens or antigens to which the animal was previously exposed.

[0071] The vaccine of the present invention comprises an immunologically effective amount of a polypeptide, cell or nucleic acid as described above in a pharmaceutically acceptable vehicle. As a result of vaccination with the composition of the present invention, the animal becomes at least partially immune or completely immune to PCV2 infection, or becomes resistant to developing moderate or severe PCV2 infection. PCV2 vaccines can be used to elicit humoral responses and / or cellular responses.

[0072] PCV2 infection or related diseases include, inter alia, post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), reproductive failure, granulomatous enteritis, exudative epidermitis, necrotizing lymphadenitis, and congenital tremor. Preferably, the non-human animal subject (e.g., pig) is protected to a degree in which one to all of the adverse physiological symptoms or effects of PCV2 infection are significantly reduced, ameliorated, or completely prevented.

[0073] The present invention also relates to a combination vaccine comprising a polypeptide, nucleic acid or cell of the present invention in combination with at least one additional protein antigen [Gupi PS Nanyar et al. (Can. Vet. J, Vol. 38, 1997: 385-387) and Clark EG (Proc. Am. Assoc. Swine Prac. 1997; 499-501)].

[0074] In practice, the exact amount required for an immunologically effective dose will vary from subject to subject, depending on factors such as the age and general condition of the subject, the nature of the formulation, and the mode of administration. An appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. For example, methods are known in the art for determining or titrating an appropriate vaccine dose to find the minimum effective dose based on the weight of the non-human animal subject, the concentration of the vaccine, and other typical factors.

[0075] In a typical embodiment, the vaccine comprises a unitary dose of 0.1-50 μg, preferably 0.1-25 μg, even more preferably 1-15 μg, usually about 10 μg of the polypeptide or nucleic acid antigen of the present invention.

[0076] The dose of the vaccine, the concentration of the components therein and the schedule of administration of the vaccine that elicits an appropriate immune response can be determined by methods such as by antibody titration of serum, e.g., by ELISA and / or serum neutralization assay analysis and / or by vaccination challenge evaluation.

[0077] In a specific embodiment, the vaccine comprises a polypeptide of the invention in purified form, optionally in combination with any suitable excipient or carrier.

[0078] In another embodiment, the vaccine comprises a nucleic acid as defined above, optionally in combination with any suitable excipient or carrier. The most preferred vaccine comprises a viral vector comprising a nucleic acid as defined above. Yet another preferred vaccine comprises a swine pox virus comprising a nucleic acid as defined above.

[0079] Depending on the route of administration, the vaccine may contain other ingredients known per se to those of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze-drying stabilizers, wetting agents or emulsifiers, pH buffers, gelling or viscosity enhancing additives, and preservatives.

[0080] Examples of pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited to, demineralized or distilled water; saline solutions; plant-based oils, such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenylpolysiloxane, and methylphenylpolysiloxane (polysolpoxane); volatile silicones; mineral oils, such as light liquid paraffin oil or heavy liquid paraffin oil; squalene; cellulose derivatives, such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropylmethylcellulose; cellulose; lower alkanols, such as ethanol or isopropanol; lower aryl alkanols; lower polyalkylene glycols or lower alkylene glycols, such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerol; fatty acid esters, such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinyl pyrrolidone; agar; carrageenan; tragacanth gum or gum arabic; and petrolatum. Typically, the carrier will constitute 10 wt%-99.9 wt% of the vaccine composition and can be buffered by conventional methods using agents known in the art (e.g., sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, and mixtures thereof, etc.).

[0081] Examples of adjuvants include, but are not limited to, oil-in-water emulsions, aluminum hydroxide (alum), immunostimulatory complexes, nonionic block polymers or copolymers, cytokines (such as IL-1, IL-2, IL-7, IFN-α, IFN-β, IFN-γ, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptide (MDP), etc. Other suitable adjuvants include, for example, potassium aluminum sulfate, heat-labile or heat-stable enterotoxins isolated from Escherichia coli, cholera toxin or its B subunit, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete adjuvant or complete adjuvant, etc. Toxin-based adjuvants (such as diphtheria toxin, tetanus toxin and pertussis toxin) can be inactivated before use, for example, by formaldehyde treatment.

[0082] Examples of freeze-drying stabilizers include carbohydrates, such as sorbitol, mannitol, starch, sucrose, dextran or glucose; proteins, such as albumin or casein, and derivatives thereof.

[0083] The vaccine may additionally comprise at least one immunogen from at least one additional pathogen, for example, a porcine pathogen, for example, Actinobacillus pleuropneumoniae; adenovirus; alphavirus, such as Eastern equine encephalitis virus; Balantidium coli; Bordetella bronchiseptica; Brachyspira, preferably Brachyspira hyodysenteriae, Brachyspira pilosulae, non-pathogenic Brachyspira, Brucella suis (preferably biovars 1, 2 and 3); classical swine fever virus, African swine fever virus Viruses; Chlamydia, preferably Chlamydia veterinariae and Chlamydia abortus; Clostridium, preferably Clostridium difficile, Clostridium perfringens types A, B and C, Clostridium novyi, Clostridium septicum, Clostridium tetani; digestive and respiratory coronaviruses; Cryptosporidium parvum; Eimeria; Eperythrozoon suis, currently named Mycoplasma suis; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtype 1, subtype 7 and subtype 14; hemagglutinating encephalomyelitis Viruses; Isospora suis; Japanese encephalitis virus; Lawsonia intracellularis; Leptospira, preferably Leptospira australis, Leptospira canis, Leptospira typhi, Leptospira icterohemorrhagica, Leptospira interrogans, Leptospira bomonae and Leptospira tarasovii; Mannheimia hemolytica; Mycobacterium, preferably Mycobacterium avium, Mycobacterium intracellularis and Mycobacterium bovis; Mycoplasma pneumoniae; Parvovirus; Pasteurella multocida; Porcine cytomegalovirus; porcine parvovirus, porcine reproductive and respiratory syndrome virus; pseudorabies virus; rotavirus; Lushan virus; Salmonella, preferably Salmonella typhimurium and Salmonella choleraesuis; Staphylococcus, preferably Staphylococcus suis; Streptococcus, preferably Streptococcus suis; porcine cytomegalovirus; porcine herpes virus; swine influenza virus; swine pox virus; Toxoplasma gondii; porcine vesicular stomatitis virus and exanthema virus; or other isolates and subtypes of porcine circovirus.

[0084] Vaccine compositions of the present invention can be liquid preparations, such as aqueous solutions, water-in-oil or oil-in-water emulsions, syrups, elixirs, tinctures, preparations for parenteral administration, subcutaneous administration, intradermal administration, intramuscular administration or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such preparations are known in the art and are usually prepared by dissolving the antigen and other typical additives in a suitable carrier or solvent system. Liquid preparations may also include suspensions and emulsions containing suspending agents or emulsifiers.

[0085] The administration route may be transdermal, transmucosal, or by a parenteral route (intradermal, intramuscular, subcutaneous, intravenous or intraperitoneal). The vaccine composition according to the present invention may be administered alone, or may be co-administered or sequentially administered with other treatments or therapies.

[0086] The present invention also relates to a method for immunizing or inducing an immune response in a non-human animal (eg, a pig), the method comprising administering to the animal a polypeptide, nucleic acid, cell, vector or vaccine as described above.

[0087] The present invention also relates to a method for treating and / or preventing a PCV2-related disease in a non-human animal (eg, a pig), the method comprising administering to the animal a polypeptide, nucleic acid, cell, vector or vaccine as described above.

[0088] As mentioned above, PCV2 infection or related diseases include, inter alia, post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), reproductive failure, granulomatous enteritis, exudative epidermitis, necrotizing lymphadenitis and congenital tremor.

[0089] The vaccine of the present invention can be conveniently administered intranasally, transdermally (i.e., applied on or at the surface of the skin for systemic absorption), parenterally, ocularly, etc. Parenteral administration routes include, but are not limited to, intramuscular, intravenous, intraperitoneal, etc.

[0090] The dosage of the vaccine of the present invention will depend on the species, breed, age, size, vaccination history, health status of the animal to be vaccinated; and the route of administration (e.g., subcutaneous, intradermal, oral, intramuscular or intravenous).

[0091] The vaccines of the present invention may be administered as a single dose or in repeated doses. The vaccines of the present invention may be administered alone, or may be administered simultaneously or sequentially with one or more additional components (such as, for example, other porcine immunogenic compositions or vaccine compositions). In the case where the compositions are administered at different times, the administration may be separate or overlapping in time.

[0092] In one embodiment, the vaccine composition of the present invention is administered to a subject susceptible to PCV2 infection or at risk of PCV2 infection to enhance the subject's own immune response. In one embodiment, the subject to whom the vaccine is administered is a pig. The animal may be susceptible to infection by PCV2 or a closely related virus.

[0093] The vaccine of the present invention is preferably administered to pigs, adult pigs, but is also administered to young pigs, piglets or to pregnant females, or to other types of non-human animals. Vaccination of pregnant females is particularly advantageous because it confers passive immunity to newborns via the transmission of maternal antibodies. The pigs may be less than 7, 6, 5, 4, 3, 2 or 1 week old; 1-6 weeks old; 2-5 weeks old; or 3-4 weeks old. For example, a "test" animal may be administered the vaccine of the present invention to evaluate the performance of the vaccine for the purpose of final use or development of a pig vaccine. Ideally, the vaccine is administered to a subject who has not yet been exposed to the PCV2 virus. Preferably, the subject is a pig that needs to be vaccinated against post-weaning multisystemic wasting syndrome (PMWS) and / or porcine dermatitis nephropathy syndrome (PDNS).

[0094] The invention also includes a combination vaccine comprising a vaccine according to the invention and at least one immunogenic active component effective against another pathogenic organism in pigs, such as, for example, Actinobacillus pleuropneumoniae; adenovirus; Balantidium coli; Bordetella bronchiseptica; Brachyspira, preferably Brachyspira hyodysenteriae, Brachyspira pilosulae, Brucella suis (preferably biovars 1, 2 and 3); classical swine fever virus, African swine fever virus; Chlamydia, preferably Chlamydia veterinaria and Chlamydia abortus; Clostridium, preferably Clostridium difficile, Clostridium perfringens; porcine respiratory coronavirus; Cryptosporidium parvum; Eimeria; Eperythrozoon suis, currently named Mycoplasma suis; Erysipelothrix rhusiopathiae coli; Haemophilus parasuis; hemagglutinating encephalomyelitis virus; Isospora suis; Lawsonia intracellularis; Leptospira, preferably Leptospira bomonae; Mannheimia haemolytica; Mycobacterium, preferably Mycobacterium avium; Mycoplasma pneumoniae; Pasteurella multocida; porcine cytomegalovirus; porcine parvovirus, porcine reproductive and respiratory syndrome virus; pseudorabies virus; rotavirus; Salmonella, preferably Salmonella typhimurium and Salmonella choleraesuis; Staphylococcus, preferably Staphylococcus suis; Streptococcus, preferably Streptococcus suis; porcine cytomegalovirus; swine influenza virus; swine poxvirus; Toxoplasma gondii; porcine vesicular stomatitis virus and vesicular exanthema virus; or other isolates and subtypes of porcine circovirus.

[0095] The present invention also provides a container containing an immunologically effective amount of a polypeptide, nucleic acid or vaccine as described above. The present invention also provides a vaccination kit, comprising an optional sterile container containing an immunologically effective amount of a vaccine, a means for administering the vaccine to an animal, and an optional instruction manual containing information on administering an immunologically effective amount of the composition to treat and / or prevent PCV2-related diseases.

[0096] Further aspects and advantages of the present invention are provided in the following section, which should be regarded as illustrative only.

[0097] Example

[0098] Example 1: Construction of a nucleic acid encoding an antigenic polypeptide containing a B5R signal peptide and PCV2 ORF2

[0099] The two synthetic double-stranded DNAs shown in SEQ ID NO: 6 and SEQ ID NO: 7 were ordered from Takara Bio (Japan). The restriction enzyme sites at their 5' and 3' ends were BamHI / SalI and XbaI / SalI, respectively. The DNAs were cloned into plasmids pMD18-Ess (B / X / S) and pMD18-ORF2 (X / S), respectively.

[0100] Since there is an XbaI site in front of the SalI site at the 3' end of SEQ ID NO: 6, pMD18-Ess (B / X / S) was cut with two restriction enzymes (XbaI and SalI). The 561 bp DNA fragment obtained by cutting pMD18-ORF2 (X / S) with XbaI and SalI was inserted into the XbaI / SalI site of pMD18-Ess (B / X / S). The resulting plasmid pMD18-Ess_ORF2 contains the antigenic peptide gene of SEQ ID NO: 5.

[0101] Plasmid pGTPs40K-S (described in USP 7,348,422) was cut with two restriction enzymes (BamHI and SalI). Figure 2 The plasmid was named pGTPs-Ess_ORF2. The plasmid contains a strong poxvirus promoter (Ps) and the antigenic peptide gene of SEQ ID NO: 5.

[0102] Example 2: Plasmids for producing recombinant SPV

[0103] First, prepare SPV genomic DNA as follows:

[0104] SPV kasza strain (VR-363) and embryonic porcine kidney ESK-4 cells (CL-184) can be purchased from the American Type Culture Collection (ATCC). ESK-4 cells were routinely cultured in Ham's F-12K medium (Gibco, catalog number: 21127-022) supplemented with the following substances: 1% streptomycin-penicillin (Gibco, catalog number: 15140-122) and 5% FBS (Gibco, catalog number: 10437-028) at 37°C in 5% CO2. For SPV genomic DNA preparation, 225 cm 2 Confluent ESK-4 cells in flasks were infected with SPV and incubated for 6 days until the cells showed 100% cytopathic effect (CPE). The infected cells were then harvested by scraping the cells into the culture medium and centrifuging at 1300 rpm for 5 min. The culture medium was poured off and the cell pellet was gently resuspended in 2 ml of phosphate buffered saline (PBS: 1.5 g Na2HPO4, 0.2 g KH2PO4, 0.8 g NaCl and 0.2 g KCl per liter of H2O) and subjected to two consecutive freeze-thaw cycles. Cell debris was then removed by centrifugation at 3000 rpm for 5 minutes at 4°C. The SPV virus particles present in the supernatant were then precipitated by centrifugation at 20,000×g for 20 min at 4°C. The resulting pellet was then suspended with 10 mM Tris (pH 7.5). SPV genomic DNA was then extracted from SPV virions by suspending with lysis buffer (20 mM Tris, pH 9, 0.1 M NaCl2, 5 mM EDTA, 0.1% SDS, 0.2 mg / ml proteinase K) and incubating at 60°C for 5 min. Two phenol:chloroform (1:1) extractions were performed, and the samples were precipitated by adding 2 volumes of ethanol and centrifuged. The supernatant was decanted, and the pellet (SPV DNA) was air dried and rehydrated in 10 mM Tris pH 7.5, 1 mM EDTA at 4°C.

[0105] Next, the TK flanking region in the SPV genome was cloned by polymerase chain reaction (PCR). The two primers (synthetic oligonucleotides) SP54242F and SP57617R shown in SEQ ID NO: 8 and SEQ ID NO: 9 were purchased from TakaraBio. PCR reactions were performed using LA Taq polymerase (Takara Bio) and primer sets (SP54242F and SP57617R) and SPV DNA as a template according to the manufacturer's protocol.

[0106] The amplified DNA of about 3.4 kbp was confirmed by 0.8% agarose gel electrophoresis and purified from the gel using the QIAquick gel extraction kit (Qiagen). The purified DNA fragment was cloned into the pCR4-TOPO vector (Invitrogen) according to the manufacturer's protocol. 14 white ampicillin-resistant transformants were picked and grown in LB broth, and each plasmid was prepared using the QuickLyse Miniprep kit (Qiagen). Each plasmid was digested with SpeI, and two candidate plasmids (inserted DNA in both directions) were selected. Their inserted DNA was sequenced using the dye terminator cycle sequencing reagent (Dye Terminator Cycle Sequencing reagent) (DTCS) and the CEQ2000XL sequencer (Beckman Coulter). One of the candidate plasmids, pCR-SPV54242 / 57617 (#2), was confirmed to contain a DNA fragment of 54,242 nt to 57,617 nt of SPV genomic DNA (GeneBank accession number: NC_003389) and was used as a basic plasmid ( Figure 1 ).

[0107] Next, PCR mutagenesis was performed using pCR-SPV54242 / 57617 (#2) as a template and two primer sets [(1) SEQ ID NO: 10 and SEQ ID NO: 11 or (2) SEQ ID NO: 12 and SEQ ID NO: 13] to delete a portion of the TK gene and introduce multiple restriction enzyme sites.

[0108] Each PCR product was subjected to 0.8% agarose gel electrophoresis and purified using a QIAquick gel extraction kit. The purified DNA fragment (amplified by PCR using primer set SEQ ID NO: 10 and SEQ ID NO: 11) was digested with two restriction enzymes KpnI and HindIII and ligated with pBluescript KS(+) (Stratagene) cut with the same restriction enzymes. The resulting plasmid pBS-TKR (Kpn.Hin) ( Figure 1 ) was digested with Sad and PstI, and a DNA fragment cleaved with the same restriction enzymes (amplified by PCR using the primer set SEQ ID NO: 12 and SEQ ID NO: 13) was inserted therein. The resulting plasmid was named pSP70 ( Figure 1 ).

[0109] The sequence between the EcoRI and HindIII sites of the multiple restriction enzyme sites of pSP70 was replaced with an oligonucleotide adaptor prepared by annealing two synthetic DNA oligonucleotides SEQ ID NO: 14 and SEQ ID NO: 15. The resulting plasmid was named pSP71 ( Figure 1 ).

[0110] The DNA fragment of the 'P7.5 promoter-LacZ' gene cassette from pNZ76 (pNZ76 was cut with HindIII and SmaI and then blunt-ended by DNA polymerase (described in USP5,387,519)) was ligated into the SmaI site of pSP71. The resulting plasmid was named pSP72 ( Figure 1 ), the 'P7.5-LacZ' gene cassette was inserted into the TK gene (55625nt-56170nt in the SPV genome).

[0111] The 0.8 kb BglI-cleaved fragment derived from pGTPs-Ess_ORF2 (Example 1) was inserted into the SfiI site of pSP72, and the resulting plasmid was named pSP72-Ess_ORF2 ( Figure 1 ). This plasmid contains the 'strong poxvirus promoter (Ps)-Ess_ORF2' gene cassette (also in the TK gene) and was used as a homology plasmid to generate recombinant SPV SVR7.

[0112] The natural PCV2-ORF2 gene was inserted into another homology plasmid instead of Ess_ORF2 to be used as a reference. Although many sequence data of PCV2-ORF2 have been reported, one of them (double-stranded DNA of SEQ ID NO: 16) was synthesized. SEQ ID NO: 16 is a complementary DNA encoding ORF2 of PCV2 isolated from France (GenBank: AF055393), and a BamHI site and a SalI site are connected to its 5' end and 3' end, respectively. This synthesized DNA was cut with BamHI and SalI, and replaced the BamHI / SalI region of pSP72-Ess_ORF2. The resulting plasmid was named pSP72-ORF2 and used as a homology plasmid to generate recombinant SPV SVR3.

[0113] Example 3: Production of recombinant SPVs (SVR3 and SVR7)

[0114] (1) Production of recombinant SPV (SVR3 and SVR7)

[0115] Recombinant SPVs were generated in ESK-4 cells by homologous recombination between the wild-type SPV genome and a homologous vector. Sub-confluent ESK-4 cells in 6-well plates were infected with wild-type SPV 4 hours before transfection with 2 μg each of pSP72-Ess_ORF2 or pSP72-ORF2 using Lipofectamin Plus reagent (Invitrogen) and incubated at 37°C for 5 days until cytopathic effect (CPE) occurred. Recombinant plaques expressing β-galactosidase were screened in cell lysates from infected and transfected cells by adding 0.5 mg / ml Bluo-gal (Invitrogen catalog number: 15519-028) to nutrient agarose overlays. Wild-type-free recombinant viruses were purified by 4-6 rounds of screening. The recombinant SPVs produced using pSP72-Ess_ORF2 or pSP72-ORF2 were named SVR7 and SVR3, respectively.

[0116] (2) Black plaque analysis (BPA)

[0117] ESK-4 cells in 24-well plates were infected with SVR3, SVR7 or wild-type SPV and incubated at 37°C. After 6 days, the monolayers were fixed with acetone and methanol (2:1). Neutralizing anti-PCV2 monoclonal antibodies (Ingenase clone 36F1) were diluted in 5% dry milk powder in PBS (1:500 dilution) and applied to the infected cells and incubated for 2 hours at room temperature (RT). The infected cells were washed with PBS and reacted with biotin-conjugated rabbit anti-mouse IgG (1:1000 dilution) and VECTASTAIN ABC Standard Kit (Vector Laboratory PK-4000). Alkaline phosphatase substrate (NBT / BCIP stock solution, Roche, catalog number: 11681451001) was diluted in 0.1M Tris (pH 9.5), 0.1M NaCl, 50mM MgCl2 and added to the infected cells and incubated at RT for 5-30min until the plaques of SVR3 or SVR7 turned purple / black. Plaques from wild-type SPV were white (negative), while all plaques from SVR3 and SVR7 were confirmed to be black (positive) ( Figure 2 ).

[0118] Example 4: Detection of polyclonal antibodies for PCV2-ORF2

[0119] In order to produce polyclonal antibodies against PCV2-ORF2, a glutathione S-transferase (GST) fusion protein with the C-terminal region of PCV2-ORF2 was expressed in E. coli. First, a DNA fragment was produced by PCR using pMD18-ORF2 (X / S) (Example 1) as a template and a primer set (SEQ ID NO: 17 and SEQ ID NO: 18).

[0120] The 0.45 kbp amplified DNA was cut with BamHI and SalI and inserted into the BamHI / SalI site of pGEX-6P-3 (GE Healthcare, 28-9546-51). The candidate plasmid was sequenced using sequencing primers pGEX-5'-SP or pGEX-3'-SP (GE Healthcare, 27-1410-01 or 27-1411-01, respectively) and confirmed to be consistent with the construction plan. The resulting plasmid was named pGEX-TGXR2.

[0121] E. coli host cells BL21 (GE Healthcare, 27-1542-01) were transformed with pGEX-TGXR2, and the transformants were confirmed to express a fusion protein of about 40 kDa by induction with isopropyl-β-D-thiogalactoside (IPTG).

[0122] To produce fusion proteins for animal immunization, a single colony of the transformant was inoculated into a 5 ml aliquot of LB broth (containing 50 μg / ml ampicillin (Amp)) and cultured overnight at 37°C with shaking. The next morning, the overnight culture was inoculated into 1 L of LB broth containing Amp and cultured at 37°C with shaking until OD600 was 0.7, at which point IPTG was added to the culture at a final concentration of 0.1 mM. The culture was incubated at 37°C for another 3 hours with shaking. The bacteria were collected by centrifugation at 3500 × g for 20 min and resuspended in 20 ml of PBS lysis buffer (1% Triton X-100 in PBS). The bacterial suspension was sonicated three times on ice with a burst of 10 sec and resting of 10 sec. The lysate was centrifuged at 17,000 × g for 20 min at 4°C, and the supernatant was transferred to a new tube.

[0123] Since the target GST fusion protein formed inclusion bodies, it was purified using Mini Whole Gel Eluter (Bio-Rad Laboratories, catalog number: 165-1255) according to the supplier's instructions.

[0124] The target GST fusion protein of about 40 kDa was purified from fraction No. 7 from the Mini Whole Gel Eluter ( Figure 3 b). The solution of part No.7 was dialyzed against PBS. Polyclonal antibodies against the fusion protein were produced by immunizing rats. Three Wister female rats (SPF) aged 5 weeks were subcutaneously immunized with 0.1 mg of GST-ORF2 protein (as an emulsion in complete Freund's adjuvant), and three weeks later, they were boosted three times at 3-week intervals with 0.05 mg of the same antigen mixed with incomplete Freund's adjuvant. Two weeks after the last immunization, the rats were bled (exanguinated) and about 1 ml of serum was collected from each rat.

[0125] Example 5: Characterization of Antigenic Peptides Expressed by Recombinant SPV

[0126] Immunofluorescence analysis (IFA) was performed to confirm the localization of ORF2 protein expressed by SVR3 or SVR7. ESK-4 cells were infected with rSPV (SVR3 or SVR7) or parental SPV. Five days later, the infected cells were washed twice with PBS and treated with (a) acetone / methanol (2:1) or (b) PBS for 5 min at room temperature. The acetone / methanol or PBS was removed, and PBS was added to prevent the samples from drying out. They were washed twice with PBS and reacted with primary antibodies [rabbit anti-β-galactosidase (α-βGal) (CAPPEL, catalog number: 0631-0002) and rat anti-PCV2-ORF2 (α-ORF2) (Example 4) (1:1000 dilution in PBS)] for 30 min at 37°C. Next, the sample was washed three times with PBS and reacted with secondary antibodies [goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Life technology, A-11006) and anti-rat IgG (H+L) Alexa Fluor 546 (A-11081) (1:1000 dilution in PBS)] for 30 min at 37° C. The sample was washed three times with PBS and observed under a fluorescence microscope.

[0127] In the case of (a) acetone / MeOH-treated cells, both β-galactosidase (green signal) and ORF2 (red signal) signals were observed in cells infected with either SVR3 or SVR7, respectively, because the cell membrane was permeable ( Figure 3 a). In contrast, in the case of (b) intact cells, both β-galactosidase (green) and ORF2 (red) were not detected on cells infected with SVR3, but ORF2 (red) was clearly observed on cells infected with SVR7 ( Figure 3 b) These results strongly suggest that the localization of the ORF2 protein changes from intracellular where the original ORF2 expressed by SVR3 is located, for example, to extracellular and on the cell surface as achieved by SVR7.

[0128] Therefore, the vaccine efficacy of SVR7 is stronger than that of SVR3, and the B5R-derived targeting signal can improve the immune response against PCV2-ORF2.

[0129] Example 6: Efficacy of in vivo vaccination

[0130] Pigs were vaccinated with a vector encoding the polypeptide SVR7 of the present invention. As a comparative example, pigs were vaccinated with SVR3. For immunization, 3-5×10 4 TCID50 / dose. The pigs were then challenged with PCV2.

[0131] More specifically, 24 piglets (PCV2 free or PCV2 negative) at 3 weeks of age were settled before vaccination and divided into different groups as shown below:

[0132] SVR3 group: 7 piglets, ♂4, ♀3.

[0133] SVR7 group: 7 piglets, ♂4, ♀3.

[0134] Non-immunized group: 7 piglets, ♂4, ♀3.

[0135] Non-immune and non-challenged group: 3 piglets, ♂3.

[0136] Vaccination was performed at 4 weeks of age and PCV2 challenge was performed 2 weeks after immunization. For PCV2 challenge, 6 × 10 5 TCID50 of PCV2 / dose. At necropsy (4 weeks after PCV2 challenge), several organs were collected for PCV2 detection, including tree lymphoid organs, tonsils, inguinal-lymph nodes, intestinal lymph and thymus.

[0137] The PCV2 genome copy number in the serum was determined 1 week, 2 weeks and 3 weeks after vaccination. The results given below show that the vaccine of the present invention induces a strong reduction in the PCV2 genome copy number. This result shows that the vaccine of the present invention is more effective than the vaccine based on the natural ORF2 sequence.

[0138] Table: Percentage of PCV2-qPCR positive pigs in serum (positive pigs / total pigs)

[0139] Group Before immunization Before excitation (6W) 1wpc(7W) 2wpc(8W) 3wpc(9W) SVR3 0％(0 / 7) 0％(0 / 7) 17％(1 / 6) 33％(2 / 6) 100％(6 / 6) SVR7 0％(0 / 7) 0％(0 / 7) 0％(0 / 6) 0％(0 / 6) 67％(4 / 6) NI Excitation 0％(0 / 7) 0％(0 / 7) 0％(0 / 7) 29％(2 / 7) 100％(7 / 7) NINC 0％(0 / 3) 0％(0 / 3) 0％(0 / 3) 0％(0 / 7) 0％(0 / 3)

[0140] also, Figure 4 The results shown in clearly demonstrate that SVR7 induces a strong anti-PCV2 antibody response. The results indicate that SVR7 induces a large amount of antibodies against PCV2 ORF2. The antibody response is stronger than that elicited by SVR3. The induction of a large antibody response is associated with a significant reduction in the PCV2 genome copy number.

[0141] These data clearly demonstrate the vaccination efficacy of the polypeptides and vectors of the present invention.

[0142] Sequence Listing

[0143] SEQ ID NO:1:KTISVVTLLCVLPAVVYSTCTV

[0144] SEQ ID NO:2:KTISVVTLLCVLPAVVYSTCTV PTMNNAKLTSTETS

[0145] SEQ ID NO:3:

[0146] GVLNTRLSRTFGYTIKRTTVKTPSWAVDMMRFNINDFVPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTAGNVDHVGLGTAFENSKYDQEYNIRVTMYVQFREFNLKDPPLNP

[0147] SEQ ID NO:4:

[0148] KTISVVTLLCVLPAVVYSTCTV PTMNNAKLTSTETS WKKEKGVLNTRLSRTFGYTIKRTTVKTPSWAVDMMRFNINDFVPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTAGNVDHVGLGTAFENSKYDQEYNIRVTMYVQFREFNLKDPPLNP

[0149] SEQ ID NO:5:

[0150] ATGAAAACGATTTCCGTTGTTACGTTGTTATGCGTACTACCTGCTGTTGTTTATTCAACATGTACTGTACCCACTATGAATAACGCTAAATTGACGTCTACCGAAACATCGTGGAAAAAAGAGAAAGGAGTCTTGAACACCAGATTGTCTAGAACCTTCGGTTACACCATTAAGAGAACCACCGTCAAAACCCCATCTTGGGCTGTCGATATGATGAGATTCAACATCAACGATTTCGTCCCACCTGGTGGTGGATCAAACCCTAGATCCGTTCCATTCGAGTACTACAGAATCAGAAAAGTCAAAGTCGAGTTCTGGCCATGCTCTCCTATTACTCAGGGTGATAGAGGAGTTGGATCAACTGCCGTCATCTTGGATGACAACTTCGTCACTAAGGCTACTGCCTTGACCTACGATCCTTACGTCAATTACTCTAGTAGACACACCATCACCCAACCATTCTCATACCATTCCAGATACTTCACTCCAAAACCTGTCTTGGACTCAACCATCGATTACTTTCAACCAAACAACAAGAGAAACCAATTGTGGTTGAGATTGCAAACTGCCGGTAACGTCGATCATGTCGGATTGGGAACCGCCTTCGAAAACTCCAAATACGACCAGGAGTACAACATTAGAGTCACCATGTACGTCCAATTCAGAGAGTTCAACTTGAAGGACCCACCATTGAACCCATAA

[0151] SEQ ID NO:6:

[0152] GGATCCACCATGAAAACGATTTCCGTTGTTACGTTGTTATGCGTACTACCTGCTGTTGTTTATTCAACATGTACTGTACCCACTATGAATAACGCTAAATTGACGTCTACCGAAACATCGTGGAAAAAAGAGAAAGGAGTCTTGAACACCAGATTGTCTAGACATTGAACCCATAAGTCGAC

[0153] SEQ ID NO:7:

[0154] TCTAGAACCTTCGGTTACACCATTAAGAGAACCACCGTCAAAACCCCATCTTGGGCTGTCGATATGATGAGATTCAACATCAACGATTTCGTCCCACCTGGTGGTGGATCAAACCCTAGATCCGTTCCATTCGAGTACTACAGAATCAGAAAAGTCAAAGTCGAGTTCTGGCCATGCTCTCCTATTACTCAGGGTGATAGAGGAGTTGGATCAACTGCCGTCATCTTGGATGACAACTTCGTCACTAAGGCTACTGCCTTGACCTACGATCCTTACGTCAATTACTCTAGTAGACACACCATCACCCAACCATTCTCATACCATTCCAGATACTTCACTCCAAAACCTGTCTTGGACTCAACCATCGATTACTTTCAACCAAACAACAAGAGAAACCAATTGTGGTTGAGATTGCAAACTGCCGGTAACGTCGATCATGTCGGATTGGGAACCGCCTTCGAAAACTCCAAATACGACCAGGAGTACAACATTAGAGTCACCATGTACGTCCAATTCAGAGAGTTCAACTTGAAGGACCCACCATTGAACCCATAAGTCGAC

[0155] SEQ ID NO:8:

[0156] AATATTACGGGTGCTGTTT

[0157] SEQ OD NO:9:

[0158] AAAAACATCGTATTCCTG

[0159] SEQ ID NO:10:

[0160] CGTTCATGTTAAGCTTAACCTGAAATATTG

[0161] SEQ ID NO:11:

[0162] GTTTAAACGAATTCGGTACCCTTAAAAACATCG

[0163] SEQ ID NO:12:

[0164] CGCCGAGCTCGAGAATATTACGGGTGCTGTTTTTAC

[0165] SEQ ID NO:13:

[0166] CCAGACTGCAGAGAACATAGGTCCTAATATAAG

[0167] SEQ ID NO:14:

[0168] AATTGCCCGGGTACCGTCGATCGACTTTTTATGGCCCCCCCGGCCA

[0169] SEQ ID NO:15:

[0170] AGCTTGGCCGGGGGGGCCATAAAAAGTCGATCGACGGTACCCGGGC

[0171] SEQ ID NO:16:

[0172] GGATCCACCATGACGTATCCAAGGAGGCGTTACCGGAGAAGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCGTCCACCCCCGCCACCGTTACCGCTGGAGAAGGAAAAATGGCATCTTCAACACCCGCCTCTCCCGCACCTTCGGATATACTGTCAAGCGAACCACAGTCAGAACGCCCTCCTGGGCGGTGGACATGATGAGATTCAATATTAATGACTTTCTTCCCCCAGGAGGGGGCTCAAACCCCCGCTCTGTGCCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCGATCACCCAGGGTGACAGGGGAGTGGGCTCCAGTGCTGTTATTCTAGATGATAACTTTGTAACAAAGGCCACAGCCCTCACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTCCCGCTACTTTACCCCCAAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAACCAGCTGTGGCTGAGACTACAAACTGCTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCGAAAACAGTATATACGACCAGGAATACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAACCCTTAATAGTCGAC

[0173] SEQ ID NO:17:GGTGGTGGATCCAACCCTAGATCCG

[0174] SEQ ID NO:18:GGTGGGTCGACCAAGTTGAACTCTCTG

Claims

1. A polypeptide comprising a cell membrane addressing peptide operably linked directly to a heterologous antigenic polypeptide, wherein: The cell membrane addressing peptide consists of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the antigenic polypeptide is the ORF2 antigenic polypeptide of the PCV2 virus.

2. The polypeptide according to claim 1, wherein The cell membrane addressing peptide consists of SEQ ID NO:

1.

3. The polypeptide according to claim 1, wherein The cell membrane addressing peptide consists of SEQ ID NO:

2.

4. The polypeptide according to any one of claims 1 to 3, wherein The antigenic polypeptide comprises the sequence of SEQ ID NO:

3.

5. The polypeptide according to claim 4, wherein The antigenic polypeptide consists of SEQ ID NO:

3. The polypeptide according to claim 1 , which consists of the amino acid sequence of SEQ ID NO:

4.

7. The polypeptide of any one of claims 1-3, further comprising an N-terminal methionine residue.

8. The polypeptide of any one of claims 1-3, which is glycosylated.

9. A cell expressing the polypeptide of any one of claims 1 to 8 on its surface.

10. A nucleic acid encoding the polypeptide of any one of claims 1 to 7.

11. A vector comprising the nucleic acid according to claim 10.

12. A recombinant virus comprising the nucleic acid of claim 10 in its genome.

13. The recombinant virus according to claim 12, wherein The recombinant virus is a recombinant swine pox virus, and the recombinant swine pox virus contains the nucleic acid according to claim 10 in its genome.

14. A composition comprising the polypeptide of any one of claims 1 to 8, the cell of claim 9, the nucleic acid of claim 10, the vector of claim 11 or the recombinant virus of claim 12 or 13.

15. A vaccine comprising the polypeptide of any one of claims 1 to 8, the cell of claim 9, the nucleic acid of claim 10, the vector of claim 11 or the recombinant virus of claim 12 or 13, and optionally an adjuvant.

16. Use of the polypeptide of any one of claims 1 to 8, the cell of claim 9, the nucleic acid of claim 10, the vector of claim 11 or the recombinant virus of claim 12 or 13 in the preparation of a medicament for treating or preventing a PCV2-related disease in a non-human animal.

17. The use according to claim 16, wherein The non-human animal is a pig.

18. Use of the polypeptide of any one of claims 1 to 8, the cell of claim 9, the nucleic acid of claim 10, the vector of claim 11 or the recombinant virus of claim 12 or 13 in the preparation of an agent for vaccinating non-human animals against PCV2 infection.

19. The use according to claim 18, wherein The non-human animal is a pig.

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