WO2013030608A1 - Nanoparticle-based veterinary vaccine - Google Patents

Abstract

The present invention provides for a porcine circovirus (PCV) vaccine comprising a recombinant cucumber mosaic virus (CMV) capable of producing virions in plants comprising said recombinant CMV, wherein the recombinant virus coat protein (CP) of said CMV comprises one or more epitopes of the coat protein (CP) of a PCV. The invention also relates to plants edible by animals expressing said recombinant CMV, and an animal feed comprising the vaccine or the recombinant CMV. The invention is applicable in the prevention or treatment of PCV associated diseases. ˙

Description

Nanoparticle-based veterinary vaccine

FIELD OF THE INVENTION

The present invention provides for a porcine circovirus (PCV) vaccine comprising a recombinant cucumber mosaic virus (CMV) capable of producing virions in plants comprising said recombinant CMV, wherein the recombinant virus coat protein (CP) of said CMV comprises one or more epitopes of the coat protein (CP) of a PCV.

The invention is useful in controlling PCV associated infections in mammals, in particular in pig herds. BACKGROUND ART

Porcine circovirus (PCV) infections are present worldwide causing major economic losses in the pig industry, estimated to be above 900 million Euros annually in the EU only. The virus, first recognized as a contaminant of pig kidney cell cultures, belongs to the Circovirus genus of the Circoviridae family. It was considered to be a non-pathogenic virus until connection between PCV and the postweaning multisystemic wasting syndrome (PMWS) of pigs had been discovered (Ellis et al., 1998). PCV is currently divided into two groups: non-pathogenic viruses of the PCV1 group and members of the PCV2 group, the causative agents of PMWS (Allan et al., 1998) and other PCV associated diseases (PCVDs, Segales et al., 2004, Chae, 2005).

PMWS affects weaned, 5- to 12-week-old piglets, and it is characterized by weight loss, dyspnoea and jaundice, combined with the pathological findings of interstitial pneumonia, enlarged lymph nodes, hepatitis and nephritis (Allan et al., 1998). Since the appearance of PMWS the number of PCVDs has increased. The picture is not clear though, the primary role of PCV2 has only been proven in cases of PMWS, the porcine respiratory disease complex (PRDC, Kim et al., 2003) and reproductive failure (RF, Park et al., 2005). Besides PMWS, PRDC and RF, PCV2 is also detected and believed to be involved in several other diseases, such as the porcine dermatitis nephropathy syndrome (PDNS, Wellenberg et al., 2004), necrotizing lymphadenitis (Chae, 2005), congenital tremors (Stevenson et al., 2001), exsudative dermatitis (Wattrang et al., 2002) or granulomatous enteritis (Chae, 2005), but PCV2 can also be present as a subclinical infection (Larochelle et al., 1999). Although PCV2 has been demonstrated in each of the listed conditions it is not known if the virus by itself is responsible for the disease. The case of PDNS is more or less clear as PCV2 can trigger the syndrome, but similar clinical signs can also be observed whenever immune complexes are deposited, leading to a type III hypersensitivity reaction (Chae, 2005, Segales et al., 2005).

PCV2 has a single stranded, circular DNA genome of 1.76 kilobases and a size of approximately 17 nm. The virion itself is a relatively simple construct; the genome has only 2 major open reading frames with a complex transcriptional profile (Cheung, 2003) to solve the problems of attachment, replication, assembly and spread. One of the proteins is involved in the replication of the genome (Rep protein), and the capsid (Cap) protein is the only structural one of the virion. The later is also the one responsible for inducing immunity, including protective immunity in the animals. Because of its simplistic design the virus has to rely on the machineries of the cells, so the best environment for replication is the actively dividing cell. Besides young animals in general, dividing cells are always provided, regardless the age, by the activated immune system of a host. PCV has developed into a virus finding and using the immune system (Krakowka et al., 2001) efficiently for replication, so besides manifesting in respiratory, reproductive or wasting problems, the infection is also a great danger when other pathogens are to invade the animal. PCV2 infection significantly reduces the efficacy of the host defense mechanisms, innate and acquired immunity alike, resulting in economic losses not caused directly by PCVDs, but by diseases due to other pathogens. These indirect losses are estimated to be even more potential than the direct ones.

Applicant's researchers were the first to prove the presence of PCV2 and PC VI in wild boars in Hungary (Csagola et al., 2006) and to provide genetic analysis of these PCV sequences, during which the first genetic evidence for naturally occurring recombination of PCV2 genomes was also provided

Currently PCV2 strains are divided into 2 major genotypes, with no obvious differences in their geographical distribution. Difference in their pathogenicity is being studied and limited evidence indicated that genotype 1 (PCV2B) viruses may be more pathogenic than genotype 2 viruses (PCV2A) (Grau-Roma et al., 2007). Antigenic differences of the Cap protein detected by monoclonal antibodies had also shown some correlation with differences in the clinical background of the isolates (Lefebvre et al., 2008). Recently a shift has been reported from PCV2A to PCV2B worldwide (Cheung et al, 2007) and a new type, only reported in Denmark so far, had been added (Dupont et al., 2007). Sequence of and baculovirus based vaccines against PCV2B are disclosed by (Jestin et al., US2009/00926227, 2009). Two additional strains of PCV2B have been isolated and identified by Wu, S. Q. who suggested that these strains were suitable for the preparation of vaccines and immunizing pigs against PMWS (Wu S Q, WO2009/085912 2009).

The control of PCV2 is crucial for the pig industry and besides following certain measures suggested by several scientists (most importantly the "Madec Principles" recommended by dr. Francoise Madec in 1997, Madec and Waddilove, 2002), vaccines are also being developed. One of them (Circovac, Merial) with the first full license to be used in Europe shows promising results, but further improvements are necessary to control the disease. Circovac is only the first in a row of recently marketed vaccines or vaccine candidates. The primary purpose of these vaccines is to provide an extra protection for the piglets in the most susceptible weaning age, by elevating the antibody level, either through colostral immunity (vaccinating the sow) or by actively immunizing piglets at the youngest age possible.

The current vaccines or vaccine candidates generally represent two main types. One is the traditional type of vaccine, manufactured by inactivation of the in vitro propagated virus. Another group of vaccines, also acting like the inactivated type (generating only humoral immunity) is based on genetic engineering using some vector (from insect viruses to bacterial expression systems). There are however several limitations of such vaccines no matter what the process of manufacturing is. The main problem with the production of an efficient inactivated PCV2 vaccine is that the virus replicates poorly in the generally used cell lines, usually not exceeding a virus titer of 10⁵ TCID50 / ml (Meerts et al., 2005), making the vaccine production costly, as expensive adjuvants are needed and also the dose of the antigen in the vaccine has to be increased by including virus concentration steps in the production process. The problem of low antigen concentration can easily be overcome by the use of expression vectors (as baculovirus for example in the CircoFLEX vaccine of Ingelvac) carrying the capsid gene.

The capsid protein of only 233 amino acids is an easy target for any expression system, but the first 44 amino acids at the amino terminal end of the protein are generally toxic for cells, from bacteria to the so far tested eukaryotic cells alike. That is 19% of the entire protein, and it has to be excluded from the expression process. The size of this sequence, rich in basic amino acids, would be less important if it were not a sequence carrying one of the few epitopes involved in immunogenicity (Mahe et al., 2000, Troung et al., 2001 , Lekcharoensuk et al„ 2004). Live virus vaccines would be the best solutions probably, as they can not only induce a specific humoral immune response but the cytotoxic T lymphocytes are also triggered when using such vaccines, resulting a more potent antiviral response. Still, attenuated PCV2 vaccines are not yet available and considering the quickly mutating nature of the PCV2 genome (Hughes and Piontkivska, 2008), and the possibility of intergenomic recombinations (Csagola et al., 2006, Ma et al., 2007) it seems to be risky to introduce such vaccines. The elegant design of PCV1-PCV2 chimeric vaccine candidates although carrying lower risks (Gillespie et al., 2008), are also potential recombination partners for any PCV2 in the massively infected pig herds, therefore they are only considered also as inactivated vaccines such as the Suvaxyn PCV2 of Fort Dodge.

Live non-pathogenic virus vectors are possible targets for future developments, and some of these like adenoviruses (Wang et al., 2007) or the Aujeszky's disease virus (Song et al., 2007) are already showing promising results, but further developments will be needed to enhance the safety and efficacy of these vectors.

A different approach to produce efficient vaccines is through the generation of virus like particles (VLP). Baculoviruses are the best known vectors for the production of such particles using the selected gene of a target virus (Jestin A et al., US 2009/0092627 Al, 2009). A more sophisticated design is the expression of the immunogenically important epitopes only, on the surface of a vector like the recently developed chimeric porcine parvovirus VP2 VLPs generated by a recombinant adenovirus (Pana et al., 2008). Besides insect baculoviruses or mammalian adenoviruses there are indications that plant viruses may also be suitable for the production of important epitopes, carried on the surface of a virion structure. However, to the best of the present Inventor's knowledge, no proposal has been made in the art to prepare PVC vaccines comprising plant virus as a vector.

The Inventors have now unexpectedly found that PCV vaccines can be prepared using a recombinant cucumber mosaic virus (CM V) capable of producing virions in plants wherein the virus coat protein of the CMV comprises PCV epitopes. While CMV has been suggested as tool to present viral epitopes (Natilla A and Nemchinov LG, 2008), it has not been used or suggested to provide porcine circovirus vaccines, and the success of the development of a useful vaccine cannot be foreseen based on the prior art.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a porcine circovirus (PCV) vaccine comprising a recombinant cucumber mosaic virus (CMV), said vaccine being capable of producing virions in plants comprising said recombinant CMV, wherein the recombinant virus coat protein (CP) of said CMV comprises one or more epitopes of the coat protein (CP) of a PCV, said virions being capable of eliciting an immune response in mammals.

In a preferred embodiment, the CP of CMV comprises inserted one or more, preferably one or two peptide segment(s) having 7 to 22, preferably at least 8, 9, 10, 1 1 , 12, 13, 14 or 15, preferably at most 21 , 20, 19, 18, 17, 16, 15 or 14 amino acids length, said one or more peptide segments comprising a sequence of amino acids selected from the following group of epitopes or epitope sequences:

- a sequence of amino acids from positions 37 to 43 of a PCV2 protein, RWRRKMG (SEQ ID NO: 1) and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution,

- a sequence of amino acids from positions 90 to 96 of a PCV2 protein, S I PFEYY (SEQ ID NO: 2) and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution,

- a sequence of amino acids from positions 126 to 145 of a PCV2 protein, DDNFVTKATALTYDPYVNYS (SEQ ID NO: 3) and a sequence variant thereof comprising at most 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof

- a sequence of amino acids from positions 169 to 186 of a PCV2 protein, STIDYFQPNNKRNQLWLR (SEQ ID NO: 4)

and a sequence variant thereof comprising at most 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof

- a sequence of amino acids from positions 224 to 233 of a PCV2 protein, FNL DPPLKP (SEQ ID NO: 5) and a sequence variant thereof comprising at most 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least

7 amino acid long fragment thereof,

- a sequence of amino acids from positions 52 to 62 of a PCV2 protein, TFGYTVKRTTV (SEQ ID NO: 9) and a sequence variant thereof comprising at most 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 76 to 83 of a PCV2 protein, IDDFVPPG (SEQ ID NO: 10) and a sequence variant thereof comprising at most 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 103 to 1 1 1 of a PCV2 protein, VEFWPCSPI (SEQ ID NO: 1 1) and a sequence variant thereof comprising at most 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least

7 amino acid long fragment thereof,

- a sequence of amino acids from positions 122 to 141 of a PCV2 protein, AVILDDNFVTKATALTYDPY (SEQ ID NO: 12)

and a sequence variant thereof comprising at most 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 161 to 167 of a PCV2 protein, FTPKPVL (SEQ ID NO: 13) and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution,

- a sequence of amino acids from positions 169 to 189 of a PCV2 protein,

STIDYFQPNN R QLWLRLQT (SEQ ID NO: 14)

and a sequence variant thereof comprising at most 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 200 to 205 of a PCV2 protein, TAFENS or AAFENS (SEQ ID NO: 15)

and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution.

A fragment may be either a fragment of the original sequence or that of the sequence variant.

In a further preferred embodiment the CP of C V comprises inserted one or more preferably one or two peptide segment(s) having 7 to 15 amino acids length, said one or more peptide segments comprising a sequence of amino acids selected from the following group of epitopes or epitope sequences:

sequence of amino acids from positions 224 to 233 of a PCV2 protein, FNLKDPPLKP (SEQ ID NO: 5) and a sequence variant thereof comprising at most 6, 5, 4, 3, 2 or 1 conservative substitution.

In a preferred embodiment the epitopes are inserted into the CP of CMV at an insertion site are selected from the following group of insertion sites:

- an insertion site between amino acids 131 and 132 of the CP of CMV, or - an insertion site between amino acids 83 and 84 of the CP of CMV.

Preferably, the amino acid 131 is a glycine (G), amino acid 132 is a serine (S) or glycine (G), amino acid 83 is a glycine (G) and/or amino acid 84 is a serine (S),

Preferably, one or both of the insertion sites comprises inserted one or more, preferably one or two peptide segment(s).

In a preferred embodiment the recombinant CMV comprises any of the following sequences:

1 11 21 31 41

MDKSGSPNAS RTSRRRRPRR GSRSASGADA GLRALTQQML KLNRTLAIGR

51 61 71 81 91

PTLNHPTFVG SESCKPGYTF TSITL PPEI EKGSYFGRRL SLPDSVTDYD

101 111 121 131 141

KKLVSRIQIR INPLPKFDST VWVTVRKVPS GFNLKDPPLK PXDLSVAAIS

151 161 171 181 191

AMFGDGNSPV LVYQYAASGV QANNKLLYDL SEMRADIGDM RKYAVLVYSK

201 211 221

DDNLEKDEIV LHVDVEHQRI PISRMLPT (SEQ ID NO: 6)

1 11 21 31 41

MDKSGSPNAS RTSRRRRPRR GSRSASGADA GLRALTQQML KLNRTLAIGR

51 61 71 81 91

PTLNHPTFVG SESCKPGYTF TSITLKPPEI EKGFNLKDPP LKPSYFGRRL

101 111 121 131 141

SLPDSVTDYD KKLVSRIQIR INPLPKFDST VWVTVRKVPS GFNLKDPPLK

151 161 171 181 191

PXDLSVAAIS AMFGDGNSPV LVYQYAASGV QANNKLLYDL SEMRADIGDM

201 211 221 231

RKYAVLVYSK DDNLEKDEIV LHVDVEHQRI PISRMLPT (SEQ ID NO: 7)

1 11 21 31 41

MDKSGSPNAS RTSRRRRPRR GSRSASGADA GLRALTQQML KLNRTLAIGR

51 61 71 81 91

PTLNHPTFVG SESCKPGYTF TSITLKPPEI EKGFNLKDPP LKPSYFGRRL

101 111 121 131 141

SLPDSVTDYD KKLVSRIQIR INPLPKFDST VWVTVRKVPS SSDLSVAAIS

151 161 171 181 191

AMFGDGNSPV LVYQYAASGV QANNKLLYDL SEMRADIGDM RKYAVLVYSK

201 211 221

DDNLEKDEIV LHVDVEHQRI PISRMLPT (SEQ ID NO: 8);

wherein X is serine (S) or glycine (G),

or a mutant or sequence variant thereof having at least 70%, 80%, 90% or 95% sequence identity thereof, id mutant or variant being capable of eliciting an immune response against PCV in animals, preferably in pigs. In a preferred embodiment the vaccine is formulated as an injectable preparation and comprising the virus in a purified form. In a more preferred embodiment the vaccine of the invention is formulated for oral or parenteral administration to the animals. In a highly preferred embodiment plants edible by animals are infected with the recombinant CMV virions, the plants propagating the virus, or appropriate parts thereof, e.g. leaves or fruits are processed to animal feed, and the aminals are vaccinated by feeding.

The invention also relates to a nucleic acid encoding the recombinant CP of CMV protein as defined herein.

The invention also relates to a recombinant cucumber mosaic virus (CMV) comprising the recombinant CP of CMV or a nucleic acid encoding it.

Preferably, the recombinant CMV is a virus like particle (VLP). More preferably, in the recombinant CMV said virions are capable of eliciting an immune response upon oral administration to pigs.

The invention also relates to a plant edible by animals expressing said recombinant CMV. The invention also relates to an animal feed. Preferably, the animal feed comprises the vaccine or the recombinant CMV or the plant of the invention. Preferably, said plant is present in a processed form.

The invention also relates to the recombinant CMV or vaccine as defined herein for use in the prevention and treatment of a PCV associated disease (PCVD), preferably a PCV2 associated disease (PCV2D).

Preferably, the PCV associated disease is selected from postweaning multisystemic wasting syndrome (PMWS), porcine respiratory disease complex (PRDC), reproductive failure (RF), porcine dermatitis nephropathy syndrome (PDNS), necrotizing lymphadenitis, congenital tremors, exsudative dermatitis and granulomatous enteritis.

The invention also relates to method for prevention or treatment of a PCV2 related disease in a mammal said method comprising the step of administering a vaccine as defined herein in a pharmaceutically effective amount to said mammal. Preferably, the mammal is pig, more preferably a piglet.

Preferably, the vaccine is in the form selected from an injectable solution, a plant extract and an animal feed comprising a plant as defined above.

In a preferred embodiment said vaccine is administered orally. Highly preferably a boosting vaccination is administered after 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 weeks after the first vaccination.

The dose of the virions is at least 0.05 μg per kg body weight or at least 0.1 or at least 0.2 or at least 0,5 μg per kg body weight as administered or as present in the vaccine dose unit.

Preferably, the dose of the virions is not more than 100 μg per kg body weight, preferably not more than

50 or 30 or 20 μg per kg body weight, more preferably not more than 10, 5 or 1 μ per kg body weight as administered or as present in the vaccine dose unit.

DEFINITIONS

An epitope is a molecular region in, preferably the surface portion of an antigenic molecule capable of eliciting an immune response to which an antibody can bind and preferably against which an antibody can be produced in a living organism. Preferably an epitope is capable of eliciting an immune response at least by a B- cell receptor response in an animal, preferably in a mammal. Preferably, an epitope is an antigenic determinant, i.e. a site in the antigen molecule to which a single antibody molecule binds; preferably an immunogenic determinant the part of an immunogenic molecule that interacts with a helper T cell in triggering antibody production. A vaccine is a pharmaceutical preparation comprising a biological material that is administered to produce or artificially increase immunity to a disease or for the prevention, amelioration or treatment of a disease. The biological material may comprise a macromolecule, e.g. protein or nucleic acid or a combination thereof, virus or part thereof, virus particle, or virus like particle, killed microorganisms, living attenuated organisms, or living fully virulent organisms, said biological material comprising or capable of eliciting the production of antigenic material having at least one epitope.

Porcine circovinis (PCV) is an approx. 17 nm diameter single-stranded nonenveloped DNA virus of the virus family Circoviridae, having an unsegmented circular genome; and an icosahedral viral capsid. PCVs are the smallest viruses replicating autonomously in the infected eukaryotic cells, using the host polymerase for genome amplification.

Cucumber mosaic virus (CMV) is a linear positive-sense, single-stranded plant pathogenic R A virus virus in the family Bromoviridae, genus Cucumovirus; CMV is an isodiametric RNA virus of about 30 nm diameter consisting of three single-stranded RNAs (RNA1 , RNA2 and RNA3), all necessary for infectivity, the coat protein (CP) gene being present both in the genomic RNA3 and in the subgenomic RNA4. Preferably, its total genome size is 8500 to 8700 kb and is broken into three parts; typically, the RNA is surrounded by a protein coat consisting of 32 copies of a single structural protein which form isometric particles.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 The predicted partial surface of the PCV2 virion pentamer. A: The plan view of the outer surface; B: The side view of the pentamer, showing the outer surface and the inner surface part.

Figure 2 A: Location of epitope insertion sites G131/S132 (medium grey) and D176/I177 (light grey) in

CMV. Both sites are found in the asymmetric unit. B: Location of epitope insertion sites G83/S84 (dark grey) and G13 1/S 132 (medium grey) in CMV. G83/S84 is present on the outer surface of the virion pentamer and hexamer whereas site G131/S 132 is present in asymmetric unit.

Figure 3 Immunglobulin serum titers after experimental vaccination of mice. 1 : negative control; 2: intraperitoneal inoculation (40μg virion/animal); 3: intraperitoneal inoculation (CMV infected plant extract, lg leaf extract/animal); 4: subcutan inoculation (CMV infected plant extract, 1 g leaf extract/animal); 5: oral administration (CMV infected plant, lg /animal; IgG titer); 6: oral administration (CMV infected plant, 4 g /animal; IgG titer); 7: oral administration (CMV infected plant, lg /animal; IgA titer); 6: oral administration (CMV infected plant, 4 g /animal; IgA titer).

Figure 4 Indirect immunofluorescence image of insect cells infected by a recombinant baculovirus encoding PCV2 capside protein. Panel A: mouse serum immunized with recombinant CMV; panel B: mouse serum inoculated with wild type CMV. In each case a 1 :80 dilution of the sera has been applied.

Figure 5 Visualization of some epitopes on the X-ray structure of the PCV2 virion (PDB ID code: 3R0R, Khayar et al. 201 1 ). Panel (A) represents the external (A) while (B) illustrates the inner surface of the PCV2 virion. The epitope sequence positions are indicated on the figure. The last seven residues are not present in the X-ray structure but it is well visible that the C-terminal tail (black beads) of the PCV2 capsid protein is located at the edge of the CP pentamer.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have suggested a new approach for PCV vaccination, i.e. to express PCV epitopes on the surface of a plant virus, CMV. A major advantage of this solution is that there is no need to mimic the virion structure such as in VLPs, the recombinant plant virus can readily be used for vaccination. Earlier, cowpea mosaic virus has been utilized in chimeric virus technology for epitope presentation (Usha et al., 1993, Porta et al. , 1994, Chatterji et al., 2002).

CMV has the reputation of having the widest host range of any known plant virus (191 hosts in 40 families), among others but not limited to edible plants (celery, lettuce, cucumber, tomato, carrot, pepper and banana), and is widespread in tropical, subtropical and temperate regions.

Construction of porcine circovirus vaccines based on a plant virus expression system.

Overview

Below the invention is further illustrated by non-limiting examples.

PCV2 viruses, mainly of PCV2B type are readily available not only as virus isolates but also as clones of complete genome sequences in our laboratory at the SZIU. A skilled person can readily obtain PCV2 viruses by isolating the virus from infected pigs in a tissue culture, amplifying the virus nucleic acid and cloning it according to standard methodologies. As an exemplary method, the viruses were isolated using porcine kidney (PK- 15) and swine testicle (ST) continuous cell lines by infecting the cells with homogenates of the infected organs and subculturing infected cells at least 3 times, using standard cell culture procedures. The virus genomes were amplified in two overlapping fragments by polymerase chain reaction (PCR) as described by Csagola et al. (2006), and cloned by standard molecular biology methods.

Sequence of various strains of PCV2 viruses are disclosed e.g. by Wu, SQ et al., in WO2009/085912A1.

In the present examples the following sequence has been used (Meehan B. M. 1998;

SEQ ID NO: 16; GenBank No: AAC35310):

1 11 21- 31 41

MTYPRRRYRR RRHRPRSHLG QILRRRPWLV HPRHRYRWRR KNGIFNTRLS

51 61 71 81 91

RTFGYTVKRT TVTTPSWAVD MMRFKIDDFV PPGGGTNKIS IPFEYYRIRK

101 111 .121 131 141

VKVEF PCSP ITQGDRGVGS TAVILDDNFV TKATALTYDP YVNYSSRHTI

151 161 171 181 191

PQPFSYHSRY FTPKPVLDST I DYFQPNNKR NQL LRLQTS GNVDHVGLGA

201 211 221 231

AFENSKYDQD YNIRVTMYVQ FREFNLKDPP LKP

The selection of the epitopes inserted into the CMV vector was determined based on computer generated three dimensional images of the selected amino acid sequences to make sure that the inserted PCV2 fragment will be even structurally identical to the original one when produced by the plant virus. A PCV2 CP pentamer was created with Symmdock (Schneidman-Duhovny et al. 2005). This PCV2 CP pentamer mimicked a partial external and internal surface of PCV2 virion. Five outer loop regions were identified on the basis of visual observation of the PCV2 pentamer three-dimensional model. These PCV2 CP loops can act as potential epitopes. The predicted PCV2 epitope sequences are follows: PCV2 37-43(7): RWRRKNG (SEQ ID NO: 1), PCV2_90- 96(7): S1PFEYY (SEQ ID NO: 2), PCV2J 26- 145(20): DDNFVTFC ATALTY DPYVNYS (SEQ ID NO: 3), PCV2_169- 186(18): STIDYFQPNNKRNQLWLR (SEQ ID NO: 4), PCV2_224-233(10): FNL DPPLKP (SEQ ID NO: 5).

Based on the three dimensional structure of a PCV2 protein (Khayat R. et al., 201 1) the following further epitopes have been predicted: PCV2_52-62( 1 1): TFGYTVKRTTV (SEQ ID NO: 9), PCV2_76-83(8): IDDFVPPG (SEQ ID NO: 10), PCV2_103- 11 1 (9): VEFWPCSPI (SEQ ID NO: 1 1), PCV2_122- 141(20): AVILDDNFVTKATALTYDPY (SEQ ID NO: 12), PCV2_161-167(7): FTP PVL (SEQ ID NO: 13), PCV2 169- 189(21): STIDYFQPNNKRNQLWLRLQT (SEQ ID NO: 14), PCV2_200-205(6): TAFENS or AAFENS (SEQ ID NO: 15).

Altogether twelve hybrid CMV CPs were constructed. Two predicted PCV2 epitope sequences were used to replace the original five CMV CP loop regions one by one and all five at once. The PCV2 epitopes were as follows: PCV2_126-145(20): DDNFVTKATALTYDPYVNYS (SEQ ID NO: 3), PCV2_224-233(10): FNLKDPPLKP (SEQ ID NO: 5). The exchanged CMV CP loop regions were the following sequence fragments: 1. loop (βΒ-pC) 76-83, 2. loop (pD-βΕ) 1 13-1 18, 3. loop (βΕ-aEF) 129- 136, 4. loop (βΡ-βΰ) 156- 163 and 5. loop (βΗ-βΙ) 193-199.

Initial experiments were unsuccessful as replacement of the CMV CP loop regions, an obvious choice due to structural similarity of the two virus' coat proteins, resulted in recombinant CMVs unable to move. This would render high scale propagation in plants impossible.The selected epitope coding sequences were tagged with common restriction enzyme recognition sequences in order to facilitate the insertion into the CMV genome. The antigenicity of the nanoparticles generated by the partners was tested by immunological techniques (ELISA, Western blot, immune fluorescence). The final testing of promising particles was performed by immunizing animals. The first phase of the package gave a picture about the effectiveness of the different epitopes and combinations of the epitopes produced on CMV surfaces.

Recombinant CMV infected plants can be processed differently and used for the testing of different routes of immunization, for example:

a) infected plants without any processing directly fed to mice,

b) crude extracts of the plants fed to groups of mice,

c) crude extracts injected intramuscularly,

d) purified viruses given orally,

e) purified viruses given parenterally.

Based on the evaluation of results pigs are used and both parenteral and oral vaccination regimes are applied. Pregnant sows are treated with the antigen and maternal immunity is tested by measuring the PCV2 specific colostral antibody titers and the antibody levels of the offspring up to the weaning age. Previously non- immunized piglets, from both PCV2 infected and non-infected sows are dosed with the recombinant antigens and tested for PCV2 antibody levels. To evaluate the success of oral immunizations particular attention is paid to mucosal immunity and besides the systemic IgG response intestinal contents are checked for the presence of PCV2 specific secretory (s)IgA molecules.

Administration of the vaccine is possible both parenterally and orally. To set up an oral administration regime in general is within the skills of a person skilled in the art (O'Hagan, D.T. 2000). Evaluation of the vaccine may be carried out as described in Example 4. Preferred parenteral administration routes are administration subcutane (sc) or intramuscularly (im), preferably by injection. Technology of parenteral administration is well known for a person skilled in the art. Adjuvants suitable for veterinary application and vaccine formulation are also well-known in the art [see e.g. Aunins, J. G. et al. (2000), O'Hagan, D.T. (2000)]. In theory, any adjuvant known for sc. or im. administration are applicable in the subject invention. Selection of the most appropriate vaccine is within the skills of a person skilled in the art. In this variant of the invention expediently the purified virion is applied as an active agent in the inventive vaccines.

In a preferred embodiment the vaccine according to the invention is an oral vaccine wherein administration is carried out by mixing the vaccine into food or drinking water of the animals. The following preferred embodiments are contemplated.

- The purified virion can be mixed into the drinking water of animals.

- The purified virion can be admixed to the feed of the animals.

- The virus can be expressed in a plant which is applicable as an animal feed e.g. a fodder crop, the plants are harvested and processed and feed the animals thereby.

Setting doses and administration schedules are as described herein. Based on the present teaching the person skilled in the veterinary science will be able to set doses and schedules in the knowledge of the need of the animals.

The tools (PCV2 from each genotype, in case of cell cultures both cultures free of PCV and cultures permanently infected with PCV2, primers and non-radioactively labeled oligonucleotides; [see eg. Tebu-Bio (France and Portugal); the PCV2B isolate is also available (example: GenBank accession number AF20131 l)]are all available to a person skilled in the art as well as methodologies (direct and indirect immune-fluorescence, Western blotting, in situ hybridization, Northern blotting, PCR and cloning) as well as the necessary equipment (thermocyclers for PCR, centrifuges, fluorescent microscope; fully equipped animal housing and post mortem facilities) required. Monoclonal antibodies to PCV2 Cap epitopes are readily available at different research and diagnostic laboratories. The polyclonal antisera were produced at the SZIU by hyperimmunizing pigs. A group of pigs at the age of 2 months were infected orally with 10⁴ TCID₅₀ (tissue culture infectious dose) of PCV2B, and boosted twice, 3 weeks apart with the same amount of virus injected simultaneously intramuscularly and intraperitoneally. Animals were exterminated 2 weeks after the second boost, blood sera were collected and titered for PCV2 specific antibodies using a standard indirect immune fluorescence test on PCV2 infected cell cultures. The titer of the pooled sera was at least 1 : 10000.

Experimental part

EXAMPLE 1 - Three dimensional model of PCV2 coat protein

Three dimensional structure of the PCV2 coat protein has not been experimentally determined so far. Therefore the present inventors applied the protein structure prediction method of Roy Z et al. and applying it the present task has prepare theoretical models of the PCV2 coat protein. The PCV2 coat protein (CP) structure was generated with I-TASSER (Roy et al. 2010, Zhang 2008). The model was built using the PCV2A CP sequence (NCBI/GenBank accession number: AAC35310. The following experimentally determined templates were used to thread the PCV2 CP structure: PDB ID codes: 2EIG (Lotus tetragonolobus seed lectin), 1 V6I (Peanut lectin), 1C8N (Tobacco necrosis virus CP), INTO (CUB 1 -EGF-CUB2 region of mannose-binding protein), 1NG0 (Cocksfoot mottle virus CP), 1JOD (pituitary adenylate cyclase-activating polypeptide), 1 E4B (L-fuculose-1- phosphate aldolase). The model structure was refined with energy minimization in order to eliminate the steric conflicts between the protein side chain atoms.

The obtained fold of the most probable model structure is nearly identical with the fold of the CMV coat protein (CP) with the difference that the outer loops of the beta barrel are much longer in the PCV2 coat protein.

EXAMPLE 2 - Engineering recombinant CMV virions

Two experimental routes have been selected:

(A) Preparation of CMV CP chimeras designed based on the epitopes predicted using the PCV2 coat protein model and virion structure. Altogether 8 chimeric coat protein structures have been designed. They were denominated based on their inserstion site and the site of origin of the epitope as follows:

l . RCMV131- PCV2_169- 186

2. RCMV 131- PCV2J 69-186-RCMV 176- PCV2J 69- 186

3. RCMV131- PCV2J26- 145

4. RCMV131- PCV2_224-233

5. RCMV131- PCV2_224-233- RCMV176- PGV2_224-233

6. RCMV131-PCV2_37-43

7. RCMV131-PCV2J7-43-RCMV176- PCV2_37-43

8. RCMV131- PCV2_90-96.

(B) Preparation of CMV CP chimeras designed based on experimentally determined epitopes. From these chimeras altogether 4 constructs have been designed:

l . RCMV131- PCV2_65-87

2. RCMV131- PCV2J 17- 131

3. RCMV131- PCV2J46- 166

4. RCMV131 -PCV2J46- 166-RCMV176- PCV2 146

(C) Further epitopes have been proposed by the 3D (X-ray) structure of PCV2 coat protein:

1. RCMV131 - PCV2 52-62

2. RCMV 131- PCV2_76-83

3. RCMV131- PCV2_103- 1 1 1

4. RCMV131 - PCV2J22- 141

5. RCMV 131 - PCV2 161 - 167

0. RCMV131 - PCV2 169- 189 7. RCMV131- PCV2 200-205.

Replacing loops ofCMV CP with PCV2 CP loops

As a first trial predicted PCV2 CP loops have been inserted to replace CMV CP loops. By protein structure prediction those regions of the R-CMV and PCV coat proteins, which form loop regions in homologous positions have been determined. These segments of the CMV coat protein were replaced by the corresponding PCV loops, one by one. Thus the part of the CMV coat protein from amino acid (aa) residue 1 13 to residue 120 was replaced by a 13 amino acid long segment of PCV origin, and part from aa 129 to aa 136 was replaced by an 8 aa long segment, the part from aa 153 to aa 163 was replaced by a 12 aa long segment, wheras the part from aa 189 to aa 199 was replaced by an 1 1 aa long segment. After checking the nucleotide sequence the mutant clones were introduced into infectious clones and effecting a co-transfection together with RNA 1 and 2 transcripts test plants were inoculated. The recombinant virus could be detected in the inoculated leaves but it did not occur in the uninfected systemic leaves. Probably the movement of the virus is inhibited in lack of the coat protein segments removed. In the inoculated leaves the regions of PCV origin were not stable and were deleted soon.

Base on the present experimental results CMV outer loops should not be altered as the virus becomes unable to move, which blocks high scale propagation of the virus in plants. It is not obvious therefore which site is appropriate for stable insertion of epitopes into the CMV virion, and it could .not be known whether PCV2 epitopes, once inserted into sites different from outer loop regions, will or will not be able to elicit immune response.

Insertin experimentally identified epitopes into further insertion sites

In spite of the negative results the present inventors have gone ahead and sought further insertion sites.

Three further insertion sites emerged as possibilities [see Fig. 2: G131/S 132 (medium grey), D176/I177 (light grey) and G83/S84 (dark grey), c.f. Nuzzaci M. et al. 2007].

Two of these sites are present in the middle of the asymmetric unit. Based on the modeling of the symmetry properties of the virion the epitope inserted into the G131/S 132 sites (medium grey) should appear on the outer surface of the virion, epitopes inserted into D 176/1177 sites should be expressed towards the inner side of the virion. Epitope expression towards the inside of the virion has an electrostatic limit, i.e. that due to RNA binding only a peptide segment with positive overall charge can be inserted here expediently. About the G83/S84 site it has been reported that it has a destabilizing effect on the virion [Nuzzani et al. 2007].

From this point new constructs were built from which the CMV loop regions were not lacking, thus it was assumed that the systematic movement of the virus is not hindered.

In these cases after position aa 131 of the coat protein PCV regions of various length were inserted., Codons defining the amino acids were selected so as to minimize stem-loop structures in the RNA, thereby reducing the possibility of RNA recombination. Clones of recombinant CMV coat protein were, after checking the nucleotide sequence, inserted into infectious clones. With transcripts made from recombinant RNA 3, and the native RNA 1 and 2 plants have been infected. In case of CMV comprising the shortest, 10 aa insert the virus could be determined by RT/PCR from the systemically infected leaves. The recombinant virion was purified, and the nucleotide sequence analysis proved the stability of the inserted region. Further analysis of stability in different host species during long term maintenance and a study on the further constructs are underway. EXAMPLE 3 - Eliciting and assessing immune response in mice

So as to precisely define the immune response inducible by the recombinant nanoparticles it was necessary to test the following features:

1) What kind of vaccination methods are suitable for use with a vaccine of this type?

2) What is the strength and type of the immune response expectable from the various ways of administration?

3) Is the antibody detecting method suitable for the measurement of local and/or systemic antibody levels? The following experiments were carried out with a wild type virus.

Mice (8 weeks old SPF outbred females, CRL: NMRI BR, Charles River, USA) were divided into 8 groups comprising 5-5 animals per groups, except groups 7 and 8 comprising 10-10 animals. Experimental setting was as follows: Group 1 : negative control, Group 2: intraperitoneally (40 μ /πιου56), Group 3: intraperitoneally (CMV infected plant extract, 1 g leaf extract/mouse), Group 4: subcutane inoculation (CMV infected plant extract, 1 g leaf extract per mouse), Groups 5 and 7: per os (1 g CMV infected plant/mouse), Groups 6 and 8: 4 g CMV infected plant/mouse. From groups 7 and 8 5-5 mice were euthanized on day 14 and the last day of the experiment so as to detect secretory IgA. Inoculations (feedings) were repeated on day 14 after the start of the experiment without using adjuvant. Antibody response was measured by ELISA using a purified CMV antigen, specific mouse IgG or IgA conjugates. Serum dilution series were started with dilution ration 1 :50 in case of serum samples, whereas in case of intestinal content samples with a 1 :2 dilution ratio, and were halved there from. Results are seen in Figure 3 without standard error values which were negligible. In each inoculated and fed group of mice detectable specific immune response was elicited. Circulating IgG could be measured on week 2 after the virus administration in case of feeding experiments, but only in the group which received a higher dose. Appearance of IgA in parallel groups showed a similar pattern. Interestingly, in aminals treated per os a dose related difference in circulating antibody levels was not found, but in local IgA level a stronger immune response was measured at the higher dose.

Immunogenicity testing of the recombinant CMV viruses expressing PCV epitopes can be made analogously.

So as to test the stable recombinant virus in mice the following experiment was carried out.

Epitopes appeared on stable recombinant virions (CMVcp l31-PCV224-233) were studied to decide whether they were capable of reacting with PCV2-specific polyclonal antibodies of swine origin. Multiple methods were used for this purpose among which indirect ELISA proved to be the most appropriate to obtain an answer. During ELISA studies purified and concentrated recombinant and wild type CMV particles were transferred into aqueous solution and various dilutions of the solutions were reacted with sera positive or negative to multiple PCV2 viruses. In an experiment pig serum exhausted by the wild type CMV was used The results showed that on the CMV surface the epitope appears in an appropriate conformation, i.e. it could react with antibodies elicited due to the PCV2 infection.

Subsequent testing of the PCV2 specific immunogenity of the recombinant nanoparticles was carried out in mice, in two phases. In each case 8 weeks old SPF female laboratory mice were used (CRL: NMRI BR, Charles River, USA) and earlier immunization results with non-recombinant viruses were taken into account. First inoculation experiment aimed at deciding whether the PCV2 epitope presented on the CMV2 surface and detected by ELISA was capable of inducing a PCV2 capsid specific immune response. As a negative control purified wild type CMV was used. For this purpose two groups of laboratory mice were immunized, the concentrated virus (recombinant or wild type) was inoculated intraperitoneally into the mice with a Freund adjuvant (1 : 1 ratio) and on day 14 after the first inoculation vaccination was repeated. Mice were exterminated on day 30 of the experiment and sera were separated. PCV2 specificities of antibodies from the sera were measured in multiple experimental systems. Among these the PCV2-capsid variant prepared by baculovirus proved to be the most successful, when insect cells infected by recombinant recombinant baculoviruses were fixed by standard methods and the presence of antibodies was shown in an indirect immunefluorescence test (Figure 4). In each other method high background values were found which made assessment difficult.

EXAMPLE 4 - Eliciting and assessing immune response in piglets

PCV2 challenge of immunized pigs

Thirteen conventional PCV2 free piglets were weaned at 2 weeks of age and divided into 3 groups. The experiments started at the age of 4 weeks. Five piglets (group 1) were injected intramuscularly with 2 mg of the recombinant CMV (epitope CMVcp l31-PCV224-233; the same as with mice), mixed with incomplete Freund's adjuvant, in a total volume of 0.5 ml. Five piglets (group 2) were vaccinated with inactivated PCV2 vaccine (Circovac, Merial). Immunizations were repeated after 10 days. The remaining 3 animals were mock immunized (group 3). Ten days after the second immunization all of the piglets were challenged with virulent live PCV2 (strain R15, kindly provided by dr. A. Csagola, Szent Istvan University, Budapest, Hungary, isolated from a PMWS affected pig and cultured in PCV free swine testicle cells in vitro), administered both orally (5x103 TCID50) and intraperitoneally (2x103 TCID50). Blood samples were collected weekly for PCV2 antibody testing. The piglets were euthanized following general anesthesia on the 23rd day after challenge. Post mortem examinations were carried out; organ samples (heart, lung, liver, spleen, kidney, tonsil, mediastinal and inguinal lymph nodes) were collected from each animal and stored at -20°C until processing.

Detection ofPCV2 DNA in infected pigs

Samples were homogenized using TissueLyser II (Quiagen) and viral DNA was extracted using the InnuPREP Virus DNA/RNA Kit (Analytik Jena AG) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qPCR) was used to detect PCV2 DNA. Primers (KCV-F: 5 '- AAGTAGCGGG AGTGGTAGG A-3 ' , SEQ ID NO: 16, and KCV-R: 5 '-GGGCTCC AGTGCTGTTATTC-3 ' , SEQ ID NO: 17) and the TaqMan probe (KCV-P: 5'FAM- TCCCGCCATACCATAACCCAGC-3'BHQ1 , SEQ ID NO: 18) were designed for the PCV2-R15 capsid gene sequence with the online tool (https://www.genscript.com/ssl-bin/app/primer: FAM: 6-carboxyfluorescein; BHQ1 : black hole quencher 1). To determine copy numbers, a serial dilution of pET6xHN plasmid (Clontech) with the PCV2 capsid gene insert was used as internal standard. PCR reactions were performed in 50 μΐ reactions, each containing 36.3 μΐ water, 5 μΐ DreamTaq buffer (Fermentas), 1 μΐ (0.5 mM) MgC12 (Fermentas), 1 μΐ (0.2mM) dNTP mixture (Fermentas), 0.5 μΐ of each primer and the probe (0.1 μΜ), 0.2 μΐ (1 unit) polymerase (DreamTaq, Fermentas) and 5 μΐ sample DNA. Reactions were performed in the Mastercycler Realplex thermocycler (Eppendorf) as follows: 94 °C for 2 minutes, 30 cycles of 94 °C, 30 s, 60 °C 30 s and 72 °C, 45 s, followed by cooling to 25 °C.

Antigenicity and immunogenicity of the recombinant PCV2 epitopes

The ELISA test showed that the PCV2 epitope was present in the CMV capsid, as the polyclonal PCV2 specific pig antibodies reacted with the recombinant construct but not with the wild type virus. The titer ( 1 : 10000) of the pig serum measured with the original PCV2 virus antigen was lower ( 1 : 100) when using the recombinant virus.

The immune sera collected from mice vaccinated with the recombinant CMV construct induced PCV2 specific antibodies, as indicated by the immune fluorescence test (Fig. 6). The titer of the antibodies was between 1 :80 and 1 :320.

PCV2 specific antibodies in pigs appeared after the second immunization in group 1 , after the first immunization in group 2 and only after challenge in group 3. Table 1 shows the results of PCV2 detection in different organ samples. No PCV2 DNA was detected in group 2 and the virus was demonstrated in only 2 animals after challenge in group 1. The DNA copy numbers measured by qPCR varied between 10³ (in lungs of one piglet) and 10⁵ (mediastinal lymph node of another piglet) in group 3. In group 1 both the lungs and the mediastinal lymph nodes of one piglet contained PCV2 DNA, but the rest of the organs were free of the virus. PC V2 was also detected in another piglet of the same group but the virus in this animal was only present in the lungs.

Table 1. The presence of PCV2 DNA in tissue samples (positive sample/total of pigs in the group).

INDUSTRIAL APPLICABILITY

It has been shown by the present inventors that it is possible to prepare an effective porcine circovirus (PCV) vaccine comprising a recombinant cucumber mosaic virus (CMV) capable of producing virions in plants comprising said recombinant CMV.

SEQUENCE LISTING

MTA Mezogazdasagi Kutatointezet

Mezogazdasagi Biotechnologiai utatokozpont

Szent 1st an Egyetem Allatorvos-tudomanyi Kar

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Claims

1. A porcine circovirus (PCV) vaccine comprising a recombinant cucumber mosaic virus (CMV) capable of producing virions in plants comprising said recombinant CMV,

wherein the recombinant virus coat protein (CP) of said CMV comprises one or more epitopes of the coat protein (CP) of a PCV, said virions being capable of eliciting an immune response in mammals.

2. The PCV vaccine of claim 1 wherein,

the CP of CMV comprises inserted one or more peptide segment(s) having 7 to 22 amino acids length, said one or more peptide segments comprising a sequence of amino acids selected from the following group of epitope sequences:

- a sequence of amino acids from positions 37 to 43 of a PCV2 protein, RWRRKNG (SEQ ID NO: 1) and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution,

- a sequence of amino acids from positions 90 to 96 of a PCV2 protein, S I PFEYY (SEQ ID NO: 2) and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution,

- a sequence of amino acids from positions 126 to 145 of a PCV2 protein, DDNFVTKATALTYDPYVNYS (SEQ ID NO: 3)

and a sequence variant thereof comprising at most 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof

- a sequence of amino acids from positions 169 to 186 of a PCV2 protein, S I DYFQPNNKRNQLWLR (SEQ ID NO: 4)

and a sequence variant thereof comprising at most 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof

- a sequence of amino acids from positions 224 to 233 of a PCV2 protein, FNLKDPPLKP (SEQ ID NO: 5) and a sequence variant thereof comprising at most 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 52 to 62 of a PCV2 protein, TFGYTVKRTTV (SEQ ID NO: 9) and a sequence variant thereof comprising at most 7, 6, 5, 4,

3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 76 to 83 of a PCV2 protein, IDDFVPPG (SEQ ID NO: 10) and a sequence variant thereof comprising at most 5,

4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 103 to 11 1 of a PCV2 protein, VEFWPCSPI (SEQ ID NO: 1 1 ) and a sequence variant thereof comprising at most 6,

5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 122 to 141 of a PCV2 protein, AVILDDNFVTKATALTYDPY (SEQ ID NO: 12)

and a sequence variant thereof comprising at most 9, 8, 7,

6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 161 to 167 of a PCV2 protein, FTPKPVL (SEQ ID NO: 13) and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution, - a sequence of amino acids from positions 169 to 189 of a PCV2 protein, STIDYFQPNN RNQLWLRLQT (SEQ ID NO: 14)

and a sequence variant thereof comprising at most 9, 8,

7, 6, 5, 4, 3, 2 or 1 conservative substitution, or an at least 7 amino acid long fragment thereof,

- a sequence of amino acids from positions 200 to 205 of a PCV2 protein, TAFENS or AAFENS (SEQ ID NO: 15)

and a sequence variant thereof comprising at most 4, 3, 2 or 1 conservative substitution.

The PCV vaccine of claim 1 or claim 2 wherein,

the CP of CMV comprises inserted one or more (one or two) peptide segment(s) having 7 to 15 amino acids length, said one or more peptide segments comprising a sequence of amino acids selected from the following group of epitope sequences:

sequence of amino acids from positions 224 to 233 of a PCV2 protein, F LKDPPLKP (SEQ ID NO: 5) and a sequence variant thereof comprising at most 6, 5, 4, 3, 2 or 1 conservative substitution.

The PCV vaccine of any of claims 1 to 3, wherein the epitopes are inserted into the CP at an insertion site selected from the following group of insertion sites:

- an insertion site between amino acids 131 and 132 of the CP of CMV, or

- an insertion site between amino acids 83 and 84 of the CP of CMV.

The PCV vaccine of claim 4, wherein the

amino acid 131 is a glycine (G),

amino acid 132 is a serine (S) or glycine (G),

amino acid 83 is a glycine (G),

amino acid 84 is a serine (S),

The PCV vaccine of claim 3 or 5 wherein one or both of the insertion sites comprises inserted one or more peptide segment(s).

The PCV vaccine of claim 6, wherein the recombinant CMV comprises any of the following sequences:

1 11 21 31 41

MDKSGSPNAS RTSRRRRPRR GSRSASGADA GLRALTQQML LNRTLAIGR

51 61 71 81 91

PTLNHPTFVG SESCKPGYTF TSITLKPPEI EKGSYFGRRL SLPDSVTDYD

101 111 121 131 141

KKLVSRIQIR INPLPKFDST VWVTVRKVPS GFNLKDPPLK PXDLSVAAIS

151 161 171 181 191

AMFGDGNSPV LVYQYAASGV QANNKLLYDL SEMRADIGDM RKYAVLVYSK

201 211 221

DDNLEKDEIV LHVDVEHQRI PISRMLPT (SEQ ID NO: 6)

1 11 21 31 41

MDKSGSPNAS RTSRRRRPRR GSRSASGADA GLRALTQQML KLNRTLAIGR

51 61 71^' 31 91

PTLNHPTFVG SESCKPGYTF TSITLKPPEI EKGFNLKDPP LKPSYFGRRL SLPDSVTDYD KKLVSRIQIR INPLPKFDST VWVTVRKVPS GFNLKDPPLK

151 161 171 181 191

PXDLSVAAIS AMFGDGNSPV LVYQYAASGV QANNKLLYDL SEMRADIGDM

201 211 221 231

RKYAVLVYSK DDNLEKDEIV LHVDVEHQRI PI SRMLPT (SEQ ID NO: 7)

1 11 21 31 41

. MDKSGSPNAS RTSRRRRPRR GSRSASGADA GLRALTQQML KLNRTLAIGR

51 61 71 81 91

PTLNHPTFVG SESCKPGYTF TSITL PPEI EKGFNLKDPP LKPSYFGRRL

101 111 121 131 141

SLPDSVTDYD KKLVSRIQIR INPLPKFDST VWVTVRKVPS SSDLSVAAIS

151 161 171 181 191

AMFGDGNSPV LVYQYAASGV QANNKLLYDL SEMRADIGDM RKYAVLVYSK

201 211 221

DDNLEKDEIV LHVDVEHQRI PISRMLPT (SEQ ID NO: 8)

wherein X is serine (S) or glycine (G),

or a mutant or sequence variant thereof having at least 70%, 80%, 90% or 95% sequence identity thereof, said mutant or variant being capable of eliciting an immune response against PCV in animals, preferably in pigs.

8. The vaccine of any of the previous claims formulated for oral or parenteral administration to the animals.

9. A recombinant cucumber mosaic virus (CMV) comprising the recombinant CP of CMV or a nucleic acid encoding it.

10. The recombinant CMV as defined in any of claims 1 to 9 wherein said virions are capable of eliciting an immune response upon oral administration to pigs.

11. The recombinant CMV or the PCV vaccine as defined in any of the previous claims for use in the prevention or treatment of a PCV associated disease (PCVD), preferably a PCV2 associated disease (PCV2D),

12. The recombinant CMV or the PCV vaccine according to claim 1 1 wherein the PCDV is postweaning multisystemic wasting syndrome (PMWS).

13. A plant edible by animals expressing a recombinant CMV of any of claims 9 to 1 1.

14. An animal feed comprising the vaccine of any of claims 1 to 8 or a recombinant CMV of any of claims 9 to 1 1.

15. The animal feed of claim 14 comprising the plant of claim 13 in a processed form.