CA2383367A1 - Prevention of myocarditis, abortion and intrauterine infection associated with porcine circovirus-2 - Google Patents

Abstract

What is described is a recombinant poxvirus, such as avipox virus, containin g foreign DNA from porcine circovirus (2). What are also described are immunological compositions containing the recombinant poxvirus for inducing an immunological response in a host animal to which the immunological compositi on is administered. Also described are methods of treating or preventing diseas e caused by porcine circovirus (2) by administering the immunological compositions of the invention to an animal in need of treatment or susceptib le to infection by porcine circovirus (2).

Description

Prevention of myocarditis, abortion and intrauterine infection associated with porcine circovirus-2.
FIELD OF THE INVENTION
The invention relates to methods and/or compositions for the prevention and/or treatment of PCV-2-caused myocarditis, and/or abortion and/or intrauterine infection, as well as pathologic sequelae including but not limited to post-weaning multisystemic wasting syndrome; and, to methods for preparing such compositions and kits for preparing such compositions or for performing such methods, inter alia.
BACKGROUND OF THE INVENTION
Porcine circovirus-2 (PCV-2) was recently identified as an agerit -that has been consistently associated with post-weaning multisystemic wasting syndrome (PMWS) in swine populations in several parts of the world (Allan et al. 1998; Ellis et al., 1998). Isolates of PCV-2 obtained from infected pigs in several countries are virtually identical genetically, and are distinctly different from the PCV (CCL33, PCV-1) that was originally identified in the 1970's as a noncytopathic contaminant of porcine kidney (PK/15) cell line (Meehan et al.
1998; Tischer et al. 1974). Pigs with naturally acquired or experimentally induced PCV-2 infections present with progressive weight loss, tachypnea, dyspnea, and jaundice (Allan et al.
1998; Allan et al. 1999; Ellis et al. 1998; Ellis et al. 1999). Gross pathologic findings that have been directly associated with PCV-2 antigen include, lymphadenopathy, interstitial pneumonia, hepatitis and nephritis (Allan et al. 1998; Allan et al. 1999;
Ellis et al. 1998; Ellis et al. 1999). See also WO-A-99 18214, WO-A-00 01409, WO-A-99 29717 and WO-A-99 29871. PCV-2 has not heretofore been directly linked to abortion or lesions in fetal pigs.
Thus, heretofore, it has not been proposed to address the issue of PCV-2-caused myocarditis, and/or abortion and/or intrauterine infection.
OBJECTS AND SUMMARY OF THE INVENTION
It has surprisingly been found that PCV-2 is a causative agent of myocarditis, abortion and intrauterine infection, as well as post-weaning multisystemic wasting syndrome.
By definition, a PCV-2 immunogen is intended to encompass live attenuated or inactivated PCV-2, or subunit(s) from PCV-2 obtained by in vitro expression or by extraction, or fragments) comprising at least one epitope of interest which can be obtained by chemical synthesis or by in vitro recombinant expression, as well as recombinant vectors) comprising and expressing in vivo sequences) or fragments) or epitope(s) of PCV-2 genome as herein disclosed or as in documents cited or referenced herein.
A similar definition applies for an immunogen of another porcine pathogen as disclosed herein.
By definition, an immunogenic composition elicits an immunogenic response -local or systemic. A vaccine composition elicits a local or systemic protective response. The term "immunogenic composition" include a "vaccine composition" (as the former term can be protective composition).
Thus, an object of the invention can be to provide methods and/or compositions for the prevention and/or treatment of PCV-2-caused myocarditis, and/or abortion and/or intrauterine infection, as well as post-weaning multisystemic wasting syndrome and/or pathologic sequelae including but not limited to post-weaning multisystemic wasting syndrome; and, methods for formulating such compositions (which compositions can also include a porcine parvovints (PPV) immunogen) and uses of a PCV-2 immunogen for formulating such compositions.
Another object is also the use of PCV-2 immunogens to prepare compositions for prevention and/or treatment of PCV-2 caused myocarditis, and/or abortion.
and/or intrauterine infection.
Another object of the invention is the isolation and characterisation of new strains identified 1103 (1103/1 P.2) and 1121 (1121/1 P.1), and their uses to produce immunogens, as well as antigens and antibodies for diagnostics, in relation with PCV-2-caused myocarditis, and/or abortion and/or intrauterine infection, as well as post-weaning multisystemic wasting syndrome and/or pathologic sequelae associated therewith.
The invention provides also for inoculation of female pigs (e.g., sows, gifts) with a composition comprising a (at least one) PCV-2 immunogen (which composition can also include an immunogen from porcine parvovirus) prior to breeding; and/or prior to serving, and/or during gestation (or pregnancy); and/or prior to the perinatal period or farrowing;
and/or repeatedly over a lifetime, to prevent myocarditis and/or abortion and/or intrauterine infection associated with PCV-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2; or, to elicit an immunogenic or protective response against PCV-2 and thereby prevent post-weaning multisystemic wasting syndrome and/or myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2 and/or other pathologic sequelae associated with PCV-2.
Advantageously, at .least one inoculation is done before serving. It is also advantageously followed by an inoculation to be performed during gestation, e.g., at about mid-gestation (at about 6-8 weeks of gestation) and/or at the end of gestation (at about 11-13 weeks of gestation). Thus, an advantageous regimen is an inoculation before serving and a booster inoculation during gestation. Thereafter, there can be reinoculation before each serving and/or during gestation at about mid-gestation (at about 6-8 weeks of gestation) and/or at the end of gestation (at about 11-13 weeks of gestation). Preferably, reinoculation can be during gestation only.
In another preferred embodiment, piglets, such as piglets from vaccinated females (e.g., inoculated as herein discussed), are inoculated within the first weeks of life, e.g., inoculation at one and/or two and/or three and/or four and/or five weeks of life. More preferably, piglets are first inoculated within the first week of life or within the third week of life (e.g., at the time of weaning). Even more advantageous, such piglets are then boosted two (2) to four (4) weeks later (after being first inoculated). Thus, both offspring, as well as female pig (e.g., sow, gilt) can be administered compositions of the invention and/or can be the subject of performance of methods of the invention.
Thus, the invention comprehends immunogenic or vaccine compositions comprising immunogen(s) from PCV-2 strains) 1103 and/or 1121, for preventing or treating myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2.
And, the invention further comprehends uses of a PCV-2 immunogen to formulate an immunogenic or vaccine composition for preventing or treating myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2.
Further still, the invention comprehends an immunogenic or vaccine composition for the prevention and/or treatment of PCV-2-caused myocarditis, and/or abortion and/or intrauterine infection and/or post-weaning multisystemic wasting syndrome comprising a pharmaceutically or veterinarily acceptable carrier and/ or vehicle and/or excipient and/or adjuvant, and a PCV-2 immunogen.
The composition can -additionally include at least one immunogen from at least one additional pig pathogen, e.g.: Porcine Reproductive and Respiratory Syndrome (PRRS), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, E. coli, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhusiopathiae, Pseudorabies, Hog cholera, Swine Influenza, and Porcine Parvovirus (PPV). Thus, vector-based compositions can include at least one immunogen from at least one additional pig pathogen, such as a vector expressing a sequence from this pathogen, wherein the vector can also be the vector expressing the PCV-2 immunogen. The vector expressing a PCV-2 sequence can comprise a PCV-2 sequence or fragment ; and the invention comprehends such nucleic acid molecules, vectors containing them, compositions comprising such nucleic acid molecules or vector expression products from such nucleic acid molecules, compositions comprising such expression products, probes or primers for such nucleic acid molecules, and methods for making and using any or all of the foregoing.
The vector can comprise a DNA vector plasmid, a bacteria such as an E. coli, a vims such as baculovirus, a herpesvirus including pig herpes viruses, including Aujeszky's disease virus, an adenovirus including a porcine adenovirus, a poxvirus, including a vaccinia virus, an avipox virus, a canarypox virus, a racoonpox and a swinepox vims, and the like. The vector-based compositions can comprise a vector that contains and expresses an ORF
selected from the group consisting of ORFs 1 to 13, such as an ORF selected from ORFs 4, 7, 10 and 13;
preferably ORFs 4 and/or 13, of a PCV-2, advantageously of any one of the PCV-2 strains identified herein and in particular of strains 1103 and/or 1121. And, the immunogen in compositions (either PCV-2 and/or from another pig pathogen) can be recombinantly produced.
The word plasmid is intended to include any DNA transcription unit in the form of a polynucleotide sequence comprising the PCV sequence to be expressed.
Advantageously, the plasmid includes elements necessary for its expression; for instance.
expression in vivo. The circular plasmid form, supercoiled or otherwise, is advantageous; and, the linear form is also included within the scope of the invention. The plasmid immunogenic or vaccine composition can be administered by way of a gene gun, intradermally via an needleless injector, subcutaneously or intramuscularly, or by mucosal route, or by any other means that allows for expression in vivo, and advantageously an immunogenic or protective response.
It is noted that the expression product generated by vectors or recombinants in this invention optionally can also be isolated and/or purified from infected or transfected cells; for instance, to prepare compositions for administration to pigs; however, in certain instances, it may be advantageous not to isolate and/or purify an expression product from a cell; for instance, when the cell or portions thereof enhance the immunogenic effect of the polypeptide.
And, techniques for protein purification and/or isolation are known and can be used, without undue experimentation, to purify and/or isolate recombinant or vector expression products and/or subunits of PCV-2 and/or other pig pathogens, in the practice of the invention ; such techniques, in general, can include: precipitation by taking advantage of the solubility of the protein of interest at varying salt concentrations, precipitation with organic solvents, polymers and other materials, affinity precipitation and selective denaturation; column chromatography, including high performance liquid chromatography (HPLC), ion-exchange, affinity, immunoaffinity or dye-ligand chromatography; immunoprecipitation, gel filtration, electrophoretic methods, ultrafiltration and isoelectric focusing, and their combinations, inter alicc.
The invention further envisages methods for the prevention and/or treatment of porcine circovirus-2 (PCV-2)-caused myocarditis, and/or abortion and/or intrauterine infection and/or post-weaning multisystemic wasting syndrome and/or other pathologic sequelae associated with PCV-2 comprising inducing an immunogenic or protective response against PCV-2 in a pig comprising administering to the pig an aforementioned or herein disclosed composition.
Thus, the invention comprehends a method for the prevention and/or treatment of porcine circovirus-2 (PCV-2)-caused myocarditis, and/or abortion and/or intrauterine infection and/or post-weaning multisystemic wasting syndrome and/or other pathologic sequelae associated with PCV-2 comprising inducing an immunogenic or protective response against PCV-2 in a pig comprising administering to the pig a composition comprising a pharmaceutically or veterinarily acceptable carrier or excipient or vehicle, with preferably an adjuvant, and an active agent comprising a PCV-2 immunogen. The method can be for the prevention of PCV-2-caused mycarditis and/or abortion and/or intrauterine infection comprising administering a composition comprising a pharmaceutically or veterinarily acceptable carrier and a PCV-2 immunogen. The PCV-2 immunogen can be an attenuated live whole PCV-2 or inactivated PCV-2. The method can involve a composition that is a subunit immunogenic, or vaccine composition. The method can involve the composition additionally including at least one immunogen from at least one additional pig pathogen , including a vector expressing such an immunogen or epitope; e.g., the at least one additional pig pathogen can be selected from the group consisting of PRRS, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, E. coli, Pseudorabies, Hog cholera, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhusiopathiae, Swine Influenza, and PPV
and combinations thereof. The method can involve a vector that is a DNA vector plasmid, a bacteria such as an E. coli, a virus such as baculovirus, a herpesvirus including Aujeszky's disease virus, an adenovirus including a porcine adenovirus, a poxvinis, including a vaccinia virus, an avipox virus, a canarypox virus, and a swinepox virus, and the like.
The method can involve a vector-based composition additionally including at least one sequence, fragment or epitope from at least one additional pig pathogen, or a vector expressing such a sequence, fragment or epitope, wherein the vector can also be the vector expressing the PCV-2 sequence, fragment or epitope. The method can involve a vectbr that contains and expresses an ORF
selected from the group consisting of ORFs 1 to 13. e.g., an ORF selectred from ORFs 4, 7, 10 and 13; preferably ORFs 4 and/or 13. The method can also involve an immunogen -based composition wherein one or more of the immunogen(s) is recombinantly produced.
In this method, females and/or piglets are preferably inoculated as described above.
In another embodiment, the invention involves a method for preparing any of the aforementioned or herein disclosed compositions comprising admixing the pharmaceutically or veterinarily acceptable carrier and possibly the adjuvant, and the PCV-2 immunogen. The method can further include transfecting or infecting a cell or host with a recombinant vector that contains DNA encoding a PCV-2 immunogen and expresses that immunogen; and optionally purifying and/or isolating the immunogen from the cell. Similarly the method can include isolating and/or purifying a PCV-2 immunogen from PCV-2, or isolating PCV-2 from a sample.
The invention also provides a kit for preparing any of the aforementioned or herein disclosed compositions or, for performing any of the aforementioned or herein disclosed methods comprising in a first container the pharmaceutically or veterinarily acceptable carrier or vehicle or excipient and in a second container the active agent comprising the PCV-2 immunogen, wherein the first and second containers are optionally packaged together, and the kit optionally includes instructions for admixture of ingredients of the composition and/or administration of the composition.
In yet another embodiment, the invention provides for administering any of the aforementioned or herein disclosed compositions to male and/or female pigs; to prevent transmission of PCV-2 and prevent or treat or control myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2.
Administration is preferably done as described above.
The term "comprising" in this disclosure can mean "including" or can have the meaning commonly given to the term "comprising" in U.S. Patent Law.
Other aspects of the invention are described in or are obvious from (and within the ambit of the invention) the following disclosure.
DETAILED DESCRIPTION
Porcine circovinis-2 (PCV-2) is an agent associated with post-weaning multisystemic wasting syndrome (PMWS) in swine populations. As shown in Examples 1 and 2, the potential spectrum of disease associated with PCV-2 is expanded by evidence of vertical transmission and associated reproductive failure.
In particular, Example 1 shows that PCV-2 was isolated from a litter of aborted piglets from a farm experiencing late term abortions and stillbirths. Severe, diffuse myocarditis was present in one piglet associated with extensive immunohistochemical staining for PCV-2 antigen. Variable amounts of PCV-2 antigen were also present in liver, lung and kidney of multiple fetuses. The presence of other agents that have been associated with fetal lesions and abortion in swine including porcine parvovirus, porcine reproductive respiratory syndrome virus, encephalomyocarditis vines and enterovirus could not be established.
More in particular, Example 2 shows that tissues obtained from 30 high health herds over a four-year period, arid tested in routine cases of abortion or reproductive failure, were positive for PCV-2 in two submissions involving several stillborn piglets and non-viable neonates presenting with severe diffuse myocarditis, cardiac hypertrophy and evidence of chronic passive congestion. The two positive submissions were from the same farm, but occurred at two different times. The presence of PCV-2 in the hearts and other tissues of affected piglets was confirmed by immunohistochemistry and virus isolation.
Failure to detect porcine circoviruses in cases of reproductive failure prior to 1999 in areas of endemic infections supports the view that reproductive disease is a new clinical manifestation of PCV-2 infection, and further suggests that sexual, as well as vertical, modes of transmission are responsible for viral dissemination in the pig population.
Accordingly, inoculation of pigs, e.g., female pigs, such as sows or gilts, with a composition including at least one PCV-2 immunogen (e.g. from at least one strain chosen among strains Imp1008, Imp1010, Imp999, Imp1011-4828, Imp1011-48121, 1103 and 1121) (which composition can also include at least one immunogen from at least one other porcine pathogen such as at least one porcine parvovims, wherein when a vector is used the vector can co-express both the PCV-2 immunogen(s) and the at least one immunogen of the at least one other porcine pathogen, e.g., PPV immunogen(s), inter alia), in particular in a schedule of immunization as described above, can prevent myocarditis and/or abortion andlor intrauterine infection associated with PCV-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2.
Thus, the invention involves methods and compositions using PCV-2 immunogen for preventing myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2. In particular, immunogen from strain 1103 and/or strain 1121 is useful for methods and compositions using PCV-2 immunogen for preventing myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2.
The invention also involves the use of any known PCV-2 strain or immunogen therefrom to formulate an immunogenic or vaccine composition for preventing or treating myocarditis and/or abortion and/or intrauterine infection associated with PCV-2, as well as post-weaning multisystemic syndrome and other pathologic sequelae associated with PCV-2 (these strains include strains Imp1008, Imp1010, Imp999, Imp1011-48285, Imp1011-48121, 1103 and 1121), in particular when the composition is intended to be administered to piglets such as to piglets from vaccinated females and more particularly to female pigs, in particular to pregnant females or females which will be subjected to serving ; and more particularly according to the inoculation schemes define above.
More particularly, the use is to formulate such compositions intended to prevent or treat myocarditis and/or abortion and/or intrauterine infection associated with PCV-2, in particular when the composition is intended to be administered to piglets such as to piglets from vaccinated females and more particularly to female pigs in particular to pregnant females or females which will be subjected to serving ; and more particularly according to the inoculation schemes defined above.
The at least one immunogen from at least one other porcine pathogen can be as used in known porcine vaccines or immunogenic compositions, or as in WO 98/03658.
Compositions for use in the invention can be prepared in accordance with standard techniques well known to those skilled in the veterinary or pharmaceutical or arts. Such compositions can be administered in dosages and by techniques well known to those skilled in the veterinary arts taking into consideration such factors as the age, sex, weight, condition and particular treatment of the pig, and the route of administration. The compositions can be administered alone, or can be co-administered or sequentially 'administered with other compositions of the invention (e.g., other compositions comprising a PCV-2 immunogen) or with other prophylactic or therapeutic compositions (e.g., other porcine immunogenic or vaccine compositions). Thus, the invention also provides multivalent or "cocktail" or combination compositions and methods employing them. In this regard, reference is made to U.S. Patent No. 5,843,456 directed to rabies compositions and combination compositions and uses thereof.
Compositions of the invention may be used for parenteral or mucosal administration, preferably by intradermal or intramuscular routes. In particular for intradermal route, injection can be done using a needleless injector. When mucosal administration is used, it is possible to use oral, nasal, or ocular routes.
In such compositions the immunogen(s) may be in a mixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like, and/or preferably with an adjuvant. The compositions can also be lyophilized or frozen. The compositions can contain auxiliary substances such as pH buffering agents, adjuvants, preservatives, polymer excipients used for mucosal routes, and the like, depending upon the route of administration and the preparation desired.
Standard texts, such . as "REML'~iGTOI~T'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, may be consulted to prepare suitable preparations, without undue experimentation. Suitable dosages can also be based upon the text herein and documents cited herein.
Adjuvants are substances that enhance the immune response to immunogens.
Adjuvants, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycerol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic~
products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.). John Wilev and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 ( 1997).
For example, it is possible to use the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.
For example the adjuvant-containing vaccine is prepared in the following way:
67%
v/v of aqueous phase comprising the immunogen are emulsified in 2,3% w/v of anhydromannitol oleate, 2,6% w/v of oleic acid ethoxylated with 11 EO
(ethylene oxide) and 28,1 % v/v of light liquid paraffin oil (European Pharmacopea type) with the aid of an emulsifying turbomixer.

An alternative method for preparing the emulsion consists in emulsifying, by passages through a high-pressure homogenizer, a mixture of 5% w/v squalane, 2.5% w/v Pluronic~
L 121. 0.2% w/v of an ester . of oleic acid and of anhydrosorbitol ethoxylated with 20 EO, 92.3% v/v of the aqueous phase comprising the immunogen.
It is also possible to formulate with synthetic polymers (e.g.,homo- and copolymers of lactic and glycolic acid, which have been used to produce microspheres that encapsulate immunogens, see Eldridge et al., hlol. Immunol. 28:287-294 (1993), e.g., biodegradable microspheres), with cytokines such as IL-2 and IL,-12 (see, e.g., U.S. Patent No. 5,334,379), and GMCSF, advantageously porcine GMCSF (granulocyte macrophage-colony stimulating factor; see, generally, U.S. Patents Nos. 4,999,291 and 5,461,663, see also Clark et al., Science 1987, 230:1229; Grant et a.1.. Drugs, 1992, 53:516), inter alia.
Certain adjuvants can be expressed in vivo with immunogen(s) and/or epitope(s); e.g., cytokines, GMCSF (see, e.g., Inumani and Takamatsu, Immunol. Cell. Biol., 1995, 73:474-76 concerning a plasmid encoding and expressing porcine GM-CSF).
A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of malefic anhydride and alkenyl derivative.
Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols.
These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Patent No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol.~R, (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol~ 974P, 934P and 971P. Among the copolymers of malefic anhydride and alkenyl derivative, the copolymers EMAO (Monsanto) which are copolymers of malefic anhydride and ethylene, linear or cross-linked, for example cross-linked with divinyl ether, are preferred. Reference may be made to J. Fields et al., Nature. 186 : 778-780, 4 June 1960.

From the point of view of their structure, the polymers of acrylic or methacrylic acid and the copolymers EMA~ are preferably formed of basic units of the following formula C (CH2)X C (CH2)y COOH COOH
in which:
Ri and R~, which are identical or different, represent H or CH3;
x = 0 or 1, preferably x = 1; and y= 1 or2,withx+y=2.
For the copolymers EMA~, x = 0 and y = 2. For the carbomers, x = y =1.
The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated. The carboxyl groups of the polymer are then partly in COO- form.

Preferably, a solution of adjuvant according to the invention, especially of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. This stock solution is diluted by adding it to the desired quantity (for obtaining the desired final concentration), or a substantial part thereof, of water charged with NaCI, preferably physiological saline (NaCL 9 g/1) all at once in several portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pH will be used as it is for mixing with the vaccine, which may be especially stored in freeze-dried, liquid or frozen form.
The polymer concentration in the final vaccine composition can be 0.01 % to 2%
w/v, e.g., 0.06 to 1 % w/v, such as 0.1 to 0.6% w/v.
From this disclosure and the knowledge in the art, the skilled artisan can select a suitable adjuvant, if desired, and the amount thereof to employ in an immunological, immunogenic or vaccine composition according to the invention, without undue experimentation.
The immunogenic or vaccine compositions according to the invention may be associated to at least one live attenuated, inactivated, or sub-unit vaccine, or recombinant vaccine (e.g. poxvirus as vector or DNA plasmid) expressing at least one immunogen or epitope of interest from at least one another pig pathogen.
Compositions in forms for various administration routes are envisioned by the invention. And again, the effective dosage and route of administration are determined by known factors, such as age, sex, weight and other screening procedures which are known and do not require undue experimentation. Dosages of each active agent can be as in herein cited documents and/or can range from one or a few to a few hundred or thousand micrograms, e.g., 1 ~g to lmg, for a subunit immunogenic, or vaccine composition; and, 10'~ to 101° TCIDSo 2~ advantageously 106 to 108 TCmSO for an inactivated (titre before inactivation) immunogenic, or vaccine composition. For a live attenuated immunogenic or vaccine composition, the dose can be between 101 and 108 TC>D;o advantageously 10~ and 106 TCB75o.
Recombinants or vectors can be administered in a suitable amount to obtain in vivo expression corresponding to the dosages described herein and/or in herein cited documents.
For instance, suitable ranges for viral suspensions can be determined empiracally. The viral vector or recombinant in the invention can be administered to a pig or infected or transfected into cells in an amount of about at least 103 pfu; more preferably about 104 pfu to about lOlo pfu, e.g., about 105 pfu to about 109 pfu, for instance about 106 pfu to about 108 pfu, per dose, e.g. of about 2 ml. And, if more than one gene product is expressed by more than one recombinant, each recombinant can be administered in these amounts; or, each recombinant can be administered such that there is, in combination, a sum of recombinants comprising these amounts.
In plasmid compositions employed in the invention, dosages can be as described in documents cited herein or as described herein. For instance, suitable quantities of each plasmid DNA in plasmid compositions can be 1 dug to 2 mg, preferably 50 ~g to lmg.
Documents cited herein regarding DNA plasmid vectors may be consulted by the skilled artisan to ascertain other suitable dosages for DNA plasmid vector compositions of the invention, without undue experimentation.
However, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunologenic response, can be determined by methods such as by antibody titrations of sera, e.g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation in pig. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be likewise ascertained with methods ascertainable from this disclosure, and the knowledge in the art, without undue experimentation.
The PCV-2 immunogen can be obtained from PCV-2 or can be obtained from in vitro recombinant expression of PCV-2 genes) or portions or epitopes thereof.
Methods for making and/or using vectors (or recombinants) for expression can be by or analogous to the methods disclosed in: U.S. Patent Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941;-5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 5,756,103, 5,766,599, 6,004,777, 5,990,091, 6.033,904, 5,869,312, 5,382,425, PCT
publications WO 94/16716, WO 96/39491, WO 95/30018, Paoletti, "Applications of pox virus vectors to vaccination: An update," PNAS USA 93:11349-11353, October 1996, Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety,"
PNAS USA 93:11341-11348, October 1996, Smith et al., U.S. Patent No. 4,745,051 (recombinant baculovirus), Richardson, C.D. (Editor), Methods in Molecular Bioloav 39, "Baculovims Expression Protocols" (1995 Humana Press Inc.), Smith et al., "Production of Huma Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector,"
Molecular and Cellular Biology, Dec., 1983, Vol. 3, No. 12, p. 2156-2165;
Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector," Molecular and Cellular Biology Mar. 1984, Vol. 4, No. 3, p. 399-406;
EPA 0 370 573, U.S. application Serial No. 920,197, filed October 16, 1986, EP
Patent publication No. 265785, U.S. Patent No. 4,769,331 (recombinant herpesvirus), Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors,"
PNAS USA 93:11307-11312, October 1996, Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors,"
PNAS USA 93:11313-11318, October 1996, Robertson et al. "Epstein-Ban vims vectors for gene delivery to B lymphocytes," PNAS USA 93:11334-11340, October 1996, Frolov et al., "Alphavims-based expression vectors: Strategies and applications," PNAS USA
93:11371-11377, October 1996, Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Patent Nos. 5,591,439, 5,552,143, WO 98/00166, allowed U.S. applications Serial Nos. 08/675,556, and 08/675,566 both filed July 3, 1996 (recombinant adenovirus), Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993, Ballay et al.
EMBO Journal, vol. 4, p. 3861-65, Graham; Tibtech 8, 85-87, April, 1990, Prevec et al., J.
Gen Virol. 70, 429-434, PCT W091/11525, Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259:1745-49, 1993 and McClements et al., "Immunization with DNA vaccines encoding glycoprotein D or alycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease," PNAS USA 93:11414-11420, October 1996. and U.S. Patents Nos 5,591,639, 5,589,466, and 5,580,859, as well as WO-A-90 11092, WO-A-93 19183, WO-A-94 21797, WO-A-95 11307, WO-A-95 20660, Tang et al., Nature_ 356, 152-154. 1992, and Furth et al. Analytical Biochemistry, 205, 365-368, 1992, relating to DNA expression vectors, inter alia. See also WO 98/33510; Ju et al., Diabetologia, 41:736 739, 1998 (lentiviral expression system); Sanford et al., U.S. Patent No.
4,945,050; Fischbach et al. (Intracel), WO 90/01543; Robinson et al., seminars in IMMUNOLOGY; vol.
9, pp.271 283 (1997) (DNA vector systems); Szoka et al., U.S. Patent No. 4,394;448 (method of inserting DNA into living cells); McCormick et al., LT.S. Patent No. 5,677,178 (use of cytopathic viruses); and U.S. Patent No. 5,928,913 (vectors for gene delivery), as well as other documents cited herein. A viral vector, for instance, selected from pig herpes viruses, such as Aujeszky's disease vims, porcine adenovirus, poxviruses, especially vaccinia virus, avipox virus, canarypox virus, and swinepox virus, as well as DNA vectors (DNA
plasmids) are advantageously employed in the practice of the invention .
The expression product from the PCV-2 genes) or portions thereof can be useful for generating antibodies such as monoclonal or polyclonal antibodies that are useful for diagnostic purposes. Similarly, expression products) from the PCV-2 genes) or portions thereof can be useful in diagnostic applications.
Further, one skilled in the art can determine an epitope of interest in a PCV-immunogen, or in an immunogen of another porcine pathogen, without undue experimentation, from the disclosure herein and the knowledge in the art; see, e.g., WO
98/40500, regarding general information for determining an epitope of interest or an epitopic region of a protein, inter alia.
According to the invention, advantageous immunogenic or vaccine compositions are:
An immunogenic or vaccine composition, collected from a cell culture in vitro which has been infected with a purified preparation of PCV-2, such as a purified preparation of porcine circovirus selected from the group consisting of the preparations deposited at the ECACC
(European Collection of 'Cell Cultures, Centre for Applied Microbiology &
Research, Salisbury, Wiltshire SP4 OJG, UK), under the following references: accession No. V97100219 (strain Imp.1008) , No. V971,00218 (strain Imp.1010) and accession No.
V97100217 (strain Imp.999) deposited October 2, 1997, accession No. V98011608(strain Imp.1011-48285) and No. V98011609 (strain Imp.1011-48121) deposited January 16, 1998, accession No.
00012710 (strain 1103) and No. 00012709 (strain 1121) deposited February 2, 2000, or an immunogenic or vaccine composition comprised of porcine circovims produced on, and isolated from cell culture irc vitro, these cells having been infected with a porcine circovirus capable of being isolated from a physiological sample or from a tissue sample, especially lesions, from a pig having the PMWS syndrome, e.g., such a composition wherein the porcine circovirus is produced on, and isolated from a pig kidney cell line, for instance, produced on, and isolated from PK/15 cells free from contamination with PCV-l; or such a composition comprising or prepared from a culture extract or supernatant, collected from a cell culture in vitro which have been infected with a such a circovims. Thus, porcine circovirus can be an immunogen. For instance, the vaccine or immunogenic composition can comprise the attenuated live whole immunogen (e.g., vims), advantageously, in a veterinarily or pharmaceutically acceptable vehicle or diluent and optionally a veterinarily or pharmaceutically acceptable adjuvant, as well as, optionally, a freeze-drying stabilizer. The immunogen (e.g., vims) can be inactivated and the vaccine or immunogenic composition can additionally and/or optionally comprise, a veterinarily or pharmaceutically acceptable vehicle or diluent and optionally a veterinarily or pharmaceutically acceptable adjuvant. The vaccine or immunogenic composition can comprise PCV-2 immunogens and/or immunogens of several porcine circoviruses (including PCV-2 or several strains of PCV-2, and including PCV-1), as well as optionally additional immunogens from another pig pathogen;
e.g, PRRS, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, E. coli, Pseudorabies, Hog cholera, Bordetella bronchiseptica, Pasteurella multocida, Erysipelothrix rhusiopathiae, Swine Influenza, PPV (see also WO-A-00 01409).
For the production of . circovirus antigenic preparations, the circoviruses may be obtained after passage on cells, in particular cell lines, e.g. PK/15 cells.
The culture . supernatants or extracts, optionally purified by standard techniques, may be used.
In the context of attenuated PCV, the attenuation may be carried out according to the customary methods, e.g. by passage on cells, preferably by passage on pig cells, especially cell lines, such as PK/15 cells (for example from 20 to 1~0, especially of the order of 40 to100, passages).
In the context of inactivated vaccine, the PCV, with the fractions which may be present, is inactivated according to techniques known to persons skilled in the art. The inactivation will be preferably carried out by the chemical route, e.g. by exposing the antigen to a chemical agent such as formaldehyde (formalin), paraformaldehyde, (3-propiolactone or ethyleneimine or its derivatives, and/or by physical treatment. The preferred method of inactivation will be herein the exposure to a chemical agent and in particular to ethyleneimine or to (3-propiolactone.
The immunogen in the vaccine or immunogenic composition can be expressed from a DNA fragment containing a PCV-2 nucleotide sequence or fragment thereof (advantageously encoding at least one epitope), e.g. selected from the group consisting of the sequences designated by the references SEQ ID No: l, SEQ ID No: 2, SEQ ID No: 3, SEQ ID
No: 4, as well as SEQ ID No: 6 and SEQ ID No: 7, (Figures 1-4 and 6-7). The immunogen in the vaccine or immunogenic composition can be expressed from a DNA fragment containing an ORF selected from the group consisting of ORFs 1 to 13, such as ORFs 4, 7, 10 and 13, preferably ORFs 4 and/or 13, of a PCV-2 strain, in particular of one of the above identified strains (as designated in WO-A-99 18214 - see also table 1 hereinafter). Thus, the immunogen or a portion thereof, such as an epitope of interest can be obtained by i~c vitro expression thereof from a recombinant or a vector. The immunogen may be further purified and/or concentrated by the conventional methods.
The immunogen in the vaccine or immunogenic composition can be expressed in vivo by an expression vector comprising a DNA fragment containing a PCV-2 nucleotide sequence or fragment thereof (advantageously encoding at least one epitope), e.g.
selected from the group consisting of the sequences designated by the references SEQ ID No: l, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6, as well as SEQ ID No: 6 and SEQ ID
No: 7, (Figures 1-4 and 6-7). Similarly, the immunogen in the vaccine, immunogenic or immunological composition can be expressed in vivo by an expression vector comprising a DNA fragment containing an ORF selected from the group consisting of ORFs 1 to 13, such as ORFs 4, 7, 10 and 13, preferably ORFs 4 and/or 13, of a PCV-2 strain, in particular of one of the above identified strains. That is, the vaccine or immunogenic composition can comprise and expression vector that expresses the immunogen or a portion thereof, e.g., an epitope of interest, in vivo.
The expression vector can be any suitable vector such as a vector selected from DNA
plasmids, bacteria such as E. coli, viruses such as baculovirus, herpesvirus such as Aujeszky's disease virus, adenovirus including porcine adenovirus, poxviruses, especially vaccinia virus, avipox virus, canarypox virus, and swinepox virus, inter alia (the one skilled in the art can also refer to the LT.S. applications of Audonnet et al. and Bublot et al., Serial Nos. 60/138,352 and 60/138,478, respectively, both filed June 10, 1999, in particular to the detailed description and more particularly to the examples ("DNA VACCINE-PCV", and "PORCINE
CIRCOVIRLTS RECOMBIi~IANT POXVIRUS VACCINE", respectively. attached as appendices).
Accordingly, the invention also comprehends nucleic acid molecules and vectors containing them, as well as expression products therefrom, compositions comprising such nucleic acid molecules and/or vectors and/or expression products, as well as methods for making and using any or all of these embodiments. The invention especially encompasses herein ORFs and/or fragments encoding an immunogen or epitope, as well as nucleic acid molecules of strains 1103 and/or 1121, in particular their ORFs 1 to 13, such as ORFs 4, 7, 10 and 13, preferably ORFs 4 and/or 13, and fragments thereof, as well as vectors comprising these nucleic acid molecules, compositions comprising these nucleic molecules, vectors, or expression products therefrom, compositions comprising such expression products, primers or probes for such nucleic acid molecules, and uses or methods involving these embodiments, e.g., for detecting, diagnosing, assaying for PCV-2, for inducing an immunogenic or protective response, and the like.
As earlier mentioned, embodiments of the invention can include antibodies.
Such antibodies can be polyclonal or monoclonal antibodies; for instance, prepared from the aforementioned circovirus, or from a polypeptide encoded by a DNA fragment having a PCV-2 sequence, e.g. selected from the group consisting of SEQ ID NOS. l, 2, 3, 4, 6 and 7, (Figures 1-4 and 6-7) or from a polypeptide from expression by a vector comprising a PCV-2 sequence, e.g. selected from the group consisting of SEQ ID NOS. l, 2, 3, 4, 6 and 7 ; or from a polypeptide from expression by a vector comprising DNA including an ORF
selected from the group consisting of ORFs 1 to 13. The skilled artisan may use techniques known in the art to elicit antibodies and to generate monoclonal or polyclonal antibodies. -Antibodies and antigens can be used in diagnostics.
The invention also comprehends probes or primers from PCV-2 strains 1103 and/or 1121 which can be useful, for instance, in detecting PCV-2 DNA, in particular with respect to myocarditis and/or abortion and/or intrauterine infection, as well as for amplifying PCV-2 DNA, e.g., for preparing an expression vector. A probe or primer can be any stretch of at least 8, preferably at least 10, more preferably at least 12, 13, 14, or l~, such as at least 20, e.g., at least 23 or 25, for instance at least 27 or 30 nucleotides in PCV-2 genome or a PCV-2 gene which are unique to PCV-2, and possibly to these strains, or which are in PCV-2 and are least conserved among the PCV or circovims family. As to PCR or hybridization primers or probes and optimal lengths therefor, reference is also made to Kajimura et al., GATA
7(4):71-79 ( 1990). Hybridization is advantageously under conditions of high stringency, as the term "high stringency" would be understood by those with skill in the art (see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Hames and Higgins, eds., 1985, Nucleic Acid Hybridization, IRL Press, .Oxford, U.K.). Hybridization will be understood to be accomplished using well-established techniques, including but not limited to Southern blot hybridization, Northern blot hybridization, in sitLC hybridization and, advantageously, Southern hybridization to PCR-amplified DNA fragments.
Like probes or primers, peptides which are not full-length PCV-2 proteins are part of invention and can be any stretch of at least 8, preferably at least 10, more preferably at least 12, 13, 14, or 15, such as at least 20, e.g., at least 23 or 25, for instance at least 27 or 30 amino acids in PCV-2 which are unique to PCV-2, and possibly to these strains, or which are in PCV-2 and are least conserved among the PCV and/or circovirus family.
Alternatively or additionally, the amino acids of the invention which are not full length PCV-2 proteins can be an epitopic region of a PCV-2 protein.
PCV-2 sequences are disclosed in Meehan et al., 1998 (Strain Imp.1010 ; ORFl nucleotides 398-1342; ORF2 nucleotides 1381-314; and correspond respectively to ORF4 and ORF13 in the invention terminology (see table 1), in U.S. application Serial No. 09/161,092 of September 1998 and to COL4 and COL13 in WO-A-9918214). Several PCV-2 strains and their sequences are disclosed herein and called Imp1008, Imp999, Imp1011-48285, Imp1011-48121, 1103 and 1121. Other strains are disclosed in A.L. Hamel et al.
J. Virol. June 20 1998, vol 72, 6: 5262-5267 (GenBank AF027217) and in I. Morozov et al. J.
Clinical Microb.
Sept. 1998 vol. 36, 9: 2535-2541, as well as GenBank AF086834, AF086835 and AF086836.
These sequences, or ORFs therefrom, or regions thereof encoding an antigen or immunogen or epitope of interest can also be used in the practice of this invention.
The invention also encompasses the equivalent sequences to those used or mentioned 25 herein and in documents cited herein; for instance, sequences that are capable of hybridizing to the nucleotide sequence under high stringency conditions (see, e.g., Sambrook et al. (1989).
Among the equivalent sequences, there may also be mentioned the gene fragments conserving the immunogenicity of the complete sequence, e.g., an epitope of interest.
The homology between the whole genome of PCV types l and 2 is about 75%. But within type 2, homology is generally above 95%. Thus, in the practice of the invention, use of any PCV-2 strain is encompassed by equivalence. A criteria can be that the strain is of type 2, e.g. that homology at the nucleotide level of the whole genome is equal or greater than 85%, advantageously 90% or greater, more advantageously 95% or greater, preferably 97, 98 or 99% or greater, with the strains disclosed herein, e.g. strain Imp1010.
Limits of the ORFs of strain Imp1010 are given in the following table 1 Start End Strand Size of the Protein size Name ORF
(nucleotides (amino nt acids (aa)) ( )) ORF 103 210 Sense 108 nt 35 as ORF2 1180 1317 Sense 138 nt 45 as ORF3 1363 1524 Sense 162 nt 53 as ORF4 398 1342 Sense 945 nt 314 as ORFS 900 1079 Sense 180 nt 59 as ORF6 1254 1334 Sense 81 nt 26 as ORF7 1018 704 Antisense 315 nt 104 as ORF8 439 311 Antisense 129 nt 42 as ORF9 190 101 Antisense 90 nt 29 as ORF10 912 733 Antisense 180 nt 59 as ORF11 645 565 Antisense 81 nt 26 as ORF12 1100 1035 Antisense 66 nt ~ 1 as ORF13 314 1381 Antisense 702 nt 213 as The ORFs are defined with respect to strain Imp1010. The invention also encompasses the use of the corresponding ORFs in any other PCV-2 strain, and any of the PCV-2 strains as defined herein or in documents cited herein. Thus, from the genomic nucleotide sequence, it is-routine art to determine the ORFs using a standard software, such as MacVectorQ. Also, alignment of genomes with that of strain 1010 and comparison with strain 1010 ORFs allows the one skilled in the art to readily determine the ORFs on the genome for another strain (e. g.
those disclosed in WO-A-99 18214, say Imp1008, Imp1011-48121, Imp1011-48285, Imp999, as well as the new strains 1103 and 1121 ). Using software or making alignment is not undue experimentation and directly provides access to these ORFs.

For example, referring to figures 6 and 7, the corresponding ORFs of strains 1103 and 1121 are as given in the following table 2 Start End Strand Size of the Protein size Name ORF (amino (nucleotides acids (aa)) (nt)) ORF1 1524 1631 Sense 108 nt 35 as ORF2 833 970 Sense 138 nt 45 as ORF3 1016 1177 Sense 162 nt 53 as ORF4 51 995 Sense 945 nt 314 as ORF5 553 732 Sense 180 nt 59 as ORF6 907 987 Sense 81 nt 26 as ORF7 671 357 Antisense 315 nt 104 as ORFB 92 1732 Antisense 129 nt 42 as ORF9 1611 1522 Antisense 90 nt 29 as ORF10 565 386 Antisense 180 nt 59 as ORF11 298 218 Antisense 81 nt 26 as ORF12 753 688 Antisense 66 nt 21 as ORF13 1735 1037 Antisense 702 nt ~ 13 as Also equivalent and useful in the practice of the invention are the nucleotide sequences which change neither the functionality nor the strain specificity (say of strain type 2) of the gene considered or those of the polypeptides encoded by this gene. The sequences differing through the degeneracy of the code are, of course, be included in the practice of the invention.
For ORF4, homology between PCV-1 and PCV-2 is about 86%, and for ORF13, the homology between PCV-1 and PCV-2 is about 66%. Thus, also equivalent sequences useful in the practice of the present invention, for ORF4, are those sequences having an homology equal or greater than 88%, advantageously 90% or greater, preferably 92% or 95% or greater homology with ORF4 of strain Imp1010, and for ORF13, those sequences having an homology equal or greater than 80%, advantageously 85% or greater, preferably 90% or 95%
or greater than ORF13 of strain Imp1010 (Using the terminology of table 1).
For homology regarding the other ORFs, one can determine those sequences which come from a PCV strain having an ORF4 and/or an ORF13 which have an homology as defined above with the corresponding ORF of strain 1010. For ORF7, sequences useful in the practice of the invention include those sequences having an homology that is advantageously equal to or greater than 80%, more advantageously 85% or greater, preferably 90% or 95% or greater with ORF7 of strain Imp1010. For ORF10, sequences useful in the practice of the invention include those sequences having an homology that is advantageously equal to or greater than 86%, more advantageously 90% or greater, preferably 95% or greater with ORF10 of strain Imp 1010 (Using the terminology of table 1 ).
Also, equivalent sequences useful in the practice of this present invention, for ORF4 are those sequences having an homology equal or greater than 88 %:
advantageously 90% or greater, preferably 92% or 95% or greater with ORF4 of strain Imp1010, and for ORF13, are those sequences having an homology equal or greater than 80%, advantageously 85% or greater, preferably 90% or 9~% or greater with ORF13 of strain Imp1010.
ORF4 and ORF13 has the potential to encode proteins with predicted molecular weights of 37.7 kD and 27.8 kD respectively. ORF7 and ORF10 (correspond to ORF3 and ORF4 in Meehan et al. 1998) has the potential to encode proteins with predicted molecular weights of 11.9 and 6.5 kD respectively. Sequences of these ORFs are also available in Genbank AF 055392. They can also be incorporated in plasmids and be used in accordance with the invention alone or in combination, e.g. with ORF4 and/or ORF13.
The other ORFs 1-3 and 5, 6, 8-9, 11-12 disclosed in the above table (COLs 1-3 and 5, 6, 8-9, 11-12 in WO-A-9918214), or regions) thereof encoding an antigen or epitope of interest, may be used in the practice of this invention, e.g., alone or in combination or otherwise with each other or with the ORFs 4 and/or 13 or regions) thereof encoding antigens) or epitope(s). Similarly, for homology, one can determine that there are equivalent sequences which come from a PCV strain having an ORF13 and/or an ORF4 which have an homology as defined above with the corresponding ORF of strain 1010 as defined herein or in Meehan et al., 1998. For ORF7, an equivalent sequence has homology thereto that is advantageously, for instance, equal or greater than 80%, for example 85% or greater, preferably 90% or 95% or greater with ORF7 of strain Imp1010. And, for ORF10, advantageously an equivalent sequence has homology that is equal or greater than 86%, advantageously 90% or greater, preferably than 95% or greater with ORF10 of strain Imp 1010.
Nucleotide sequence homology can be determined using the "Align" program of Myers and Miller, ("Optimal Alignments in Linear Space", CABIOS 4, 11-17, 1988, and available at NCBI. Alternatively or additionally, the term "homology" or "identity", for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nref - Ndrf)*100/Nref, wherein Nd;f is the total number of non-identical residues in the two sequences when aligned and wherein N,ef is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence similarity of 75%
with the sequence AATCAATC (Nref = 8; Nd;~2).
Alternatively or additionally, "homology" or "identity" with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman, 1983 PNAS USA 80:726, for instance, using a window size of nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics TM
Suite, Intelligenetics Inc. CA).. When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA
sequence is considered equal to uracil (U) in the RNA sequence.
RNA sequences within the scope of the invention can be derived from DNA
sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in R.~lA sequences.
Additionally or alternatively, amino acid sequence similarity or identity or homology can be determined using the BlastP program (Altschul et al., Nucl. Acids Res.
25, 3389-3402) and available at NCBI. The following references provide algorithms for comparing the relative identity or homology of amino acid residues of two proteins, and additionally or alternatively with respect to the foregoing, the teachings in these references can be used for determining percent homology or identity: Needleman SB and Wunsch CD, "A
general method applicable to the search for similarities in the amino acid sequences of two proteins,"
J. Mol. Biol. 48:444-453 (1970); Smith TF and Waterman MS, "Comparison of Bio-sequences," Advances in Applied Mathematics 2:482-489 (1981); Smith TF, Waterman MS
and Sadler JR, "Statistical characterization of nucleic acid sequence functional domains,"
Nucleic Acids Res., 11:2205-2220 (1983); Feng DF and Dolittle RF, "Progressive sequence alignment as a prerequisite to correct phylogenetic trees," J. of Molec.
Evol., 25:351-360 (1987); Higgins DG and Sharp PM, "Fast and sensitive multiple sequence alignment on a microcomputer," CABIOS, 5: 151-153 (1989); Thompson JD, Higgins DG and Gibson TJ, "ClusterW: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positions-specific gap penalties and weight matrix choice, Nucleic Acid Res., 22:4673-480 ( 1994); and, Devereux J, Haeberlie P and Smithies O, "A
comprehensive set of sequence analysis program for the VAX," Nucl. Acids Res., 12: 387-395 (1984).
The invention further comprehends uses of a PCV-2 immunogen, either alone or in further combination with an immunogen of another porcine pathogen to generate compositions according to the invention, e.g., admixing the ingredients; and, the invention also therefore comprehends kits wherein components are individually contained and optionally the containers are packaged together for admixture and/or administration, wherein the kit can also optionally include instructions for admixture and/or administration.
While the invention has been discussed in terms of administering to female pigs immunogenic or vaccine compositions comprising a PCV-2 immunogen, the invention can also comprehend administering such compositions to sow or gilt and/or to boar as described herein; Thus, both mother and offspring (e.g., sow, gilt) and boar can be administered_ compositions of the invention and/or can be the subject of performance of methods of the invention. Accordingly, populations of pigs can be administered compositions of the inventions and/or can be the subject of performance of methods of the invention.
According to the present invention, immunogenic and vaccine compositions may comprise immunogens from more than one PCV-2 strain. For example, it is possible to combine immunogens from strains 1121 and 1103, from one or both of these strains with at least one other strain disclosed herein, or any other combination.

The present invention provides for methods allowing the one skilled in the art to evaluate the efficacy of vaccines against PCV-2. A first method is an ELISA
method or with seroneutralization. A second. method is a vaccination followed by challenge with a virulent PCV-2 strain, e.g. one of the strains disclosed herein. In other words, the invention allows one to check for PCV immunogens, including PCV-1 immunogens, able to elicit an immunogenic or protective response against PCV-2. Such PCV immunogens are then useful to prevent or treat pigs against in particular myocarditis and/or abortion and/or intrauterine associated with PCV-2, as well as against PMWS and/or other pathologic sequelae associated therewith.
Thus one aspect of the invention is to provide immunogenic or vaccinal compositions comprising a PCV immunogen and able to elicit an immunogenic or protective response against PCV-2. The invention relates also to methods of immunization or vaccination using such an immunogen, as well as to the use of such an immunogen to produce such an immunogenic or vaccinal composition.
The invention shall be further described by way of the following Example And Results, provided for illustration and not to be considered a limitation of the invention.
Figure 1 depicts SEQ ID No. l, the PCV DNA sequence of the genome of the Imp.
1011-48121 strain.
Figure 2 depicts SEQ ID No. 2. the DNA sequence of the. genome of the Imp.

48285 strain.
Figure 3 depicts SEQ ID No. 3, the DNA sequence of the genome of the Imp. 999 strain.
Figure 4 depicts SEQ ID No. 4. the DNA sequence of the genome of the Imp. 1010 strain.
Figure 5 depicts SEQ ID No. ~, the DNA sequence of the genome of the PK/15 strain.
Figure 6 depicts SEQ ID No. 6., the DNA sequence of the genome of the 1103 strain, isolated in Alberta, Canada. k means g (G) or t (T), y means c (C) or t (T).
These variations of sequence were observed in the viral population.
Figure 7 depicts SEQ ID No. 7, the DNA sequence of the genome of the 1121 strain, isolated in Saskatoon. Canada.

EXAMPLE AND RESULTS
EXAMPLE/RESULT 1: 1VIYOCARDITIS, ABORTION AND INTRAUTERINE

Late term abortions and farrowings with both stillborn and mummified piglets occurred in a new 450-female pig swine facility as it was brought into production.
Pseudopregnancy was also observed in several gilts. Gilts received two doses of an inactivated vaccine containing parvovirus and leptospiral immunogens prior to breeding.
A litter received for postmortem examination consisted of nine fetuses that appeared to have died at various stages of gestation. There were 2 mummified, 2 macerated, 3 autolysed and 2 fresh, stillborn piglets. Lesions were observed on gross pathological examination in one partially autolysed fetus only. In this fetus both ventricles of the heart were dilated, the liver was enlarged and firm and there was both hydrothorax and ascites.
Histopathologically, there were extensive areas of myocardial degeneration or necrosis with edema and mild fibrosis, and a diffuse moderate -infiltration of lymphocytes and macrophages. There was marked generalized hepatic congestion and hepatocellular loss. The spleen and kidneys were also congested. Significant histological lesions were not detected in the other fetuses.
Immunohistochemical staining for PCV-2 was performed as previously described using a rabbit polyclonal antiserum and a monoclonal antibody that were raised against PCV-2. on sections of formalin-fixed, routinely processed and embedded tissue (Ellis et al., 1998;
Ellis et al., 1999). In the fetus with dilated cardiomyopathy there was extensive staining for PCV-2 antigen throughout the affected myocardium. Staining was most extensive in areas of necrosis and appeared to involve primarily myocytes. Both cytoplasmic and nuclear staining was present. In multiple fetuses there was extensive staining in the liver. In some sections it appeared to involve primarily sinusoidal endothelium and Kupfer cells, while in other fetuses, including the one with myocarditis, there was also nuclear and cytoplasmic staining of-hepatocytes. Positively stained cells were scattered throughout the lung, and multifocally in the kidney. Polymerase chain reaction for PCV-2 was performed as previously described using frozen tissue (Ellis et al., 1999). PCR product of the expected size for PCV-2 was amplified from fetal tissue. PCV-2 was isolated from the fetus with myocarditis and a pool of tissues from other fetuses in the litter by inoculating tissue homogenates onto PCV-free PK-15 cells.

Fetal tissues were also examined for other viral pathogens that have been associated with fetal injury and abortions in swine, including, porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalomyocarditis (EMCV), and enteroviruses. PPV antigen was not detected by fluorescent antibody testing (FAT) on frozen sections of lung, liver, and spleen from the mummified or stillborn fetuses.
Homogenates of liver, lung, and spleen from the aborted fetuses were also inoculated into cultures of PCV-free PK-15 cells, primary porcine fallopian tube cells and Vero cells. Cytopathic viruses were not detected after three passages. Tissues were negative for PPV using PCR. PRRSV
antigen was not detected by immunohistochemical staining.
Thus, there were fetal lesions and abortion directly associated with PCV-2.
These results also show vertical transmission of the virus.
In a previous study, PCV-1 was isolated from 2 of 160 pig fetuses examined, implying that this group of viruses can be vertically transmitted; however, PCV-1 antigen could not be associated with any lesions in the tissue (Allan et al., 1995). The exclusion of other agents that have been associated with fetal lesions and abortion in swine, including, PPV (Bolt et al., 1997; Molitor et al., 1991), PRRSV (Lager et al., 1996), EMCV (Kim et al., 1989), and enterovinls (Molitor et al., 1991) indicate that PCV-2 can cause significant fetal pathology and subsequent abortion.
However, PCV-1 immunogens (still according to the general definition given at the beginning) may elicit an immunogenic or protective response against myocarditis and/or abortion and/or intrauterine infection as well as post-weaning multisystemic wasting syndrome and ergo PCV-1 immunogens can also be used in the practice of this invention (e.g., in the methods, compositions, uses, etc.) - either alone or in conjunction with PCV-2 immunogens (the vector can contain and express DNA encoding for both a PCV-1 immunogen and/or epitope and a PCV-2 immunogen and/or epitope) and/or alone or in conjunction an immunogen and/or epitope of other porcine pathogen ( if a vector is used, the vector can contain and express DNA encoding for both a PCV-1 immunogen and/or epitope and an immunogen and/or epitope of another porcine pathogen, or for a PCV-1 immunogen and/or epitope and a PCV-2 immunogen and/or epitope and an immunogen and/or epitope of another porcine pathogen). Thus, one skilled in the art may alternatively or additionally use a PCV-1 immunogen, and/or epitope and/or vector encoding such an immunogen and/or epitope in the practice of this invention without any undue experimentation; for instance, to so do, one need only read the text herein prior to this Example and at the conclusion of (after) this Example, and substitute --PCV-1-- for ".PCV-2" with any modification minor based on teachings herein.
The wasting syndrome associated with PCV-2 infection most often occurs in 5-12 week old pigs (Allan et al., 1998; Ellis et al., 1998). Experimental infection of neonatal swine indicates a relatively long prodromal period between infection and the development of clinical signs associated with PCV-2 (Allan et al. 1999; Ellis et al. 1999). The findings herein show that the virus is transmitted vertically or in the perinatal period. Not only may interuterine vertical transmission of PCV-2 result in abortion, but it is possible that sublethally in actero infected piglets may be the animals that subsequently develop PNIWS.
Furthermore, these results show that inoculation of female pigs with a composition comprising an PCV-2 immunogen (which composition can also include an immunogen from another porcine pathogen, e.g., porcine parvovirus), prior to breeding or serving, or prior to the perinatal period and/or during gestation can prevent myocarditis and/or abortion and/or intrauterine infection associated with porcine circovirus-2, as well as post-weaning multisystemic wasting syndrome and other pathologic sequelae associated with PCV-2, by eliciting an immunological response or antibodies against PCV-2.
Of course, compositions, methods, and other aspects of the invention can be used or practiced in animals other than pigs, e.g., sheep. bison, cattle, wild boar;
for instance, if PCV-2 infects such other animals.
EXAMPLE/RESULT 2: 1VIYOCARDITIS, ABORTION AND INTRAUTERINE

The presence of PCV-2 in neonatal piglets suggests that vertical transmission may be an important means of viral transmission. This mode of transmission may be related not only to reproductive failure. but also to the development of multisystemic disease later in life. It is of interest to determine whether previously undetected PCV-2 (and PCV-1) has been vertically transmitted in pork producing areas where PMWS, and by extension PCV-2 infection, has been endemic for at least several years.
Thirty eight submissions involving reproductive failure received in the diagnostic laboratory at the Western College of Veterinary Medicine (WCVM), University of Saskatchewan, Saskatoon, Canada, over a four-year period from a total of 30 high health herds in Canada were evaluated. Five of the farms from which the samples were obtained had diagnosed cases of PMWS. Twenty-seven of the thirty-eight submissions (71 %) were classified as abortions; five of-these (13%) also involved at least one mummified fetus. Of the remaining 10 cases: 5 involved stillborn piglets along, with nonviable piglets (13%); 2 with stillborn and one or more mummified feti (5%); 2 with only stillborn piglets (5%); and one with only mummified feti (2.5%). Routine diagnostics for pathogens other than circovims revealed 4 cases ( 11 %) in which the etiology was determined to be porcine parvovirus and 2 cases (5 %) in which the etiology was determined to be of bacterial origin.
Gross necropsies were performed and tissues were collected and fixed in buffered formalin (fixat'ion time 24-72 hrs) and, in most cases, fresh tissues were also submitted for routine microbiological evaluation. None of these cases had been previously tested for PCV-2.
The PCR technique used for the detection of PCV-l and PCV-2 was performed as previously described (Tischer et al. 1974). PCV-1 was not detected by PCR in any submissions comprising reproductive failure from the four-year period. PCV-2 was detected by PCR in two different submissions that originated from the same multi-site pork production unit on two separate occasions in the spring of the last year in the four-year period. The first of these submissions comprised a litter of piglets with gross evidence of myocarditis, cardiac hypertrophy, and chronic passive congestion.
Immunohistochemical identification of PCV-2 in tissues was performed as previously described (Tischer et al. 1974). Immunohistochemical staining (IHC) for PCV-2 was positive in hearts from all six of the piglets that were submitted, while 4 of 6 were positive by PCV-2 PCR (see following Table).
Table: Detection of PCV-2 in the formalin fixed hearts of porcine with myocarditis by PCR, IHC and viral isolation in cell culture.
PCV-2 postive tissues PCR IHC Virus Isolation Fixed 5/6 6/6 N/A

Frozen 4/4 N/A 2/4 The second submission from the same farm consisted of a litter of four piglets in which 2 were stillborn and 2 others died shortly after birth. All four piglets also had gross evidence of a severe, difuse myocarditis, cardiac hypertrophy, and chronic passive congestion. Only fresh frozen heart, and pooled lung/spleen tissues were submitted for analysis. PCV-2 PCR
was positive in the hearts of 2 of 4 piglets and in the pooled lung and splenic tissues of 4 of 4 piglets. Isolation of PCV-2 from affected hearts and/or pooled lung and splenic tissue was positive in 2 of the 4 cases that were PCV-2 positive by PCR. Based on serology and/or PCR, other agents associated with reproductive failure in swine, including porcine reproductive and respiratory syndrome virus and porcine parvovirus were apparently circulating in the breeding herd. However, these agents could not be shown to be associated with the severe cardiac (or other) lesions in the affected piglets; but, they may contribute to PMWS.
PCV-2 was not detected by PCR or IHC in any representative cases of reproductive failure submitted during the first three years of the four-year period (it was detected in cases of reproductive failure submitted during the last year of the four-year period).
In order to rule out damage to DNA due to formalin fixation as a possible adverse factor limiting the ability to detect PCV-2 by PCR, PCR was performed on tissues collected from four weanling piglets with PMWS, PCV-2 DNA was amplified in all fixed tissues tested, including;
lung, liver, kidney and bronchial lymph node, from all four individuals. Moreover, the sensitivity of the PCR PCV-2 was independent of the length of time that each tissue was fixed in formalin.
These results confirm and extend the previous observation (West et al. 1999) that PCV-2 can be vertically transmitted and can be present in large amounts within lesions from piglets infected ih utero. Vertical transmission of PCV-2 virus and resultant fetal damage, such as myocarditis, is an additional disease manifestation of PCV-2.
Furthermore, the failure to detect PCV-2 in cases of reproductive failure prior to the last year of the four-year period-from an endemic area of PCV-2 infection may indicate that vertical transmission was not the primary mechanism responsible for the initial dissemination of viral infection. Sexual, as well as vertical, modes of transmission can be attributed to the spread of PCV-2 infection in pigs.
EXAMPLE/RESLTLT 3: CULTURE AND ISOLATION OF THE
PORCINE CIRCOVIRUS STRAINS

Viruses 1103 and 1021 were isolated respectively in Alberta, respectively Saskatoon, Canada, from abortive cases according to the method described in J. Ellis et al. Can. J. Vet.
1998, vol 39, 44-51.
Viral culture is carried out on PK/15 cell cultures, known to be uncontaminated with the porcine circovirus (PCV), pestiviruses, porcine adenoviruses and porcine parvovinises (Allan G. et n1 Pathogenesis of porcine circovirus experimental infections of colostrum-deprived piglets and examination of pig foetal material. Vet. Microbiol. 1995, 44, 49-64).
Monolayers of PK/15 cells are dissociated by trypsinization (with a trypsin-versene mixture) from confluent cultures, and taken up in MEM-SA medium containing 15%
foetal calf serum not contaminated by pestivinis (= MEM-G medium) in a final concentration of about 400,000 cells per ml. 10 ml aliquot fractions of this cell suspension are then mixed with 2 ml aliquot fractions of the inocula described above, and the final mixtures is aliquoted in 6 ml volumes in two Falcon flasks of 25 cm'. These cultures are then incubated at +37°C for 18 hours under an atmosphere containing 10% CO~.
After incubation, the culture medium of the semi-confluent monolayers were treated with 300 mM D-glucosamine (Cat # 648175, Sigma-Aldrich~Company Limited, Poole, UK) (Tischr I. et al., Arch. Virol., 1987 96 39-57), then incubation was continued for an additional period of 48-72 hours at +37°C. Following this last incubation, one of the two Falcons of each inoculum was subjected to 3 successive freeze/thaw cycles. The PK/15 cells- of the remaining Falcon were treated with a trypsin-versene solution, resuspended in 20 ml of MEM-G
medium, and then inoculated into 75 cm'' Falcons at a concentration of 400,000 cells/ml. The freshly inoculated flasks were then "superinfected" by addition of 5 ml of the corresponding lysate obtained after the freeze/thaw cycles.
EXAMPLE/RESLTLT 4: TECHNIQUE FOR THE DETECTION OF
PCV BY IMMUNOFLUORESCENCE
The initial screening of all the cell culture preparations fixed with acetone was carried out by an indirect immunofluorescence technique (IIF) using a 1/100 dilution of a pool of adult pig sera. This pool of sera comprises sera from 25 adult sows from Northern Ireland and is known to contain antibodies against a wide variety of porcine vinises, including PCV:
porcine parvovirus, porcine adenovirus, and PRRS virus. The IIF technique was carried out by bringing the serum (diluted in PBS) into contact with the cell cultures for one hour at +37°C, followed by two washes in PBS. The cell cultures were then stained with a 1/80 dilution in PBS of a rabbit anti-pig immunoglobulin antibody conjugated with fluorescein isothiocyanate for one hour, and then washed with PBS and mounted in glycerol buffer prior to the microscopic observation under ultraviolet light.
EXAMPLE/RESULT 5: PRODUCTION OF PCV ANTIGENS
BY IN VITRO CULTURE
The culture of the noncontaminated PK/15 cells and the viral multiplication were carried out according to the same methods as in Example 1. The infected cells are harvested after trypsinization after 4 days of incubation at 37°C and enumerated.
The. next passage is inoculated with 400,000 infected cells per ml.
The various PCV-2 strains disclosed herein, e.g. strains 1103 and 1121 are so cultivated.
EXAIVIPLE/RESULT 6: TITRATION OF PCV-2 Titration is carried out in 96-well microplates. A suspension of PK/15 cells (150 000 cells per ml) is first introduced (100 ~l per well). Then dilutions of the viral culture are done and 100 ~ul thereof are introduced in the wells. Incubation is done at 37°C with C02. After 24h, there is carried out a treatment with glucosamine half an hour at 37°C (for the conditions see example 3). The culture medium is then removed and fresh medium is introduced.
Incubation is conducted 72h at 37°C. Revelation of the foci is done using an anti-PCV-2 monoclonal antibody and a FITC labelled mouse conjugate.
This method can be used to titration for preparing inactivated as well as live attenuated PCV-2.
EXAMPLE/RESULT 7: I1V'ACTIVATION OF THE VIRAL ANTIGENS
At the end of the viral culture, the infected cells are harvested and lysed using ultrasound (Branson Sonifier) or with the aid of a rotor-stator type colloid mill (UltraTurrax, IKA). The suspension is then centrifuged at 3700 g for 30 minutes. The viral suspension is inactivated with 0.1% ethyleneimine for 18 hours at +37°C or with 0.5%
beta-propiolactone for 24 hours at +28°C. If the virus titre before inactivation is inadequate, the viral suspension is concentrated by ultrafiltration using a membrane with a 300 kDa cut-off (Millipore PTMK300). The inactivated viral suspension is stored at +5°C.
EXAiVIPLE/RESULT 8: PREPARATION OF THE VACCINE IN THE FORM
OF AN EMULSION BASED ON MINERAL OIL.

The vaccine is prepared according to the following formula:
- suspension of inactivated porcine circovirus: 250 ml - Montanide~ ISA 70 (SEPPIC): 750 ml The aqueous phase and the oily phase are sterilized separately by filtration.
The emulsion is prepared by mixing and homogenizing the ingredients with the aid of a Silverson turbine emulsifier.
One vaccine dose contains about 1075 TCID50. The volume of one vaccine dose is 0.5 ml for administration by the intradermal route, and 2 ml for administration by the intramuscular route.
This vaccine is used in a vaccination programme against the multisystemic wasting syndrome in combination with the Parvovax~ vaccine.
EXAMPLE/RESULT 9: PREPARATION OF THE VACCINE IN THE FORM
OF A METABOLIZABLE OIL-BASED EMULSION
The vaccine is prepared according to the following formula:
- suspension of inactivated porcine circovirus 200 ml - Dehymuls HRE 7 (Henkel): 60 ml - Radia 7204 (Oleofina): 740 ml The aqueous phase and the oily phase are sterilized separately by filtration.
The emulsion is prepared by mixing and homogenizing the ingredients with the aid of a Silverson turbine emulsifier.
One vaccine dose contains about 1075 TCID50. The volume of one vaccine dose is ml for administration by the intramuscular route.
This vaccine is used in a vaccination programme against the multisystemic wasting syndrome in combination with the Parvovax° vaccine.
2~ EXAMPLE/RESULT 10: VACCINATION OF PIGLETS
WITH DNA (PLASMID) VECTOR
Groups of 3 or 4 piglets, caesarian-derived day 0 are placed into isolators.
The piglets are vaccinated day 2 either with (A) a plasmid comprising ORF 13 or with (B) a mixture of this plasmid and another plasmid comprising ORF 4, and with a physiological solution for the control group. Each plasmid is diluted in sterile physiological solution (NaCl 0,9%) at 250 ~g/~1 final concentration. A 2 ml volume is injected by intramuscular route in two points of 1 ml (1 point each side of the neck). A second injection of vaccine or placebo is administered day 14. Vaccination with DNA is well tolerated by piglets and no evidence for adverse reaction to vaccination is noted. The piglets are challenged day 21 by oronasal administration of PCV-2 viral suspension, 1 ml in each nostril. After challenge piglets are weighed once a week. Rectal temperatures are recorded on days 17, 21, 22, 24, 27, 29, 31, 34, 37, 41, 44. Day 44 fecal swabs are collected from each piglet for PCV-2 shedding. The virus is detected and quantified by quantitative PCR. Day 45 necropsies are performed and tissue samples are collected for virus isolation.
~ Clinical symptoms There is no significant difference for the mean body weight gains or the mean body temperatures between groups.
~ Necropsy lesions The only gross finding noted in pigs at termination is bronchial lymphadenopathy. The lesions are scored according the following criteria.
0 = no visible enlargement of lymph nodes 1 = mild lymph nodes enlargement, restricted to bronchial lymph nodes 2 = moderate lymph nodes enlargement, restricted to bronchial lymph nodes 3 = severe lymph nodes enlargement, extended to bronchial submandibullar prescapsular and inguinal lymph nodes.
std is an abbreviation for standard deviation Groups Lymphadenopathy scores mean std N
(A) 1.2 1.3 4 (B) 2.0 1.7 3 controls 3.0 0.0 3 N = number of piglets in each group A reduction of the lymph node lesions is observed in 3 out 4 piglets immunized with (A) and 1 Ollt 3 piglets immunized with (B) mixture. This difference is not significant (p>0.05) due to the high value of the standard deviations (std).
~ Virus load in lymph nodes tissues:
Quantitative virus re-isolation is performed on tissue homogenates prepared from bronchial and mesenteric lymph nodes.
The data presented correspond to the virus titers in tissue homogenates after transformation in loglo.
PCV-2 titers Groups Bronchial LN Mesenteric LN
mean std mean std N

(A) 0.9 0.8 0.9 0.8 4 (B) 0.7 0.6 0.2 0.2 3 Controls 2.0 1.1 1.8 1.1 4 Bronchial lymph nodes seem to contain the most infectious virus. A reduction of the viral load is observed in bronchial and mesenteric lymph nodes from piglets immunized with either (A) or (B) mixture. This reduction is significant (p <_ 0.05 for the plasmids mixture.
~ Viral excretion:
Post challenge fecal swabs are assessed for schedding PCV-2 by PCR based on amplification of PCV-2 ORF 13. Each assay is performed in triplicate on 2 ml of sample.
Unvaccinated controls are negative for PCV-2 prior challenge and positive after challenge confirming the validity of the PCR assay.
Value are expressed as loglo (number of molecules of PCV-2 DNA in 2 ~l sample).

Logla number of PCV-2 DNA molecules Groacps mean std N
(A) 3.3 0.3 4 (A) 2.9 0.7 3 Controls 3.6 0.6 4 The differences between groups are not significant (p > 0.05).
EXAMPLE/RESULT 11: VACCINATION OF PIGLETS WITH
CANARYPOX LIVE VECTOR AND RESULTS
Groups of 3 or 4 piglets, caesarian-derived day 0 are placed into isolators.
Day 2 the piglets are vaccinated with 10$ pfu of (C) a canarypox comprising ORF 13, or (D) of a canarypox comprising ORF 13 and ORF 4, or parental canarypox, in 1 ml of PBS, by intramuscular route on the side of the neck. A second injection of vaccine or placebo is administered at day 14. Vaccination with canarypox is well tolerated by piglets and no evidence for adverse reaction to vaccination is noted The piglets are challenged day 21 by oronasal administration of .a PCV-2 viral suspension, 1 ml in each nostril.
Day 45 necropsies are performed and samples of tissues are collected for virus isolation.
Necropsy results ~ PMWS is characterized generally by lymphadenopathy and more rarely by hepatitis or nephritis. So the gross findings in lymph nodes are scored for each piglet in the following manner : 0 = no visible enlargement of lymph nodes ; 1 = mild lymph nodes enlargement, restricted to bronchial lymph nodes ; 2 = moderate lymph nodes enlargement, restricted to bronchial lymph nodes ; 3 = severe lymph nodes enlargement, extended to bronchial, submandibullar prescapular and inguinal lymph nodes.

Groups Scores (C) 0.5 0.0 0.0 1.0 mean 0.38 standard deviation 0.48 (D) 0.0 0.5 0.5 1.0 mean 0.5 standard deviation 0.41 Controls 2.0 2.5 2.5 2.5 mean 2.38 standard deviation 0.25 Bronchial lymphadenopathy for PCV-2 is a prominent gross finding. A
significant reduction-of the lymph nodes lesion in relation to control group is observed after immunization with (C) and (D) (p <_ 0.05).
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

References 1. Allan GM, McNeilly F, Cassady JP, et al.: 1995, Pathogenesis of porcine circovirus;
experimental infections of colostrum deprived piglets and examination of pig foetal material.
Vet Microbiol 44:49-641.
2. Allan GM, McNeilly F, Kennedy S, et al.: 1998, Isolation of porcine circovinis-like viruses from piglets with a wasting disease in the United States of America and Europe. J Vet Diag Invest 10:3-10.
3. Allan GM, Kennedy S, McNeilly F, et al.: 1999, Experimental reproduction of wasting disease and death by co-infection of pigs with porcine circovirus and porcine parvovirus, J
Comp Path 121:1-11 (July 1999).
4. Bolt DM, Hani H, Muller E, and Waldvogel AS: 1997, Nonsuppurative myocarditis in piglets associated with porcine parvovirus infection. J Comp Path 117:107-118.
5. Ellis, JA, Hassard L, Clark EG, et al.: 1998, Isolation of circovirus from lesions of piglets with postweaning multisystemic wasting syndrome. Can Vet J 39:44-51 6. Ellis JA, Krakowka S, Lairmore M, et al.: 1999, Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diag Invest 11:3-14.
7, Kim HS, Jo HS and Bergeland ME: 1989, Serologic, virologic, and histopathologic observations of encephalomyocarditis virus infection in mummified and stillborn pigs- J Vet Diag Invest 1:101-104.
8. Lager Kl'VI and Halbur PG: 1996, Gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus. J Vet Diag Invest 8:275-282.
9. Meehan BM, McNeilly F. Todd D, et al.: 1998, Characterization of novel circovirus DNA's associated wasting disease syndromes in pigs. J Gen Virol 79:2171-2179.

10. Mengeling WL 1992, Porcine parvovirus. In: Diseases of swine, ed. Leman AD, 7th ed-,._ pp.299-31 1. Iowa State University Press, Ames, IA.

11. Molitor TW, Orveerakul K, Zhang ZZ, et al.: 1991, Polymerase chain reaction (PRC) amplification for detection of porcine parvovirus. J Virol Meth 32:201-211 12. Pensaert M and DeMeurichy W: 1973, A porcine enterovirus causing fetal death and mummification. Experimental infection of pregnant female pigs. Zentralbl Veterinaermed Beih 11:2025.

13. Tischer 1, Rasch R and Tochtermann G: 1974, Characterization of papovavirus- and picomavirus-like particles -in permanent piglet kidney cell lines. Zentralbl-Bakertiol-Org-A
226:153-167. .

14. West KH, Bystrom, JM, Wojnarowicz C, et al.: 1999, Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus-2. J Vet Diag Invest 1.

WO 01/16330 DNA ZTaccine - pCV PCT/EP00/08781 R~P~N ~I x -~
RELATED APPLICATIONS:
Reference is made to US Application Serial 09/082,558 filed on 21 May 1998 and to US Application Serial 09/161,092 filed on 25 September 1998 in the form of a continuation-in-part of Serial 09/082,558, these applications being incorporated herein by way of reference, and each document cited in the present application also incorporated herein by way of reference. (Reference is made to U.S. application Serial No. 09/161, 092, filed 09/25/98 as a CIP of U.S.
application Serial No. 09/082,558, filed 05/21/98, claming priority from French Application Nos.97/12382, 98/00873, 98/03707, filed 10/03/97, 1/22/98, 3/20/98 ;
each of which, and each document cited therein, incorporated herein by reference).
The present invention relates to plasmid constructs encoding and expressing porcine circovirus (PCV for Porcine CircoVirus) immunogens responsible for the PMWS syndrome (Porcine Multisystemic Wasting Syndrome or Post-Weaning Multisystemic Trusting Syndrome), to methods of vaccination and to DNA
vaccines, as well as to methods of producing and of formulating these vaccines. All documents cited herein, and all documents cited in documents cited herein are hereby incorporated herein by reference.
PCV was originally detected as a noncytopathogenic contaminant in pig kidney cell lines PK/15. This virus was classified among the Circoviridae with the chicken anaemia virus (CAV for Chicken Anaemia Virus) and the PBFDV virus (Pscittacine Beak and Feather Disease Virus). These are small nonenveloped viruses (from 15 to 24 nm) whose common characteristic is to contain a genome in the form of a circular single-stranded DNA of 1.76 to 2.31 kilobases (kb). It was first thought that this genome encoded a polypeptide of about 30 kDa (Todd et al., Arch. Virol., 1991, 117: 129-135). Recent work has however shown a more comr x. transcription (Meehan B.M. . al., J. Gen.
Virol., 1997, 78: 221-227). Moreover, no significant homologies in nucleotide sequence or in common antigenic determinants are known between the three species of circoviruses known.
The PCV derived from PK/15 cells is considered not to be pathogenic. Its sequence is known from B.M. Meehan et al., J. Gen. Virol. 1997 (78) 221-227.
It is only very recently that some authors have thought that strains of PCV could be pathogenic and associated with the PMWS syndrome (G.P.S. Nayar et al., Can. Vet.
J., 1997, 38: 385-387 and Clark E.G., Proc. Am. Assoc.
Swine Prac. 1997: 499-501). Nayar et a1, have detected PCV DNA in pigs having the PMWS syndrome using PCR
techniques.
Monoclonal and polyclonal antibodies directed against circoviruses found in pigs having the symptoms of the PMWS syndrome have been able to demonstrate differences between these circoviruses and the porcine circoviruses isolated from culture of PK-15 cells (Allan G.M. et a1. Vet Microbiol., 1999, 66: 115-123).
The PMWS syndrome detected in Canada, the United States and France is clinically characterized by a gradual loss of weight and by manifestations such as tachypnea, dyspnea and jaundice. From the pathological point of view, it is manifested by lymphocytic or granulomatous infiltrations, lymphadenopathies and, more rarely, by hepatitis and lymphocytic or granulomatous nephritis (Clark E. G., Proc. Am. Assoc.
Swine Prac. 1997: 499-501; La Semaine Veterinaire No.
26, supplement to La Semaine Veterinaire 1996 (834); La Semaine Veterinaire 1997 (857): 54; G.P.S. Nayer et al., Can. Vet. J., 1997, 38: 385-387).
These circoviruses obtained from North America and from Europe are very closely related, with a degree of identity of more than 96~ of their nucleotide sequence, whereas the degree of identity is less than 80°s when the nucleotide sequences of these circoviruses are compared with those of porcine circoviruses isolated prom PK-15 cells. Accordingly, two viral subgroups have been proposed, PCV-2 for the circoviruses associated with the PMWS syndrome and PCV-1 for the circoviruses isolated from the PK-15 cells (Meehan B.M. et al., J. Gen. Virol., 1998, 79:
2171-2179; WO-A-9918214).
The Applicant has found that plasmid constructs encoding and expressing PCV-2 immunogens can be used to immunize pigs against the PMWS syndrome.
PCV-2 immunogens can be used in combination with PCV-1 immunogens to also immunize these animals against PCV-2.
According to a less preferred mode, the PCV-1 immunogens may be used alone.
The subject of the present invention is plasmid constructs encoding and expressing a PCV-1 or PCV-2 immunogen, in particular the open reading frames (ORFs) 1 and 2 of PCV-1, and the ORFs 1 and 2 of PVC-2.
It goes without saying that the invention automatically covers the plasmids encoding and expressing equivalent nucleotide sequences, that is to say the sequences which change neither the functionality or the strain specificity of the gene considered or those of the polypeptides encoded by this gene. The sequences differing through the degeneracy of the code will, of course, be included.
The PCV-2 sequences used in the examples are derived from Meehan et a1. supra (ORF1 nucleotides 398-1342; ORF2 nucleotides 1381-314; and correspond respectively to ORF4 and ORF13 in US 09/161,092 of 25 September 1998 and to COL4 and COL13 in WO-A-9918214). Other PCV-2 sequences have also been published in WO-A-9918214 and called Imp1008, Imp999, Imp1011-48285 and Imp1011-48121, as well as in A.L. Hamel et a1. J. Virol. June 1998, vol 72, 6;
5262-5267 (GenBank AF027217) and in I. Morozov et a1.
J. Clinical Microb. Sept. 1998 vol. 36, 9: 2535-2541, as well as GenBank AF086834, AF086835 and AF086836, and give access to equivalent ORF sequences.

T invention also covers L a equivalent sequences in the sense that they are capable of hybridizing to the nucleotide sequence of the gene considered under high stringency conditions. Among these equivalent sequences, there may be mentioned the gene fragments conserving the immunogenicity of the complete sequence.
The other ORFs 1-3 and 5-12 disclosed in US 09/161,092 of 25 September 1998 (COLs 1-3 and 5-12 in WO-A-9918214), in particular the ORFs 7 and 10, may be used under the conditions described here, in combination or otherwise with each other or with the ORFs 1 and 2 as defined here.
The word plasmid is here intended to cover any DNA transcription unit in the form of a polynucleotide sequence comprising the PCV sequence to be expressed and the elements necessary for its expression in vivo.
The circular plasmid form, supercoiled or otherwise, is preferred. The linear form is also included within the scope of the invention.
The subject of the present invention is more particularly the plasmids called pJP109 (containing the ORF2 gene of PCV-2, Figure 1), pJP111 (containing the ORFl gene of PCV-2, Figure 2), pJP120 (containing the ORF2 gene of PCV-1, Figure 3) and pJP121 (containing the ORF1 gene of PCV-1, Figure 4).
Each plasmid comprises a promoter capable of ensuring, in the host cells, the expression of the inserted gene under its control. It is in general a strong eukaryotic promoter and in particular a cytomegalovirus early promoter CMV-IE, of human or murine origin, or optionally of other origin such as rat or guinea pig. More generally, the promoter is either of viral origin or of cellular origin. As a viral promoter other than CMV-IE, there may be mentioned the SV40 virus early or late promoter or the Rous Sarcoma virus LTR promoter. It may also be a promoter from the virus from which the gene is derived, for example the promoter specific to the gene. As cellular -omoter, there may be mention. the promoter of a cytoskeleton gene, such as for example the _desmin promoter, or alternatively the actin promoter. When several genes are present in the same plasmid, they may be provided in the same transcription unit or in two different units.
The plasmids may also comprise other transcription regulating elements such as, for example, stabilizing sequences of the intron type, preferably intron II of the rabbit (3-globin gene (van Ooyen et a1.
Science, 1979, 206: 337-344), signal sequence of the protein encoded by the tissue plasminogen activator gene (tPA; Montgomery et a1. Cell. Mol. Biol. 1997, 43:
285-292), and the polyadenylation signal (polyA), in particular of the bovine growth hormone (bGH) gene (US-A-5,122,458) or of the rabbit ~3-globin gene.
The subject of the present invention is also immunogenic preparations and DNA vaccines comprising at least one plasmid according to the invention, encoding and expressing one of the PCV-1 or PCV-2 immunogens, preferably one of the abovementioned ORFs, in addition a veterinarily acceptable vehicle or diluent, with optionally, in addition, a veterinarily acceptable adjuvant.
The subject of the present invention i.s more particularly immunogenic preparations and vaccines containing at least one plasmid encoding and expressing one of the PCV-1 or PCV-2 immunogens, compositions formulated with an adjuvant, in particular a cationic lipid containing a quaternary ammonium salt, e,g, preferably DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanammonium; WO-A-9634109), and preferably coupled with a neutral lipid, e,g, preferably DOPE (dioleoylphosphatidylethanolamine), to form DMRIE-DOPE. Preferably, the plasmid mixture with this adjuvant is made immediately before use and preferably, before its administration to the animal, the mixture thus produced is allowed to form a complex, for exa: ~.e over a period rangins from 10 to 60 minutes, in-particular of the order of 30 minutes.
When DOPE is present, the DMRIE.DOPE molar ratio preferably ranges from 95:5 to .5:95, more particularly 1:1.
The plasmid:DMRIE or DMRIE-DOPE adjuvant weight ratio may range. in particular from 50:1 to 1:10, in particular from 10:1 to 1:5, preferably from 1:1 to 1:2.
According to another advantageous mode of the invention, it is possible to use, as adjuvant, an adjuvant compound selected from the polymers of acrylic or methacrylic acid and the copolymers of malefic anhydride and of alkenyl derivative. The polymers of acrylic or methacrylic acid crosslinked in particular with polyalkenyl ethers of sugars or of polyalcohols are preferred. These compounds are known by the term carbomer (Pharmeuropa vol. 8, No. 2, June 1996).
Persons skilled in the art can also refer to US-A-2,909,462 (incorporated by reference) describing such acrylic polymers crosslinked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced with unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol~ (GF Goodrich, Ohio, USA) are particularly appropriate. They are crosslinked with an allyl saccharose or with allylpentaerythritol. Among them, there may be mentioned Carbopol~ 974P, 934P and 971P.
Among the copolymers of malefic anhydride and of an alkenyl derivative, the EMAs~ (Monsanto) are preferred which are copolymers of malefic anhydride and ethylene, linear or crosslinked, for example crosslinked with divinyl ether. Reference may be made to J. Fie' 's et al. Nature, 186 . 778-7f 4 June 1960 (incorporated by reference). From the point of view of their structure, the polymers of acrylic or methacrylic acid and the EMAs~ preferably consist of basic units of the following formula:
Rz OOOH OOOH
in which - R, and R2, which are identical or different, represent H or CH3 - x = 0 or 1, preferably x = 1 - y = 1 or 2 , wi th x + y = 2 For the EMAs~, x - 0 and y - 2. For the carbomers, x = y = 1.
The dissolution of these polymers in water leads to an acidic solution which will be neutralized, preferably to physiological pH, to give .the adjuvant solution into which the actual vaccine will be incorporated. The carboxyl groups of the polymer are then partly in C00- form.
For this type of adjuvant, it is preferable to prepare a solution of the adjuvant, in particular of carbomer, in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. This stock solution is diluted by adding it to the required quantity (in order to obtain the desired final concentration), or a substantial part thereof, of water loaded with NaCl, preferably physiological saline (NaCl 9 g/1), in one or more portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pH will be used as it is to mix with the plasmid, in particular stored in lyophilized, liquid or frozen form.

Tt. polymer concentration in the _inal vaccine composition will be 0 . O1 % to 2 % w/v, more particularly 0.06 to 1% w/v, preferably 0.1 to 0.6% w/v.
In a specific embodiment, the immunogenic or vaccine preparation comprises a plasmid or a mixture of plasmids encoding and expressing PCV-2 ORF1 and ORF2.
The invention also provides for combining the vaccination against the porcine circovirus with a vaccination against other pig pathogens, in particular those which may be associated with the PMWS syndrome.
The subject of the present invention is also mixtures of plasmid containing at least one plasmid according to the invention and at least another plasmid encoding and expressing a porcine immunogen, selected for example from the group consisting of the glycoproteins gB and gD of the Aujeszky's disease virus (pseudorabies virus or PRV), the haemagglutinin and the nucleoprotein of the porcine influenza virus HlNl, the haemagglutinin and the nucleoprotein of the porcine influenza virus H3N2, the ORF5 and ORF3 genes of the PRRS virus of the Lelystad and USA strains, the VP2 protein of the porcine parvovirus, the E1 and E2 proteins of the hog cholera virus (HCV), the deleted apxI, apxII and apxIII genes from Actinobacillus pleuropneumoniae (see for the plasmids for example WO-A-9803658).
These mixtures of plasmids are taken up in a veterinarily acceptable vehicle or diluent, with optionally, in addition, a veterinarily acceptable acljuvant as described above, thus forming immunogenic preparations or multivalent DNA vaccines. These preparations or multivalent vaccines may in particular be advantageously formulated with DMRIE, and preferably coupled with a neutral lipid, DOPE, to form the DMRIE-DOPE.
The preparations or monovalent or multivalent DNA vaccines according to the invention, formulated or otherwise with adjuvant as described above, may also be advantageously supplemented with a cytokine preferably of porch, origin, in particular porcin GM-CSF. This addition of porcine GM-CSF (granulocyte macrophage -colony stimulating factor; Clark S.C. et a1. Science 1987, 230: 1229; Grant S.M. et a1. Drugs, 1992, 53:
516) may be carried out by incorporating into the preparation or into the vaccine either porcine GM-CSF
protein, or a plasmid encoding and expressing the porcine GM-CSF gene (Inumaru S. and Takamatsu H.
Immunol. Cell. Biol., 1995, 73: 474-476). Preferably, the porcine GM-CSF gene is inserted into a plasmid different from those encoding the PCV immunogens or the other porcine immunogens.
In particular, the plasmid encoding and expressing the porcine GM-CSF may be the plasmid pJP058 (Figure 5).
The immunogenic preparations and the monovalent or multivalent DD?A vaccines according to the invention may also be combined with at least one conventional vaccine (attenuated live, inactivated or subunit) or recombinant vaccine (viral vector) directed against at least one porcine pathogen which is different or identical. The invention provides in particular for the combination with adjuvant-containing conventional vaccines (attenuated live, inactivated or subunit). For the inactivated or subunit vaccines, there may be mentioned those containing in particular alumina gel alone or mixed with saponin as adjuvant, or those formulated in the form of an oil-in-water emulsion.
The subj ect of the present invention is also a method of immunization which makes it possible to induce an immune response in pigs towards the circoviruses according to the invention. Its subject is in particular a method of vaccination which is effective in pigs. These methods of immunization and vaccination comprise the administration of one of the preparations or of one of the monovalent or multivalent DNA vaccines as described above. These methods of immunization and vaccination comprise the administration of one or more successive doses of these preparatio or DNA vaccines. The prepay; ~_ons and DNA
vaccines may be administered, in the context of this method of immunization or of vaccination, by various routes of administration proposed in the prior art for polynucleotide vaccination, in particular the intramuscular and intradermal routes, and by means of known administration techniques, in particular injections with a syringe having a needle, by liquid jet (Furth et a1. Analytical Bioch., 1992, 205:
365-368) or by projection of gold particles coated with DNA (Tang et a1. Nature, 1992, 356: 152-154).
This method not only allows for administration to adult pigs, but also to the young and to gestating females; in the latter case, this makes it possible, in particular, to confer passive immunity onto the newborns (maternal antibodies).
The quantity of DNA used in the vaccines according to the present invention is between about 10 ~tg and about 2000 ~.g, and preferably between about 50 ~g and about 1000 Et.g. Persons skilled in the art will have the competence necessary to precisely define the effective dose of DNA to be used for each immunization or vaccination protocol.
The dose volumes may be between 0.5 and 5 ml, preferably between 2 and 3 ml.
A preferred method of immunization or of vaccination consists in the administration of the DNA
vaccines according to the invention by the intramuscular route.
The invention will now be described in greater detail with the aid of nonlimiting exemplary embodiments, taken with reference to the drawing, in which:
Figure 1: plasmid pJP109 Figure 2: plasmid pJP111 Figure 3: plasmid pJP120 Figure 4: plasmid pJP121 Figure 5: plasmid pJP058 WO 01/16330 ~CT/EP00/08781 Sequence ing SEQ ID
l~

SEQ ID No. 1:oligonucleotide JP779 SEQ ID No. 2:oligonucleotide JP780 SEQ ID No. 3:oligonucleotide JP781 SEQ ID No. 4:oligonucleotide JP782 SEQ ID No. 5:oligonucleotide JP783 SEQ ID No. 6:oligonucleotide JP784 SEQ ID No. 7:oligonucleotide JP785 SEQ ID No. 8:oligonucleotide JP786 EXAMPLES
Example 1 Construction of the plasmid pJP109 The plasmid pGEM7Z-Imp1010 Stoon-EcoRI No. 14 containing the genome of the PCV-2 virus in the form of an EcoRI fragment (B. Meehan et a1. J. Gen. Virol.
1998. 79 2171-2179) was digested with EcoRI in order to isolate, after agarose gel electrophoresis, the EcoRI
EcoRI fragment of 1768 base pairs (bp). This fragment was self-ligated.
The ORF2 gene of the PCV-2 virus strain 1010-Stoon (B. Pdeehan et a1. J. Gen. Virol. 1998. 79. 2171-2179;
GenBank sequence accession No. AF055392) was amplified, using the template consisting of the self-ligated EcoRI-EcoRI fragment, by the polymerase chain reaction (PCR) technique with the following oligonucleotides:
JP779 (SEQ ID NO 1) (35 mer):
5'CATCATCATGTCGACATGACGTATCCAAGGAGGCG3' and JP780 (SEQ ID NO 2) (36 mer):
5'TACTACTACAGATCTTTAGGGTTTAAGTGGGGGGTC3' in order to generate a 730 by PCR fragment. This fragment was digested with SalI and BglII in order to isolate, after agarose gel electrophoresis, the 715 by SalI-BglII restriction fragment. This fragment was then ligated with the plasmid pVR1012 (Hartikka J. et al.
Human Gene Therapy. 1996. 7. 1205-1217), digested beforehand with Sall and BglII, to give the plasmid pJP109 (5567 pb) (Figure 1).

WO .01/16330 Example 2: 'onstruction of the plasmid pJ L1 A polymerase chain reaction was carried out with the plasmid pGem7Z-Imp1010-Stoon (see Example 1) (B. Meehan et a1. J. Gen. Virol. 1998. 79. 2171-2179), and the following oligonucleotides:
JP781 (SEQ ID NO 3) (35 mer):
5'CATCATCATGTCGACATGCCCAGCAAGAAGAATGG3' and JP782 (SEQ ID NO 4) (36 mer):
5'TACTACTACAGATCTTCAGTAATTTATTTCATATGG3' in order to generate a 970 by PCR fragment containing the ORF1 gene of the PCV-2 virus. This fragment was digested with SalI and BglII in order to isolate, after agarose gel electrophoresis, the 955 by SalI-BglII
restriction fragment. This fragment was then ligated with the plasmid pVR1012 (Example 1) to give the plasmid pJPlll (5810 bp) (Figure 2).
Example 3: Construction of the plasmid pJP120 (PCV-1 ORF2) A polymerase chain reaction was carried out with the plasmid pPCVl (B. Meehan et al. J. Gen. Virol. 1997.
78. 221-227), and the following oligonucleotides;
JP783 (SEQ ID NO 5) (35 mer):
5'CATCATCATGTCGACATGACGTGGCCAAGGAGGCG3' and JP784 (SEQ ID NO 6) (40 mer):
5'TACTACTACAGATCTTTATTTATTTAGAGGGTCTTTTAGG3' in order to generate a 730 by PCR fragment containing the ORF2 gene of the PCV-1 virus (PK-15 strain, GenBank sequence accession No. U49186). This fragment was digested with SalI and BglII in order to isolate, after agarose gel electrophoresis, the 715 by SalI-BglII
restriction fragment. This fragment was then ligated with the plasmid pVR1012 (Example 1) to give the plasmid pJP120 (5565 bp) (Figure 3).
Example 4: Construction of the plasmid pJPl21 (PCV-1 ORF1) The plasmid pPCVl containing the PCV1 virus genome in the form of a PstI fragment (B. Meehan et a1. J. Gen.

WO 01/16330 pCT/EP00/08781 Virol. 19 , 78, 221-227) was digested ith PstI in .order to isolate, after agarose gel electrophoresis, the 1759 base pair (bp) PstI-Pstl fragment. This fragment was self-ligated.
The ORF1 gene of the PCV-1 virus strain PK-15 (B. Meehan et a1. J. Gen. Virol. 1997, 78, 221-227;
GenBank sequence. accession No. U49186) was amplified, using the template consisting of the self-ligated PstI-PstI fragment, by the polymerase chain reaction (PCR) technique with the following oligonucleotides:
JP785 (SEQ ID NO 7) (35 mer):
5'CATCATCATGTCGACATGCCAAGCAAGAAAAGCGG3' and JP786 (SEQ ID NO 8) (36 mer):
5'TACTACTACAGATCTTCAGTAATTTATTTTATATGG3' in order to generate a 965 by PCR fragment containing the ORF1 gene of the PCV-1 virus (strain PK-15). This fragment was digested with SalI and BglII in order to isolate, after agarose gel electrophoresis, the 946 by SalI-BglII restriction fragment. This fragment was then ligated with the plasmid pVR1012 (Example 1) to give the plasmid pJP121 (5804 bp) (Figure 4).
Example 5: Construction of the plasmid pJP058 (expressing porcine GM-CSF) Pig blood was collected over a tube containing EDTA by taking blood from the jugular vein. The mononucleated cells were harvested by centrifugation on a Ficoll gradient and then cultured in vitro in RPMI 1640 medium (Gibco-BRL) and stimulated by addition of concanavaline A (Sigma) at a final concentration of about 5 ~,g/ml in the culture medium. After 72 hours of stimulation, the lymphoblasts were harvested and the total RNA of these cells was extracted with the extraction kit "Micro-Scale Total RNA Separator Kit" (Clontech) following the manufacturer's recommendations. A reverse transcription reaction, carried out with the aid of the kit "1st-Strand cDNA Synthesis Kit" (Perkin Elmer), followed by a polymerase chain reaction, was carried out on t: total RNA extracted from .ese porcine lymphoblasts with the following oligonucleotides:
RG972 (33 mer):
5'TATGCGGCCGCCACCATGTGGCTGCAGAACCTG3' and RG973 (34 mer):
5'TATGCGGCCGCTACGTATCACTTCTGGGCTGGTT3' in order to generate a PCR fragment of about 450 base pairs (bp). This fragment was digested with NotI in order to isolate, after agarose gel electrophoresis, the 450 by NotI-NotI fragment. This fragment was then ligated with the plasmid pVR1012 (Example 1), preferably digested with NotI and dephosphorylated, to give the plasmid pJP058 (5405 bp) (Figure 5). The sequence of the pGM-CSF gene cloned into the plasmid pJP058 was checked and found to be identical to that available in the GenBank database (accession No.
D21074).
Example 6: Production of the purified plasmids for the vaccination of pigs Escherichia coli K12 bacteria (strains DH10B or SCS1) were transformed with the plasmids pJP109, pJP111, pJP058, pJP120 and pJP121 of Examples 1 to 5 supra. The five transformed clones obtained respectively with these five plasmids were then cultured separately, with shaking at +37°C, in Luria-Broth (LB) medium. The bacterial cultures were harvested at the end of the exponential phase and the plasmids were extracted according to the alkaline lysis technique. The extracted plasmids were then purified on a caesium chloride gradient according to the technique described by Sambrook et a1. (Molecular Biology: A Laboratory Manual, 2nd Edition, 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). After final extraction of ethidium bromide and precipitation in the presence of absolute ethanol, the purified plasmids were resuspended in TE buffer (1 mM Tris/EDTA, pH 8.0)-in order to obtain stock solutions containing 2 mg of plasmid pE ml. These stock solutions ~.e stored at -20°C before use.
Example 7: Control of the expression of ORFs 1 and 2 of the PCV-2 virus In order to control the products of expression of the PCV-2 ORF2 and PCV-2 ORF1 genes, cloned respectively into the plasmids pJP109 and pJP111, these plasmids were transfected into CHO-K1 (Chinese Hamster Ovary) cells (ATCC No. CCL-61) with the Lipofectamine Plus~
transfection kit (Gibco-BRL, Catalogue# 10964-013), following the manufacturer's recommendations for use.
48 hours after transfection, the transfected cells are washed and fixed with a 95o glacial acetone solution for 3 minutes at room temperature. Five monoclonal antibodies specific for the PCV-2 ORF1 proteins (F199 1D3GA and F210 7G5GD) and ORF2 proteins (F190 4C7CF, F190 2B1BC .and F190 3A8BC) were used as first antibodies. An anti-mouse IgG conjugate, labelled with Cy3, was used to reveal the specific labelling. A
PCV-2 specific fluorescence was observed with the 3~PCV-2 ORF2 monoclonals in the cells transfected with the plasmid pJP109, but not in those transfected with the plasmid pJPlll. In contrast, a PCV-2 specific fluorescence was observed with the two PCV-2 ORF1 monoclonals in the cells transfected with the plasmid pJP111, but not in those transfected with the plasmid pJP109. No fluorescence was detected with the PCV-2 monoclonals in CHO cells transfected with the plasmid pVR1012 alone or in the nontransfected CHO cells.
The same expression result was obtained with a polyclonal serum specific for the PCV-2 virus. In this case, a fluorescein-labelled anti-pig IgG conjugate was used to detect the specific fluorescence. No fluorescence was detected with this polyclonal serum in CHO cells transfected with the plasmid pVR1012 alone or in the nontransfected CHO cells.

Example 8: 'accination of pigs with naked JA
8.1. One-day-old piglets Groups of piglets obtained by Caesarean on DO of the protocol, are placed in an isolating unit. These piglets are vaccinated at the age of 2 days by the intramuscular route with various vaccinal solutions of plasmid. The vaccinal solutions are prepared by diluting the stock solutions in sterile physiological saline (0.9% NaCl).
The piglets are vaccinated:
either with the plasmid pJP109 alone or with the mixture of the plasmids pJP109 and pJP111 or with the mixture of the plasmids pJP109 and pJP058 or with the mixture of 'the plasmids pJP109, pJP111 and pJP058 The vaccinal solutions comprise 500 ~,g of each plasmid.
Volume: The vaccinai solutions are injected by the intramuscular route in a total volume of 2 ml. In practice, given the age of the piglets on vaccination (1-2 days), 1 injection of 1 ml is given on each side of the neck (= 2 x 1 ml).
Two injections of vaccine are carried out at two weeks' interval, that is to say on days D2 and D14 of the protocol.
A challenge is made on D21 of the protocol by oronasal administration of a viral suspension of a virulent PCV-2 strain. The piglets are then monitored for 3 weeks for the appearance of specific clinical signs of post-weaning multisystemic wasting syndrome in piglets. The signs which are monitored are:
rectal temperature: daily measurement for the first 14 days, then two measurements during the 3rd week following the challenge.
Weight: weighing of the piglets just before the challenge then once per week during the 3 weeks following the challenge.

Collection f blood samples to test fo: viremia and antibodies: blood samples taken on D2, D14, D21, D28, D35 and D42.
Autopsy: on D42, the surviving pigs are humanely killed and undergo autopsy to search for anatomicopathological lesions and to make histological preparations from the liver, the lymph nodes, the spleen, the kidneys and the thymus to search for lesions in these tissues.
8.2. 5-7-week old piglets 5- to 7-week old piglets, no longer having maternal antibodies specific for the PCV-2 virus are vaccinated by the intramuscular route:
either with the plasmid pJP109 alone, or with the mixture of the plasmids pJP109 and pJP111 or with the mixture of the plasmids pJP109 and pJP058 or with the mixture of the plasmids pJP109, pJP111 and pJP058 the vaccinal doses are the same as those indicated in Example 8.1 (500 ~.g per plasmid). The vaccinal solutions are injected by the intramuscular route in a volume of 2 ml (a single administration of 2 ml, into the neck muscles).
Two vaccinations are performed at 21 days' interval (DO and D21). A challenge is made 14 days after the last vaccination (D35) by intramuscular administration of a viral suspension of a virulent PCV-2 strain.
The pigs are then monitored for 8 weeks for the occurrence of specific clinical signs of the post weaning multisystemic wasting syndrome in piglets. The clinical monitoring of the piglets after the challenge is identical to that described in Example 8.1 except that the total duration of observation is this time 8 weeks.
Example 9: Vaccination of pigs with DNA formulated with DMRIE-DOPE
It is possible to use, in place of the naked plasmid DNA solutions described in Example 8, solutions of plasmid DN. formulated with DMRIE-DOPE. A ANA solution (containing one or more plasmids according to Example 6) at 1 mg/ml is prepared in 0.9o NaCl. A DMRIE-DOPE
solution at 0.75 mM is prepared by taking up a lyophilisate of DMRIE-DOPE in a suitable volume of sterile distilled water.
The formation of the plasmid DNA-cationic lipid complexes is achieved by diluting, in equal parts, the DMRIE-DOPE solution at 0.75 mM with the DNA solution at 1 mg/ml in 0.9o NaCl. The DNA solution is introduced gradually, with the aid of a syringe mounted with a 26G
needle, along the wall of the vial containing the cationic lipid solution so as to avoid the formation of foam. Gentle shaking is carried out as soon as the two solutions have been mixed. A composition comprising 0.375 mM DMRIE-DOPE and 500 ~,g/ml of DNA is finally obtained.
It is desirable for all the solutions used to be at room temperature for all the operations described above. The DNA/DP~IRIE-DOPE complex formation is performed at room temperature for 30 minutes before immunizing the pigs.
The pigs are then vaccinated according to the conditions described in Examples 8.1. and 8.2.
It should be clearly understood that the invention defined by the appended claims is not limited to the specific embodiments indicated in the description above, but encompasses the variants which depart from neither the scope nor the spirit of the present invention.

WO 01/16330 ~CT/EP00/OR781 CLAIPdS
1. Immunogenic preparation or vaccine comprising, on the one hand, a plasmid encoding and expressing a gene selected from the group consisting of ORF1 of PCV-2, ORF2 of PCV-2, ORFl of PCV-1 and ORF2 of -1, PCV

and, on the other hand, an element of capable increasing the immune respons e directed against the product of expression of the gene.

2. Immunogenic preparation or vaccine accordingto Claim 1, characterized in that the element capable of increasing the immune respon se comprises DMRIE as adjuvant.

3. Immunogenic preparation or vaccine accordingto Claim 2 , characterized in that the DMRIE is coupledto a neutral lipid.

4. Immunogenic preparation or vaccine accordingto Claim 3, characterized in that the DMRIE is coupledto DOPE.

5. Immunogenic preparation or vaccine accordingto Claim 1, characterized in that the element capable of increasing the immune response comprises a carbomeras adjuvant.

6. Immunogenic preparation or vaccine accordingto Claim l, characterized in that the element capable of increasing the immune respons e comprises a porcine cytokine.

7. Immunogenic preparation or vaccine accordingto Claim 6, characterized in that the porcine cytokineis GM-CSF.

8. Immunogenic preparation or vaccine accordingto Claim 6 or 7, characterized i n that it comprises a plasmid encoding and expressing the porcine cytokine.

9. Immunogenic preparation or vaccine accordingto Claim 1, characterized in that the element capable of increasing the immune response ne comprises a porci cytokine and a compound selected up from the gro comprising DMRIE, DMRIE/DOPE carbomer, as adjuvant.
and 10. Ircu Zogenic preparation or vaccim according to .any one of Claims 1 to 9, characterized in that it comprises a plasmid encoding and expressing another porcine immunogen.

Figure 1l5 Plasmid pJP109 hCMV/IE promoter Kanamycin resistance pJP~09 (5567 bp) Intron A
-oRI

polyA H~i Figure 215 Piasmid pJP111 hCM~7/IE promoter Kanamycin resistance pJP111 (5810 bp) Intron A
-oR1 polyA H~i Figure 315 Piasmid pJP120 hCMV/IE
promoter Kanamycin resistance pJP120 (5565 bp) In~'on A
-ox~

polyA ~i Figure 4l5 Plasmid pJP121 hCMV/IE romoter P
Kanamycin resistance pJP121 (5804 bp) zntron A
-ox~
PC'V-1 ORFl polyA H~i Figure 5l5 Plasmid pJP0~8 hCMV/IE promoter Kanamycin resistance pJP058 (5405 bp) pC3~I-CSF
polyA H~i >~ PP~IUD J X

Porcine Circovirus Recombinant Poxvirus Vaccine Reference is made to US Serial No. 09/082,358, filed on May 21, 1998 and to U.S.
Serial No. 09/161,092 filed on September 25, 1998 as a Continuation-in-Part of Serial No. 09/082,558, each of which is hereby incorporated herein by reference together with each of the documents cited therein.

The present invention relates to modified poxviruses and to methods of making and using the same. More in particular, the invention relates to recombinant ALVAC, which virus expresses gene products of porcine circovirus 2 (PCV2), and to immunological compositions or vaccines v~~hich induce an immune response directed to PCV2 gene products and which confer protective immunity against infection by PCV2.
Several publications are referenced in this application. Full citation to these documents is found at the end of the specification preceding the claims. These documents pertain to the field of this invention; and, each of the documents referenced in this application, as well as each document cited in each of the documents, are hereby incorporated herein by reference.
BACK ROUND OF THE INVENTION
Postweaning multisystemic blasting syndrome (PMWS) is a recently recognized disease of young pigs. PMWS is characterized clinically by progressive weight loss and other symptoms such as tachypnea, dyspnea and jaundice.

Pathologically, lymphocytic and granulomatous infiltrates, lymphadenopathy, and, more rarely, lymphocytic and granulomatous hepatitis and nephritis have been observed (Clark, 1997; Harding, 1997).
This disease has been described in different European countries as well as in North America. Treatment and prevention of this disease are not currently available.
Several lines of evidence point to porcine circovirus as the etiologic agent of PMWS (Ellis et al., 1998). Circoviruses have been recovered from pigs with PMWS, and antibodies to porcine circovirus have been demonstrated in pigs with the disease.
Circoviruses are single stranded circular DNA viruses found in a range of animal and plant species. Porcine circovirus was originally isolated as a contaminant from a continuous pig kidney cell line. The cell culture isolate has been designated PK-15 (Meehan et al., 1997). More recently, porcine circovirus obtained from pigs with PMWS has been compared to PK-15. Such viruses differ substantially from PK-15 at the nucleotide and protein sequence level, and have been designated PCV2 (Meehan et al., 1998; Hamel et al., 1998).
As many as thirteen open reading frames (ORFs) have been identified in the PCV2 genome (COL1 to COL13 in the French patent application 98 03707). Four of these ORFs share substantial homology with analogous ORFs within the genome of PK-15. ORF1 (Meehan et al., 1998; corresponding to COL4 in the French patent application 98 03707), comprising nt 398-1342 (GenBank accession number AF055392), has the potential to encode a protein with a predicted molecular weight of 37.7 kD. ORFZ (Meehan et al., 1998; corresponding to COL13 in the French patent application 98 03707), comprising nt 1381-1768 joined to 1-314 (GenBank accession number AF055392), may encode a protein v~~ith a predicted molecular weight of 27.8 kD. ORF3 (Meehan et al., 1998; con esponding to COL7 in the French patent application 98 03707), comprising nt 1018-704 (GenBank accession number AF055392), may encode a protein with a predicted molecular weight of 11.9 kD.
ORF4 (Meehan et al., 1998; corresponding to COL10 in the French patent application 98 03707), comprising nt 912-733 (GenBanh accession number AF055392), may encode a protein with a predicted molecular weight of 6.5 kD.
ORF 1 of PCV2 is highly homologous (86% identity) to the ORF1 of the PK-1 S isolate (Meehan et al., 1998). The ORF1 protein of PK-15 has been partially characterized (Meehan et al., 1997 ; Mankertz et al., 1998a). It is known to be essential for virus replication, and is probably involved in the viral DIvTA
replication.
Protein sequence identity between the respective ORF2s was lower (66%
identity) than that of the ORFls but each of the ORF2s shared a highly conserved basic IvT-terminal region, similar to that observed in the IvT-terminal region of the major structural protein of the avian circovirus chicken anemia virus (CAV) (Meehan et al., 1998). Recently, Mankertz et al. (1998b) has suggested that the ORF2 of the PK-isolate (designated ORF 1 in Mankertz et al., 1998b) codes for a capsid protein.
Greater differences were observed between the respective ORF3s and ORF4s of the PK-15 isolate and PCV2. In each case, there was a deletion of the C-terminal region of PCV2 ORF4 and ORF3 compared to the corresponding ORFs present in the genome of the PK-15 isolate. The highest protein sequence homology was observed at the N-terminal regions of both ORF3 and ORF4 (Meehan et al., 1998).
The transcription analysis of the genome of PCV2 has not been published yet.
Recent data obtained with the PK-15 isolate indicated that the ORF2 transcript is spliced (Mankertz et al., 1998b).

Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. With the eradication of smallpox, a new role for poxviruses became important, that of a genetically engineered vector for the expression of foreign genes (Panicali and Paoletti, 1982;
Paoletti et al., 1984). Genes encoding heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990). A highly attenuated strain of vaccines, designated MVA, has also been used as a vector for poxvirus-based vaccines. Use of MVA is described in U.S. Patent No. 5,185,146.
Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipox viruses. Both fowlpoxvirus (FPV; Taylor et al.
1988a, b) and canarypoxvirus (CPV; Taylor et al., 1991 & 1992) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988c). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988c). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990 ; Edbauer et al., 1990).
Other attenuated poxvirus vectors have been prepared by genetic modifications of wild type strains of virus. The NYVAC vector, derived by deletion of specific virulence and host-range genes from the Copenhagen strain of vaccinia (Tartaglia et al., 1992) has proven useful as a recombinant vector in eliciting a protective immune response against an expressed foreign antigen.
Another engineered poxvirus vector is ALVAC, derived from canarypox virus.
ALVAC does not productively replicate in non-avian hosts, a characteristic thought to improve its safety profile (Taylor et al., 1991 & 1992). Both ALVAC and NYVAC
are BSL-1 vectors.
One approach to the development of a subunit PCV2 vaccine is the use of live viral vectors to express relevant PCV2 ORFs. Recombinant poxviruses can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Patent Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587;
5,174,993;
5,494,807; and 5,505,941, the disclosures of which are incorporated herein by reference. It can thus be appreciated that provision of a PCV2 recombinant poxvirus, and of compositions and products therefrom particularly ALVAC based PCV2 recombinants and compositions and products therefrom, especially such recombinants containing ORFs 1 and/or 2 of PC~'2, and compositions and products therefrom would be a highly desirable advance over the current state of technology.
OBJECTS AND SUMMARY OF THE Il~'~'ENTION
It is therefore an object of this invention to provide compositions and methods for treatment and prophylaxis of infection with PCV2. It is also an object to provide a means to treat or prevent PMWS.
In one aspect, the present invention relates to an antigenic, immunological or vaccine composition or a therapeutic composition for inducing an antigenic or immunological response in a host animal inoculated with the composition, said vaccine including a carrier and modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The virus used in the composition according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus and more particularly, ALVAC. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from PCV2 ORFs, e.g., PCV2 ORF 1 and/or 2.
In yet another aspect, the present invention relates to an immunogenic composition containing a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The modified recombinant virus includes, with a non-essential region of the virus genome a heterologous DNA sequence which encodes an antigenic protein (e.g., derived from PCV2 ORFs, especially ORES 1 and/or 2) wherein the composition, when administered to a host, is capable of inducing an immunological response specific to the antigen.
In a still further aspect, the present invention relates to a modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that the virus has attenuated virulence, and wherein the modified recombinant virus further contains DIVA from a heterologous source in a nonessential region of the virus genome. The DNA can code for PCV2 genes such as any or all of PCV2 ORF1, ORF2, ORF3, or ORF4 (Meehan et al., 1998). In particular, the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor or by utilizing naturally host-restricted viruses. The virus used according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus or canarypox virus. Advantageously, the open reading frame is selected from the group consisting of J2R, B 13R + B 14R, A26L, A56R, - K1L, and I4L (by the terminology reported in Goebel et al., 1990); and, the combination thereof. In this respect, the open reading frame comprises a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range gene region or a large subunit, ribonucleotide reductase;
or, the combination thereof. A suitable modified Copenhagen strain of vaccinia virus is identified as NYVAC (Tartaglia et al., 1992), or a vaccinia virus from which has been deleted 12R, B13R+B14R, A26L, A56R, C7L-Kl l and I4L or a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range region, and a large subunit, ribonucleotide reductase ( ee a o U.S.
Patent No. 5,364,773). Preferably, the poxvirus vector is an ALVAC or, a canarypox virus (Rentschler vaccine strain) which was attenuated, for instance, through more than 200 serial passages on chick embryo fibroblasts, a master seed therefrom was subjected to four successive plague purifications under agar from which a plague clone was amplified through five additional passages.
The invention in yet a further aspect relates to the product of expression of the inventive recombinant poxvirus and uses therefor, such as to form antigenic, immunological or vaccine compositions for treatment, prevention, diagnosis or testing; and, to DNA from the recombinant poxvirus which is useful in constructing DNA probes and PCR primers.
In one aspect, the present invention relates to a recombinant poxvirus containing therein a DNA sequence from PCV2 in a non-essential region of the poxvirus genome. The poxvirus is advantageously an avipox virus, such as fowlpox virus, especially an attenuated fowlpox virus, or a canarypox virus, especially an attenuated canarypox virus, such as ALVAC.
According to the present invention, the recombinant poxvirus expresses gene products of the foreign PCV2 gene. Specific ORFs of PCV2 are inserted into the poxvirus vector, and the resulting recombinant poxvirus is used to infect an animal.
Expression in the animal of PCV2 gene products results in an immune response in the animal to PCV2. Thus, the recombinant poxvirus of the present invention may be used in an immunological composition or vaccine to provide a means to induce an immune response which may, but need not be, protective.
The administration procedure for recombinant poxvirus-PCV2 or expression product thereof, compositions of the invention such as immunological, antigenic or vaccine compositions or therapeutic compositions, can be via a parenteral route (intradermal, intramuscular or subcutaneous). Such an administration enables a systemic immune response, or humoral or cell-mediated responses.
More generally, the inventive poxvirus- PCV2 recombinants, antigenic, immunological or vaccine poxvirus- PCV2 compositions or therapeutic compositions can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and the route of administration. The compositions can be administered alone, or can be co-administered or sequentially administered with compositions, e.g., with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocitrtail" or combination compositions of the invention and methods employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and, the route of administration. In this regard, reference is made to U.S. Patent No. 5,843,456, incorporated herein by reference, and directed to rabies compositions and combination compositions and uses thereof.
Examples of compositions of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions. In such compositions the recombinant poxvirus or antigens may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
The compositions can also be lyophilized. The compositions can contain auxiliary WO 01/16330 .PCT/EP00/08781 substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Suitable dosages can also be based upon the Examples below.
The compositions can contain at least one adjuvant compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of malefic anhydride and alkenyl derivative.
The preferred adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol.
8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Patent No.
2,909,462 (incorporated herein by reference) which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol~ (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol~ 974P, 934P and 971P.
Among the copolymers of malefic anhydride and alkenyl derivative, the copolyners EMA~ (Monsanto) ~~hich are copolymers of malefic anhydride and ethylene, linear or cross-linked, for example cross-linked with divinyl ether, are R~
- -- -C ~~~ X C (~2~ y -- -preferred. Reference may be made to J. Fields et al., Nature, 186 : 778-780, 4 June 1960, incorporated herein by reference.
From the point of view of their structure, the polymers of acrylic or methacrylic acid and the copolymers EMA~ are preferably formed of basic units of the following formula in which - R, and R2, which are identical or different, represent H or CH, - x = 0 or 1, preferably x = 1 - y=1 or2,withx+y=2 For the copolymers EMA~, x = 0 and y = 2. For the carbomers, x = y =1.
The dissolution of these polymers in water leads to an acid solution which will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the vaccine itself will be incorporated. The carboxyl groups of the polymer are then partly in COO' form.
Preferably, a solution of adjuvant according to the invention, especially of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. This stock solution is diluted by adding it to the desired quantity (for obtaining the desired final concentration), or a substantial part thereof, of water charged with NaCI, preferably physiological saline (NaCL 9 g/1) all at once in several portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pH will be used as it is for mixing with the vaccine, which may be especially stored in freeze-dried, liquid or frozen form.
The polymer concentration in the final vaccine composition will be 0.01% to 2% w/v, more particularly 0.06 to 1% wlv, preferably 0.1 to 0.6% w/v.
The immunological compositions according to the invention may be associated to at least one live attenuated, inactivated, or sub-unit vaccine, or recombinant vaccine (e.g.
poxvirus as vector or DNA plasmid) expressing at least one immunogen from another pig pathogen.
These and other embodiments are disclosed or are obvious from and encompassed by the following detailed description.
~IZiEF DES .RIPTION OF THE DRAWINGS
A better understanding of the present invention will be had by referring to the accompanying drawings, in which:
~ FIG. 1 (SEQ ID N0:1) shows the nucleotide sequence of a 3.7 kilobase pair fragment of ALVAC DNA containing the C6 open reading frame.
~ FIG. 2 shows the map of pJP 102 donor plasmid.
~ FIG. 3 (SEQ ID N0:8) shows the nucleotide sequence of the 2.5 kilobase pair fragment from pJP102 donor plasmid from the KpnI (position 653) to the SacI
(position 3166) restriction sites.
~ FIG. 4 shows the map of pJP 105 donor plasmid.
~ FIG. 5 shows the map of pJP 107 donor plasmid.

~ FIG. 6 (SEQ ID N0:11) shows the nucleotide sequence of the 3.6 kilobase pair fragment from pTPl 07 donor plasmid from the KpnI (position 653) to the SacI
(position 4255) restriction sites.

The invention is directed to recombinant poxvinases containing therein a DNA
sequence from PCV2 in a nonessential region of the poxvirus genome. The recombinant poxviruses express gene products of the foreign PCV2 gene. In particular, ORF2 and ORF1 genes encoding PCV2 proteins were isolated, characterized and inserted into ALVAC (,canarypox vector) recombinants. The molecular biology techniques used are the ones described by Sambrook et al.
(1989).
('ell Lines and Virus Strains. The strain of PCV2 designated Imp.1010-Stoon has been previously described (Meehan et al., 1998). It was isolated from mesenteric lymph node tissues from a diseased pig originating from Canada. Cloning of the PCV2 genome was described by Meehan et al. (1998). Plasmid pGem7Z-Imp1010-Stoon-EcoRI No. 14 contains the PCV2 genome as an EcoRI fragment inserted into the EcoRI site of plasmid pGem-7Z (Promega, Madison, WI). The complete nucleotide sequence of the Imp.1010-Stoon PCV2 strain has been determined by Meehan et al. (1998) and is available under the GenBanh accession number AF055392.
The parental canarypox virus (Rentschler strain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial passages on chick embryo fibroblasts. A master viral seed was subjected to four successive plaque purifications under agar and one plaque clone was amplified through five additional passages after which the stock virus was W~ 01/16330 CA 02383367 2002-02-27 used as the parental virus in in vitro recombination tests. The plaque purified canarypox isolate is designated ALVAC. ALVAC was deposited November 14, 1996 under the terms of the Budapest Treaty at the American Type Culture Collection, ATCC accession number VR-2547.
The generation of poxvirus recombinants involves different steps: (1) construction of an insertion plasmid containing sequences ("arms") flanking the insertion locus within the poxvirus genome, and multiple cloning site (MCS) localized between the two flanking arms (e.g., see Example 1); (2) construction of donor plasmids consisting of an insertion plasmid into the MCS of which a foreign gene expression cassette has been inserted (e.g. see Examples 2 to 5); (3) in vitro recombination in cell culture between the arms of the donor plasmid and the genome of the parental poxvirus allowing the insertion of the foreign gene expression cassette into the appropriate locus of the poxvirus genome, and plaque purification of the recombinant virus (e.g. see Example 6).
PCV2 recombinant immunogens may be used in association with PCV1 immunogens, for immunization of animals against PMWS. In a least preferred approach, PCV1 immunogens may be used without PCV2 immunogens.
The present invention is additionally described by the following illustrative, non-limiting Examples.
Example 1 - CONSTRUCTION OF CANARYPOX INSERTION PLASMID

Figure 1 (SEQ ID N0:1) is the sequence of a 3.7 kb segment of canarypox DNA. Analysis of the sequence revealed an ORF designated C6L initiated at position 377 and terminated at position 2254. The following describes a C6 insertion plasmid constructed by deleting the C6 ORF and replacing it with a multiple cloning site (MCS) flanked by transcriptional and translational termination signals. A 380 by PCR
fragment was amplified from genomic canarypox DNA using oligonucleotide primers C6A1 (SEQ ID N0:2) and C6B 1 (SEQ ID N0:3). A 1155 by PCR fragment was amplified from genomic canarypox DNA using oligonucleotide primers C6C1 (SEQ
ID N0:4) and C6D1 (SEQ ID NO:S). The 380 by and 1155 by fragments were fused together by adding them together as template and amplifying a 1613 by PCR
fragment using oIigonucleotide primers C6A1 (SEQ ID N0:2) and C6D1 (SEQ ID IvT0:5).
This fragment was digested with SacI and KpoI, and ligated into pBluescript SK+
(Stratagene, La Jolla, CA, USA) digested with SacIlKpnI. The resulting plasmid, pC6L was confirmed by DNA sequence analysis. It consists of 370 by of canarypox DNA upstream of C6 ("C6 left arm"), vaccinia early termination signal, translation stop codons in six reading frames, an MCS containing SrnaI, PstI, XhoI and EcoRI
sites, vaccinia early termination signal, translation stop codons in six reading frames and 1156 by of downstream canary pox sequence ("C6 right arm").
Plasmid pJP099 was derived from pC6L by Iigating a cassette containing the vaccinia H6 promoter (described in Taylor et al. (1988c), Guo et al. (1989), and Perkus et al. (1989)) coupled to a foreign gene into the SmaIlEcoRI sites of pC6L.
This plasmid pJP099 contains a unique EcoRV site and a unique NruI site located at the 3' end of the H6 promoter, and a unique SaII site located between the STOP
codon of the foreign gene and the C6 left arm. The -4.5 kb EcoRVISaII or NruIISaII
fragment from pJP099 contains therefore the plasmid sequence (pBluescript SK+
;
Stratagene, La Jolla, CA, USA), the 2 C6 arms and the S' end of the H6 promoter until the EcoRV or Nrul site.
cP~"Pn~es of the primers:
Primer C6A1 (SEQ ID N0:2) ATCATCGAGCTCGCGGCCGCCTATCAAAAGTCTTAATGAGTT
Primer C6B 1 (SEQ ID N0:3) GAATTCCTCGAGCTGCAGCCCGGGTTTTTATAGCTAATTAGTCATTTTTTC
GTAAGTAAGTATTTTTATTTAA
Primer C6C1 (SEQ ID N0:4) CCCGGGCTGCAGCTCGAGGAATTCTT"ITTATTGATTAACTAGTCAAATGAG
TATATATAATTGAAAAAGTAA
Primer C6D1 (SEQ ID NO:S) GATGATGGTACCTTCATAAATACAAGTTTGATTAA.ACTTAAGTTG
Example 2 - CON~'i'R~,r~'TTI~IN !1F AT ~~er mn~TnR PT A~MiI) FOR

Plasmid pGem7Z-Imp1010-Stoop-EcoRI No. 14, containing the PCV2 genome as an EcoRI fragment in plasmid pGem-7Z, was digested with EcoRI, and a 1768bp fragment was isolated and ligated.
In order to insert PCV2 ORF 2 into an appropriate ALVAC insertion vector:
Primers JP760 (SEQ ID N0:6) and JP773 (SEQ ID N0:7) were used to amplify PCV2 ORF 2 from the 1768bp ligated EcoRI fragment (see above) resulting in PCR

J1304. Primer JP760 (SEQ ID N0:6) contains the 3' end of the H6 promoter from EcoRV and the 5' end of PCV2 ORF 2. Primer JP773 (SEQ ID N0:7) contains the 3' end of PCV2 ORF 2 followed by a SaII site. The product of PCR J1304 was then digested with EcoRVISaII and cloned as a --750 by fragment into a --4.5 kb EcoRVlSalI fragment from pJP099 (see above in Example 1). The resulting plasmid was confirmed by sequence analysis and designated pJP102 (see the map of pJP102 in Figure 2 and the sequence (SEQ ID N0:8) in Figure 3). The sequence of ORF 2 matches sequence available in GenBank, Accession Number AF055392. The donor plasmid pJP102 (linearized with NotI) was used in an in vitro recombination (IVR) test to generate ALVAC recombinant vCP1614 (see Example 6).
~~nence of the primers:
JP760 (SEQ ID N0:6) CAT-CAT-CAT-GAT-ATC-CGT-TAA-GTT-TGT-ATC-GTA-ATG-ACG-TAT-CCA-AGG-AGG-CG
JP773 (SEQ ID N0:7) TAC-TAC-TAC-GTC-GAC-TTA-GGG-T'TT-AAG-TGG-GGG-GTC
example 3 - CONSTRU T10N OF AN ALVAC DONOR PLASMID FOR
g('V2 ORF2 AND ORFl PCV2 ORF 1 was amplified by PCR using primers JP774 (SEQ ID N0:9) and JP775 (SEQ ID N0:10) on plasmid pGem7Z-Imp1010-Stoop-EcoRI No. 14 resulting W~ 01/16330 CA 02383367 2002-02-27 in PCR J1311. Primer JP774 (SEQ )D N0:9) contains the 3' end of the H6 promoter from NruI and the 5' end of PCV2 ORF1. Primer JP775 (SEQ ID NO:10) contains the 3' end of PCV2 ORF 1 followed by a SaII site. The product of PCR J1311 (~l Kb) was cloned into pCR2.1 (Invitrogen, Carlsbad, CA). The resulting plasmid was confirmed by sequence analysis and designated pJP104. The sequence of ORF1 matches sequence available in GenBank, Accession Number AF055392. A --970 by NruIISaII fragment was isolated from pJP104 and cloned into a --4.5 kb NruIISaII
fragment from pJP099 (see Example 1), resulting in a plasmid which was confirmed by restriction analysis and designated pJP105 (see Figure 4). The donor plasmid pJPlO~ could be used in an in vitro recombination test (described in Example 6) to generate ALVAC recombinant expressing the PCV2 ORF1.
A ~838bp BamHIlSaII from pJP102 (see Example 2) was blunted using the Klenow fragment of DNA polymerase, and was cloned into the Klenow-blunted EcoRI site of pJP 105. Clones were checked for orientation of insert by restriction analysis and a head-to-head orientation was chosen. This plasmid was confirmed by sequence analysis and designated pJP107 (see the map of pJP107 in Figure 5 and the sequence (SEQ )D I~TO:11) in Figure 6). The donor plasmid pJP107 (linearized with NotI) was used in an in vitro recombination 5 (IVR) test to generate the ALVAC
recombinant vCP 1615 (see Example 6).
)P774 (SEQ >D N0:9) CAT-CAT-CAT-TCG-CGA-TAT-CCG-TTA-AGT-TTG-TAT-CGT-AAT-GCC-CAG-CAA-GAA-GAA-TGG

JP775 (SEQ 117 NO:10) TAC-TAC-TAC-GTC-GAC-TCA-GTA-ATT-TAT-TTC-ATA-TGG
example 4 - CONSTRUCTION OF ALVAC DONOR PLASMID FOR

Plasmid pPCV 1 (B. Meehan et al. J. Gen. Virol. 1997. 78. 221-227), containing the PCV 1 genome as a PstI fragment in plasmid pGem-7Z, was used as a template to amplify the PCV 1 ORF2.
In order to insert PCV2 ORF 2 into an appropriate ALVAC insertion vector Primers JP787 (SEQ ID N0:12) and JP788 (SEQ ID NO:I3) were used to amplify PCV 1 ORF 2 from plasmid pPCV 1 (see above) resulting in PCR J 1315. Primer (SEQ ID N0:12) contains the 3' end of the H6 promoter from EcoRV and ORF 2 followed by a SaII site. The product of PCR J13I5 was then digested with EcoRVISaII and cloned as a 750 by frao~ment into a ~4.5 kb EcoRVISaII fragment from pJP099 (see above in Example 1). The resulting plasmid was confirmed by sequence analysis and designated pJP 113. The sequence of ORF 2 matches sequence available in GenBank, Accession Number U49186. The donor plasmid pJP 113 (linearized with NotT) was used in an in vitro recombination (IVR) test to generate ALVAC recombinant vCP1621 (see Example 7).
.tee ,quence of the n'~mer~s:
JP787 (SEQ 117 N0:12) CAT-CAT-CAT-GAT-ATC-CGT-TAA-GTT-TGT-ATC-GTA-ATG-ACG-TGG-CCA-AGG-AGG-CG

JP788 (SEQ ID N0:13) TAC-TAC-TAC-GTC-GAC-TTA-TTT-ATT-TAG-AGG-GTC-TTT-TAG-G
Example 5 - CONSTRUCTION OF AN A VAC DONOR PLASMID FOR
PCVI ORF2 AND ORFl Plasmid pPCV 1 (see Example 4 above), containing the PCV 1 genome as a PstI fragment in plasmid pGem-7Z, was digested with PstI, and a 1759 by fragment was isolated and ligated.
Primers JP789 (SEQ ID N0:14) and JP790 (SEQ ID N0:15) were used to amplify PCV 1 ORF1 from the 1759 by ligated PstI fragment (see above), resulting in PCR J1316. Primer JP789 (SEQ ID N0:14) contains the 3' end of the H6 promoter from NruI and the S' end of PCV 1 ORF1. Primer JP790 (SEQ ID IvT0:15) contains the 3' end of PCV 1 ORF1 followed by a SaII site. The product of PCR J1316 (--1 Kb) was cloned into pCR2.1 (Invitrogen, Carlsbad, CA). The resulting plasmid was confirmed by sequence analysis and designated pJP114. The sequence of ORF1 matches sequence available in GenBank, Accession Number U49186. A 970 by NruIlSalI fragment was isolated from pJP114 and cloned into a ~4.5 kb NruIlSalI
fragment from pJP099 (see Example 1), resulting in a plasmid which was confirmed by restriction analysis and designated pJP 115. The donor plasmid pJP 115 could be used in an in vitro recombination test (described in Example 7) to generate ALVAC
recombinant expressing the PCV 1 ORFl.

A ~838bp BamHUSaII from pJP 113 (see Example 4) was blunted using the Klenow fragment of DNA polymerase, and was cloned into the Klenow-blunted EcoRI site of pJP 115. Clones were checked for orientation of insert by restriction analysis and a head-to-head orientation was chosen. This plasmid was confirmed by sequence analysis and designated pJP 117. The donor plasmid pJP 117 (linearized with NotI) was used in an in vitro recombination (IVR) test to generate the ALVAC
recombinant vCP1622 (see Example 7).
~~"encP of the primers:
JP789 (SEQ ID N0:14) CAT-CAT-CAT-TCG-CGA-TAT-CCG-TTA-AGT-TTG-TAT-CGT-AAT-GCC-AAG-CAA-GAA-AAG-CGG
JP790 (SEQ 117 NO:15) TAC-TAC-TAC-GTC-GAC-TCA-GTA-ATT-TAT-TTT-ATA-TGG
xampie 6 - GENERATION OF AL~'AC-PCV2 RECOMBINANTS
Plasmids pJP102 (see Example 2 and Figure 2) and pJP107 (see Example 3 and Figure 5) were Iinearized with NotI and transfected into ALVAC infected primary CEF cells by using the calcium phosphate precipitation method previously described (Panicali and Paoletti, 1982 ; Piccini et al., 1987). Positive plaques were selected on the basis of hybridization to specific PCV2 radiolabeled probes and subjected to four sequential rounds of plaque purification until a pure population was achieved.
One representative plaque from each IVR was then amplified and the resulting ALVAC

recombinants were designated vCP1614 and vCP1615. The vCP1614 virus is the result of recombination events between ALVAC and the donor plasmid pJP 102, and it contains the PCV2 ORF2 inserted into the ALVAC C6 locus. The vCP1615 virus is the result of recombination events between ALVAC and the donor plasmid pJP
107, and it contains the PCV2 ORF2 and ORFl inserted into the ALVAC C6 locus in a head-to-head orientation.
In a similar fashion, a recombinant ALVAC expressing only PCV2 ORF1 can be generated using the donor plasmid pJP105 described in Example 3.
Immunofluorescence. In order to determine if the PCV2 proteins were expressed in ALVAC recombinant infected Vero cells, immunofluorescence (IF) analysis was performed. Infected Vero cells were washed with PBS 24 hrs after infection (m.o.i. of approx. 10) and fixed with 95% cold aceton for 3 minutes at room temperature. Five monoclonal antibody (MAb) preparations (hybridoma supernatant) specific for PCV2 ORF1 (PCV2 199 1D3GA & PCV2 210 7GSGD) or ORF2 (PCV2 190 4C7CF, PCV2 190 2B1BC & PCV2 190 3A8BC) were used as the first antibody.
These specific monoclonal antibodies were obtained from Merial-Lyon.
Monoclonal antibodies can also be obtained following the teachings of documents cited herein, e.g.
U.S. Ser. Nos. 09/082,358 and 09/161,092, incorporated herein by reference.
The IF
reaction was performed as described by Taylor et al. (1990).
PCV2 specific immunofluorescence with the three ORF2-specific antibodies could be detected in cells infected with vCP 1614 and cells infected with vCP
1615.
PCV2 specific immunofluorescence with the two ORF1-specific antibodies could be detected in cells infected with vCP 16I 5 only. These results indicated that, as expected, vCP1614 expresses only ORF2, whereas vCP1615 expresses both ORF1 and ORF2. No fluorescence was detected in parental ALVAC infected Vero cells, nor in uninfected Vero cells.
~cample 7 - GENERATION OF ALVAC-PC~'1 RECOMBINANTS
Plasmids pJP113 (see Example 4) and pJP117 (see Example 5) were linearized with NorI and transfected into ALVAC infected primary CEF cells by using the calcium phosphate precipitation method previously described (Panicali and Paoletti, 1982; Piccini et al., 1987). Positive plaques were selected on the basis of hybridization to specific PCV1 radiolabeled probes and subjected to four sequential rounds of plaque purification until a pure population was achieved. One representative plaque from each IVR was then amplified and the resulting ALVAC recombinants were designated vCP1621 and vCP1622. The vCP1621 virus is the result of recombination events beriveen ALVAC and the donor plasmid pJP113, and it contains the PCV1 ORF2 inserted into the ALVAC C6 locus. The vCP1622 virus is the result of recombination events between ALVAC and the donor plasmid pJP 117, and it contains the PCV1 ORF2 and ORF1 inserted into the ALVAC C6 locus in a head-to-head orientation.
In a similar fashion, a recombinant ALVAC expressing only PCV 1 ORF1 can be generated using the donor plasmid pJPl 15 described in Example 5.
~mmunofluorescence. In order to determine if the PCV 1 proteins were expressed in ALVAC recombinant infected Vero cells, immunofluorescence (1F') analysis was performed. Infected Vero cells were washed with PBS 24 hrs after infection (m.o.i. of approx. 10) and fixed with 95% cold aceton efor 3 minutes at room temperature. A specific anti-PCV1 pig polyclonal serum (Allan G. et al. Vet.

Microbiol. 1999. 66: 115-123) was used as the first antibody. The IF reaction was performed as described by Taylor et al. (1990).
PCV 1 specific immunofluorescence could be detected in cells infected with vCP1621 and cells infected with vCP1622. These results indicated that, as expected, vCP1621 and vCP1622 express PCV1-specific products. No fluorescence was detected with a PCV2-specific pig polyclonal serum in cells infected with vCP

and in cells infected with vCP1622. No fluorescence was detected in parental ALVAC
infected V ero cells, nor in uninfected V ero cells.
Exam,.ple 8 FO MULATION OF RECOMBINAhTT CANARYPOX VIRUSES
WITH .ARBOPOLTM 974P
For the preparation of vaccines, recombinant canarypox viruses vCP1614 and vCP1615 (Example 6) can be mixed with solutions of carbomer. In the same fashion, recombinant canarypox viruses vCP1621 and vCP1622 (Example 7) can be mixed with solutions of carbomer. The carbomer component used for vaccination of pigs according to the present invention is the CarbopolTM 974P manufactured by the company BF Goodrich (molecular weight of r 3,000,000). A 1.5 % CarbopolT"t stock solution is first prepared in distilled water containing 1 g/1 of sodium chloride.
This stock solution is then used for manufacturing a 4 mg/ml CarbopolT"' 974P
solution in physiological water. The stock solution is mixed with the required volume of physiological water, either in one step or in several successive steps, adjusting the pH value at each step with a 1N (or more concentrated) sodium hydroxide solution to get a final pH value of 7.3-7.4. This final Car'oopoITM 9?4P solution is a ready-to-use solution for reconstituting a lyophilized recombinant virus or for diluting a concentrated recombinant virus stock. For example, to get a final viral suspension containing 10'8 pfu per dose of 2 ml, one can dilute 0,1 ml of a 10'9 pfu/ml stock solution into 1,9 ml of the above CarbopolT"' 974P 4 mg/ml ready-to-use solution. In the same fashion, CarbopoITM 974P 2 mg/ml ready-to-use solutions can also be prepared.
Example 9 - IMMUNIZATION OF PIGS AND SUBSEQUENT CHALLENGE
9.1. IMMUNIZATION OF 1 DAI'-OLD PIGLETS
Groups of piglets, caesarian-derived at Day 0, are placed into isolators. The piglets are vaccinated by intramuscular route at Day 2 with various vaccine solutions.
Vaccine viral suspensions are prepared by dilution of recombinant viruses stocks in sterile physiological water (IvTaCI 0.9 %). Suitable ranges for viral suspensions can be determined empiracally, but will generally range from 106 to 10'°, and preferably about 10'° , pfu/dose. Vaccine solutions can also be prepared by mixing the recombinant virus suspension with a solution of CarbopoITM 974P, as described in Example 8.
Piglets are vaccinated either with Recombinant virus vCP 1614 (Example 2) Recombinant virus vCP1615 (Example 3) Recombinant virus vCP1614 mixed with Carbopol (4 mg/ml solution) Recombinant virus vCP1615 mixed with Carbopol (4 mg/ml solution) The viral suspensions contain 10'8 plaque forming units (pfu) per dose. Each viral suspension is injected by intramuscular route under a volume of 1 ml. The intramuscular injection is administered into the muscles of the neck.

Two injections of viral suspensions are administered at Day 2 and Day 14 of the experiment. A challenge is done on Day 21 by an oronasal administration of a viral suspension prepared from a culture of PCV-2 virulent strain. Afrer challenge, piglets are monitored during 3 weeks for clinical signs specific of the post-weaning multisystemic syndrome. The following signs are scored Rectal temperature : daily monitoring for 2 weeks post-challenge, then 2 measures of rectal temperature during the third week.
Weight : piglets are weighed right before the challenge, and then weekly during the first 3 weeks post-challenge.
Blood samples are taken at Day 2, day 14, Day 21, Day 28, Day 35 and Day 42 of the experiment in order to monitor viremia levels and anti-PCV-2 specific antibody titers.
Necropsies : at Day 42, all surviving piglets are humanely euthanized and necropsied to look for specific PWIvIS macroscopic lesions. Tissue samples are prepared from liver, lymph nodes, spleen, kidneys and thymus in order to look for specific histological lesions.
9.2. IMMUNIZATION OF 5-7 WEEK-OLD PIGLETS
5-7 week-old piglets, free of anti-PCV-2 specific maternal antibodies, are vaccinated by intramuscular route with various vaccine solutions. Vaccine viral suspensions are prepared by dilution of recombinant viruses stocks in sterile physiological water (NaCI 0.9 %). Vaccine solutions can also be prepared by mixing the recombinant virus suspension with a solution of CarbopolTM 974P, as described in Example 8.
Piglets are vaccinated either with Recombinant virus vCP1614 (Example 2) WO 01/16330 pCT/EP00/08781 Recombinant virus vCP1615 (Example 3) Recombinant virus vCP1614 mixed with Carbopol (4 mg/ml solution) Recombinant virus vCP1615 mixed with Carbopol (4 mg/ml solution) The viral suspensions contain 10'8 plaque forming units (pfu) per dose. Each viral suspension is injected by intramuscular route under a volume of 2 ml. The intramuscular injection is administered into the muscles of the neck.
Two injections of the viral suspensions are administered at Day 0 and Day 21 of the experiment. A challenge is done at Day 35 by an oronasal administration of a viral suspension prepared from a culture of PCV-2 virulent strain. After challenge, piglets are monitored during 8 weeks for clinical signs specific of the post-weaning multisystemic syndrome. The clinical monitoring is identical to the one described in Example 9.1. except that total duration of monitoring is 8 weeks instead of 3 weeks.
l~Tecropsies are done throughout the experiment for piglets dying from the challenge and at the end of the experiment (Day 97) for all surviving piglets. Tissue samples are the same as described in Example 9.1.

~F,FERENCES
1. Clark, E.G. Proc. Amer. Assoc. Swine Pract., pp. 499-501 (1997).
2. Edbauer, C., R. Weinberg, J. Taylor, A. Rey-Senelonge, J.F. Bouquet, P.
Desmettre and E. Paoletti, Virology 179, 901-904 (1990).
3. Ellis, J., L. Hansard, E. Clark, J. Handing, G. Allan, P. Willson, J.
Strakappe, K. Martin, F. McNeilly, B. Meehan, D. Todd, D. Haines, Can. Vet. J. 39, 44-51 (1998).
4. Goebel, S.J., G.P. Johnson, M.E. Perkus, S.W. Davis, J.P. Window, E.
Paoletti, Virology 179, 247-266, 517-563 (1990).
5. Guo, P., S. Goebel, S. Davis, M.E. Perkus, B. Languet, P. Desmettre, G.
Allen, and E. Paoletti, J. Virol. 63, 4189-4198 (1989).
6. Hamel, A.L., L.L. Lin and G. P.S. Nayar, J. Virol. 72, 5262-5267 (1998).
7. Handing J.C., Proc. Am. Assoc. Swine Pract. 28, 503 (1997).
8. Mankertz, A., J. Mankertz, K. Wolf, H.-J. Buhk, Gen. Virol. 79, 381-384 (1998a).
9. Mankertz, J., H.-J. Buhk, G. Blaess, A. Mankertz, Virus Gene 16, 267-276 (1998b).
10. Matthews, R.E.F., Intervirology 17, 42-44 (1982).
11. Meehan, B.M., J.L. Creelan, M.S. McNulty; D. Todd, J. Gen. Virol. 78, 221-227 (1997).
12. Meehan, B.M., F. McNeilly, D. Todd, S. Kennedy, V.A. Jewhurst, J.A. Ellis, L.E. Hansard, E.G. Clark, D.M. Haines, G.M. Allan, J. Gen. Virol. 79, 2171-2179 (1998).
13. Nayar, G.P.S., A. Hamel and L. Lin. Can. Vet. J. 38, 385-386 (1997).
14. Panicali, D. and E. Paoletti, Proc. Natl. Acad. Sci. USA 79, 4927-4931 (1982).
15. Paoletti, E., B.R. Lipinskaks, C. Samsonoff, S. Mercer, and D. Panicali, Proc.
Natl. Acad. Sci. U.S.A. 81, 193-197 (1984).

16. Perkus, M.E.; K. Limbach, and E. Paoletti, J. Virol. 63, 3829-3836 (1989).

17. Piccini, A., M.E. Perkus, and E. Paoletti, ~ Methods in Enzymology, Vol.
153, eds. Wu, R., and Grossman, L., (Academic Press) pp. 545-563 (1987).

.PCT/EP00/08781 18. Sambrook, J., E.F. Fritsch, and T. Maniatis, ~ Molecular cloning: A
laboratory manual, 2nd edition, (Cold Spring Harbor Press, NY) (1989).

19. Tartaglia, J., J. Winslow, S. Goebel, G.P. Johnson, J. Taylor, and E.
Paoletti, J.
Gen. Virol. 71, 1517-1524 (1990).

20. Tartaglia, J., Perkus ME, Taylor J, Norton EK, Audonnet JC, Cox WI, Davis SW, van der Hoeven J, Meignier B, Riviere M, and E. Paoletti, Virology 188, 217-32 (1992).

21. Taylor, J., R. Weinberg, B. Languet, Ph. Desmettre and E. Paoletti, Vaccine 6, 497-503 (1988a).

22. Taylor, 3. and E. Paoletti, Vaccine 6, 466-468 (1988b).

23. Taylor, J., R. Weinberg, Y. Kawaoka, R. Webster and E. Paoletti, Vaccine 6, 504-508 (1988c).

24. Taylor, J., C. Edbauer, A. Rey-Senelonge, J.F. Bouquet, E. Norton, S.
Goebel, P. Desmettre and E. Paoletti, J. Virol. 64, 1441-1450 (1990).

25. Taylor, J., C. Trimarchi, R. Weinberg, B. Languet, F. Guillemin, P.
Desmettre and E. Paoletti, Vaccine 9, 190-193 (1991).

26. Taylor J, Weinberg R, Tartaglia J, Richardson C, Alkhatib G, Briedis D, Appel M, Norton E, Paoletti E., Virology187, 321-328 (1992).

27. Todd, D., F.D. Niagro, B.W. Ritchie, W. Curran, G.M. Allan, P.D. Lukert, K.S. Latimer, W.L. Steffens, M.S. McNulty, Arch. Virol. 117, 129-135 (1991).

LAr>vt We claim:
1. A recombinant virus comprising DNA from porcine circovirus 2.
2. The recombinant virus of claim 1 which is a poxvirus.
3. The recombinant poxvirus of claim 2 which is an avipox virus.
4. The recombinant avipox virus of claim 3 which is ALVAC.
5. The recombinant ALVAC virus of claim 4, wherein the DNA from porcine circovirus 2 codes for and is expressed as the porcine circovirus major capsid protein.
6. The recombinant ALVAC virus of claim 4, wherein the DNA from porcine circovirus 2 consists of the open reading frame 2 (ORF2) of porcine circovirus 2.
7. The recombinant ALVAC virus of claim 4, wherein the DIvTA from porcine circovirus 2 consists of the open reading frame 1 (ORF1) of porcine circovirus 2.
8. The recombinant ALVAC virus of claim 4, wherein the DNA from porcine circovirus 2 consists of the open reading frames 1 and 2 (ORF 1 and 2) of porcine circovirus.
9. The recombinant ALVAC virus of claim 4 which is vCP1614 or vCP1615.
10. An immunological composition for inducing an immunological response in a host inoculated with the immunological composition, the immunological composition comprising a carrier and the recombinant virus of claim 1.
11. An immunological composition for inducing an immunological response in a host inoculated with the immunological composition, the immunological composition comprising a carrier and the recombinant virus of claim S.

12. An immunological composition for inducing an immunological response in a host inoculated with the immunological composition, the immunological composition comprising a carrier and the recombinant virus of claim 6.
13. An immunological composition for inducing an immunological response in a host inoculated with the immunological composition, the immunological composition comprising a carrier and the recombinant virus of claim 7.
14. An immunological composition for inducing an immunological response in a host inoculated with the immunological composition, the immunological composition comprising a carrier and the recombinant virus of claim 8.
15. An immunological composition for inducing an immunological response in a host inoculated with the immunological composition, the immunological composition comprising a carrier and the recombinant virus of claim 9.
16. A method for inducing an immunological response in a host comprising administering to the host the immunological composition of claim 11.
17. A method for inducing an immunological response in a host comprising administering to the host the immunological composition of claim 1?.
18. A method for inducing an immunological response in a host comprising administering to the host the immunological composition of claim 13.
19. A method for inducing an immunological response in a host comprising administering to the host the immunological composition of claim 14 20. A method for inducing an immunological response in a host compnstng administering to the host the immunological composition of claim 1~

t'od I ~
Hindlll (1) :l. AAGCTTCTATCPAziGTCTTPATGAGTTAGGTGTAGATAGTATAGATATTACTACA~.AGGTATTCATATT
7:I TCCTATCAATTCT AzyGTAGATGATATTP.ATAACTCAAAGATG.ATGATAG_AGATAATAGATACGCTCAT
14::
ATAATGACTGCAAATTTGGACGGTTCAG'T'~'I"I'AATCATCACGCGTTCATAAGT'~"I'CAACTGCATAGATC
21a AAAFTCTCACTAP.iai,AGATAGCCGATGTATTTGAGAGAGATTGGACATCTAACTACG..~TAAAGAAATTAC

28:1 AGTTATAAATAATACATAATGGATTTTGTTATCATCAGTTATATTTP.ACATAACTACAATAAAAAGTATT
3 5:. AAATAAAAATACTTACTTACGAAAAAATGTCT~TTATTACAAAPACTATATrTTACIiGAACAATCTATAGT
l~Met Ser LeuLeuGl nLysLeuTyrPheThr GI uGl nSer I I eVa 42:. AGAGTCCTTTAAGAGTTATP.ATTTAAAAGATAACCATPATGTP.ATATTTACCAC=~TCAGATGATGATACT
lES~ I GI user PheLysSer TyrAsnLeuLysAspAsnHi sAsnVa I I I ePheThr Thr SerAspAspAspThr 491 GTTGTAGTAATP.P_~TGPAGATP?TGTACTGTTATCTACAACATTATTATCATT'I"uATAAAATTCTGTTTT
3gIVaIVaIVaIIIeAsnGIuAspAsnVaILeuLeuSerThrArgLeuLeuSerPheAspLysIleLeuPheP
56J. TTAACTCCTTTAATAACGGTTTATCAAAATACGAAACTATTAGTGATACP.ATATTAGATATAG.nTACTCA
62~ heAsnSer PheAsnAsnGl yLeuSer LysTyrGl uThr I I eSerAspThr I I eLeuAsp I I
eAspThr Hi 631. TF.ATTATTATATA.~_s.TAGTTCTTCTTC~GTTAGATATTCTAAAi~i-.AP.iaGAGCGTGTGATTTAGAATTA
8~~ sAsnT y r T y r I I eP ro5er 5er Ser Ser LeuLeuAsp I I eLeuLysLys ArgAl aCysAspLeuGl uLeu 70J. GAAGATCTAAATTATGCGTTAATAGGAGACAATAGTAACTTATATTATAAAGATATC,ACTTF:C=.TGAATA
104 GI uAspLeuAsnTyrAl aLeul I eGIyAspAsnSerAsnLeuTyrTyrLysAspMetThr TyrMetAsnA
771. ATTGGTTATTTACT.'-.F-.AGGATTATTAGATTACF~GTTTGTATTATTGCGCGATC~TAGATAP~TGTTACAA
13~ t snT rpLeuPheThr LysGl yLeuLeuAspT y rLysPheVa I LeuLeuArgP,spVa IAspLysCysTy rLy Nrul (880) Ndel (901) 847. ACAGTAT AL
TAY.F~.PAGAATACTATAATAG.ATATAATACATCGCGATFACAGAC~:GTATP.ACATATGGGTT
15'_~~ sG! nTyrAsnLysLysAsn'thr I I a I I eAsp I I a 1 I eHi sArgAspAsnArgGl nTyrAsn I I eT rpVa I

AAA..ATG'i'.'ATACA~TACTGTTCTCCTG.G.~_TATATATTATGGTTACATGATCTAAAAGCCGC'_T'G...'~
VAAG
179 LysAsnVa I I I eGl uTyrCysSer P roGl yTyr I I eLeuT rpLeuHi sAspLeuLysAl aAl aAl aGl uA
981 ATGATTG~"TTAAGATACGATAACCGTATP.AACGAATTATCTGCGGATAAATTATACACTTTCGAGT'I'CAT
202 spAspT rpLeuArg T yrAspAsnArgl I eAsnGl uLeuSer AI aAspLysLeuTyrThr PheGf uPhel I
1051 AGTTATATTAGA.'~
~TAT~TATP_An~GATTTACGAGTAGGTACAATP_~TTGTACF~'CCAAACAAGATAATA
225 ~ eVa I I I eLeuGl uAsnAsn I I eLysHi s LeuArgVa I GI yThr I I a I I eVa I
Hi s P roAsnLys I I a I I a 1121 G~.~TAATC-~~ACATCTAATAATATACTTACTGATTTTCTATCTTACGTAGAAGAACTAATATATCATCATA
249~AI aAsnGl yThr SerAsnAsnl I eLeuThrAspPheLeuSer TyrVa I GI uGl uLeu I I
eTyrHi sHi sA
EcoRl (1223) 1191 ATTCATC'_ATAATATTGGCCGGATATI~'~TTAGAATTCTTTGAGACCACTATTTTATCAGF~.TTTATTTC
272 snSer Seal I e1 I eLeuAl aGl y'tyrPheLeuGl uPhePheGl uThrThr 1 I eLeuSer GI uPhel I eSe 126i ';TCATCTT~."TGAATGGGTAATGAATAGTAACTGTTTAGTACACCTGAAAACAGwTATGAAGrTATACTC
295p r Ser Ser Ser GI a T rpVaIMetAsnSerAsnCysLeuVal Hi sLeuLysThr GI yTyrGl uAl al I eLeu TTTGATG'CI'AGTT_ATTT'ITCC_T~ACTCTCTACTAAAAGCAATTATGTAAAFTF?TGGACAAAGAAAACTT
3191' PheAspAl aSer LeuPhePheGl nLeuSer Thr LysSerAsnTyrVa I Lys T yrT rpThr LysLysThr L

TGCAGTATAAGAA..~"~r~TTTAAAGACGGTAAACAGTTAGCAA.TaATF~'ATAA:'I'AAGAAAGATAGTCAGGT
3421 euGl nTyrLysAsnPhePheLysAspGl yLysGl nLeuAl-aLys T yr I I e1 I
eLysLysAspSer GI nVa WO 01/16330 ~ ~ ~ ~ ~ PCT/EP00/08781 147:: GATAGATAGAGTATGTTAiiTACACGCAGCTGTATATAATCACGTAACTTACTTAATGGATACGTTTAAA
36'i~ I I I eAspArgVa I CysTy rLeuHi sAl aAl aVa llyrAsnHi sVa I Thr T y rLeuMetAspThr PheLys 1541. ATTCCTGGTTTTGATT:TAAATTCTCCGGAATGATAGATATACTACTGTTTGGAFTATTGCATP.AGGATA
389tIleProGIyPheAspPheLysPheSerGlyMetIleAspIleLeuLeuPheGIyIleLeuHisLysAspA
1617. ATGAGAATATATTTTATCCGAAACGTGTTTCTGTAACTA.~TATAATATCAGAATCTATCTATGCAGATTT
41a:~ snGl uAsnl 1 ePheTyrProLysArgVal Ser VaIThrAsnl I e1 I e5er GI user I I
eTyrAl aAspPh 1681.
TTACTTTATATCAGATGTTAATAAATTCAGTAAAAAGATAGP.ATATAA.~ACTATG~_'I'I'CCTATACTCGCA
43'~~ eTyrPhe I I eSerAspValAsnLysPheSer LysLys I I eGl uTyrLysThrNlet PheProl I eLeuAl a 1751.
GAAAACTACTATCG~.AAAGGAAGGCCCTAT'ITI'ACACATACATCTAACGAAGATCITCTGTCTATCTGTT
459 GI uAsnT y rT y rP roLysGl yA rgP roTyrPheThr Hi sThr SerAsnGl uAspLeuLeuSer I I eCysL
1821 TATGCGAAGTAACAGTTTGTAAAGATATAAAAAATCCATTATTATATTCTF.AAAAGGATATATCAGCF.AA
48~ t euCysGl uVa I Thr Va I CysLysAsp I I eLysAsnP roLeuLeuT yr5er LysLysAsp I I eSer AI aLy 1891 ACGATTCATAGGTTTATTTACATCrGTCGATATAAATACGGCTGTTGAGTTAAGAGGATATAAAATAAGA
50'_~~ sArgPhe I I eGl yLeuPheThr Ser ValAspl I eAsnThr AI aVal GI uLeuArgGl yTyrLys I I eArg 1967 GTAATAGGATGTTTAGAATGGCCTGAAF~.GATAAAAATATTTAATTCTAATCCTACATACATTAGATTAT
529 Va l I I eGl yCysLeuGl uT rpProGl uLys I I eLys I I ePheAsnSerAsnProThr Tyrl I eArgLeuL
2D31. TACTAACAGAAAGACGTTTAGATATTCTACATTCCTATCTG..~TTAAATTTFATATAACAGAGGATATAGC
552 euLeuThr GI uArgArgLeuAspl I eLeuHi sSer TyrLeuLeuLysPheAsnl I eThr GI
uAspl I eAl 2101 TACCAGAGATGGAGTCAGAAATAATTTACCTATAATTTCTTTTATCGTCAGTTF?'TGTAGATCGTATACT
575 ~ aThr A rgAspGl yVa IA rgAsnAsnLeuP ro I I a I I eSer Phe 1 I eVa I Se r Ty rCysArgSer T yrThr Ndel (2189) TATFIjATTACTAAATTGCCATATGTACAATTCGTGTAAGATAAC~.AAGTGTAF~..TATAATC~.GGTF.ATAT
599 TyrLysLeuLeuAsnCysHi st'letTyrAsnSer CysLys I I eThr LysCysLysTyrAsnGl nVa I I I eT
2241 ATAATCCTATATAGGAGTATATATA.aTTGAAAAAGTAAAATATAAATCF~ATAATAATGAP.ACGF.AATAT
622~yrAsnProl I e~
2311 C_T~GTAATAGACAGGAACTGGCAGATTCTTC~TCTAATGAAGTAAGTACTGCTAAATCTCCAAAATTAGAT

F_AAAATGATACAGCAAATACAGC~.TCATTC~ACGAATTACCTTTTAAT'T_'T'T'TTCAGACACACCITATTAC
2 451 APACTAACTAAGTCAGATGATGAGAIiAGTF.AATATAAATTTF~CTTATGGG Tp.TF
~TATAATAAAGATTC
2521 ATGATATTAATAATTTACTTAACGAT'TrAATAGACT_TATTCCATCAACCCCT~_'CAAACCTTTCTGGATA
25°1 TTATAF.AATACCAGTTAATGATA:"TF.AAATAGATTGTTTAAGAGATGTAAATAATTATTTGGAGGTAAAG
2661 GATATAAAATTAGTCTATCTTTCACATGGAAATGAATTACCTAATATTAATP.ATTATGATAC~TITTI' 2731 TAGGATTTACAGCTGTTATATGTATCAACAATACAGGCAGATCZ'ATGGTTATGGTAAAACACTGTAACGG
28D1 GAAGCnGCATTCTATGGTAACTC~:CCTATGTTTAATAGCCAGATCATT~TA~2'CTATAAACATTTTACCA
BamHl (2880) 2871 C.~AATAATAGGATCCTCTAGATATTTAATATTATATCTAACAACAA~SITF.ACGATGTATGGC
2941 CAGis-.GTATTI~'CTACTAATAAAC.LTAAAGATAGTCTATCTTATCTACAAGATATGAF.AGAAGATAATCA
Hindlll (3058) 301'_ TTTAGTAGrAGCTACTAATATCuAAAGAAATGTATACAAhAACGTGGAAGCTTTTATATTAAATAGCATA
30c1 i"TACTAGF.P.GATTTAAAATCTAGACTTAGTATAACAF~ACAGTTAAATGCCAATATCGATTCTATATTTC

WO 01/16330 , .PCT/EP00/08781 ~°~~ c 315:1 ATCATAACAGTAGTACATTAATCAGTGATATACTGAAACGATCTACAGACTCAACTATGCAAGGAATAAG
322..
CPATATGCC~~TTATGTCTP.F:TAT:I'TAACTTTAGAACTAAla.ACGTTCTACCAATACTAAAA.ATAGGATA
3297. CGTGATAC-;n."TGT'TP.AAAGCTGCP~TAAATAGTF_T~GGATGTAGAAGAP~TAC~ITGTTCTATACCTTCGG
3367. AGGAAAGAACTTTAGAACAACTTAAGTTTAATCAAAC'TTGTATTTATGAACACTATAAAAAAATTATGGA
393?. AGATACArGTAAAAGAATGGATGi:GAATGTCGTAGTTTAGAACATPACTATACGGCTAACTTATATAAA
3507. GTGTACGGACAAAACGAATATATGATTACTTATATACTAGCTCTCATAAGTAGGATTAATAATATTATAG
3577 AP.ACTTTAAAATATAATCTGGTGGGGCTAGACGAATCTACP.ATACGTAATATAAATTATATAATTTCACA
3 64 7 P ~GAAC~s~-~AAAAAATCAAGTTTCTAATACC iTATAGATP.AACTATATfirITTACCACTGA

~$s) (»s4) (1839) 11 (1918) (2026) V (2030) Sacl ( i167) Notl (3159) (2296) Smal (2762) Stul X2641 ) Ndel (2472) EcoRl (2363) WO 01/16330 pCT/EP00/08781 Kpnl (1) 1 GGTACCTI'CATAAATACAAGTTTGATTAAACTTAAGTTGTTCTAP1~GTTCTTTCCTCCGAAGGTATAGAA
71 CAPAGTATTTCTTCTACATCCTTACTATTTATTGCAGCTTTTP.ACAGCCTATCi-CGTATCCTATTTTTAG
147. TATTGGTAGAACGTTTTAGTTCTAAAGTTAAAATATTAGACATAATTGGC.~.TATTGCTTATTCCTTGCAT
217. AGTTGAGTCTGTAGATCGTTTC~GTATATCACTGATTAATGTACTACTGTTATCaTGAAATATAGAATCG
287. ATATTGGCATTTAACTGTTTTGTTATACTAAGTCTAGATTTTAAATCTTCrAGTAATATG~TATTTAATA
351. TAAAAGCTTCCACGTTTTTGTATAC~TTTCTTTCCATATTAGTAGCTACTACTAAATGATTATCTTCTTT
421. GATATCTTGTAGATAAGATAGACTATCTTTATCTTTATTAGTAGAAAATACTTCTGGCCATACATCGTTA
BamHl (532) 491. AA~_'I'~TTTTGTTGTTGTTAGATATAATATTAAATATCTAGAGGATCCTATTAT~TGTGGTAAAATGTTTA
561 TAGAGTAAAATG_~TCTGGCTATTAAACATAGGCCAGTTACCATAGAATGCTGCTTCCCGTTACAGTGTTT
63:. TACCATAACCATAGATCTGCCTGTATTG'i"iGATACATATAACAGCTGTAAATCCTAAAAAATTCCTATCA
70:TP.ATTATTAATATTAGGTAATTCATTTCCATGTGAAAGATAGACTP.ATTT'_'ATFTCCTTTACCTCCAAAT
77_ AA'.I'ATTTF,CATCTCTTAAACFn~CTATTTTAATATCATTAACrGGTATTTTATPATATCCAGAAAGGTT
8e1 TGAAGGGV:~TGATGGAATAAGTCTATTAACATCGTTAAGTAAATTATTP.ATATCnTGAATCTTTF.TTATA
911. TTATACCCATAAGTTAAATTTATATTTACTTTCTCATCATCTGACTTAGTTAGTTTGTA$TAAGGTGTGT
981 CTGAAAAFLTTAAAAGGTAATTCGTTGr.ATG.~F:GCTGTATTTGCTGTATCATTT=TATCTAA ~"'~
iTGGAG
1057. ATT1'AGCi:GTACTTACTTCATTACAAG~-l:GAATCTGCC:~GTTCCTGTCTATTACTGATATTTCGTTTCAT
EcoRl (1 1121 TATTATA'I~ATTTATATTTTACTTTTTCAATTATATATACTCATTTGACTAGTTAATCAATrA~AAGAAT
1197 TC~:~Gv.AG.~..CCTGCAG'"TAFTTAATTP.AG..~TACAAATAGTZiCGTTTTCACCTTGTCTT-.ATAACIAATTA
8amHl (1266) '.:26i ATTP.AGGATCCCCCTyGC'!'TCT"T_iATTCTATACTTAAAFiAGTGAAAATAAATArnAAG.~"';':~;'~'GAG
GGTT
EcoRV (1378) Nrul (1374) 1331 GTGTTAP.1-..TTGAAAGCGAGAAATAATCF:TAAATTATTTCATTATCGCGATATCCGTTAAGTTTGTATCGT

AATGACG=i:TCCAAGGAGGCCTTACCGG~GAAG.n.AGACACCGCCCCCGCAGCC.TCTTG:JCCAGATCCTC
l~MetThrTyrProArgArgArgTyrArgArgArgArgHi sArgProArgSer Hi sLeuGl yGl n1 I eLeu 1471 CGCCGCCGCCCCTGGCTCGTCCACCCCCGCCACCGCTACCGTTGGAGAAGGAAP.AATGGCATCTTCAAC~:
24 ~A rgArgArgProT rpLeuVal Hi sProArgHi sArgTyrArgT rpArgArgLysAsnGl y I I
ePheAsnT
'591 CCCGCCTCTCCCGCACCTTCGGATATA'.'TGTCP.AGCGTACCACAGTCACAACGCCCTCCTGGGCGGTGGA
971~hrAroLeuSerAraThrPheGIyTyrThrVaILysArgThrThrVaIThrThrProSerTrpAlaValAs Smal (1644) C:=TGATGA:zATT'..=~AAATTGACCACTTTGTTCCCCCGGGAGGGGGGACCAA~AAATCT:.'TATACCCTT_~' 701' pP~letMetArgPheLys I I eAspAspPheUal ProProGl yGl yGl yThrAsnLys 1 I eSer I I eProPhe WO 01/16330 ~ , ~ PCT/EP00/08781 r (653) i1 (1184) ~I (1839) HI (1918) (2026) ~V (2030) Saci (3 1 (2153) Notl (3 (2320) (2376) Smal (3005) Sall (2999) Ndel (2979) EcoRV (2748) Ncol (2671) ~' ( ~~. 5 pni (653) mHl (1184) t (1958) ~Ndel (2127) Sacl (4256) C6 left azm Notl (24H) H5 Promote= ~EcoRl (2236) Smal (2303) u~, r,rrm-,re.
Smal (:;851) ~coRV (2569) Sall (:845)// ~rul (2573) Ndel (a825) amHl (2764) EcoRV (3594) Ncol (3517) Sacl (3222) 6c1! (3166) Sacl (2999) ~EcoRV (2876) 'Nrul (2872) W~ 01/16330 CA 02383367 2002-02-27 1i Kpnl (1) l GGTACCTTCATAAATACAAGZ'ITGATTAAACTTAAGTTGTTCTP.P.AGTTCZTTCCTCCGAAGGTATAGAA
71 CAAAGTATTTCTTCTACATCCTTACTATTTATTGCAGCT~'TTAACAGCCTATC1~CGTATCCTATTTTTAG
141. TATTGGTAGAACGT'1"1TAGTTCTAAAGTTAAAATATTAGACATAATTGGCATATTGCTTATTCCTTGC'AT
211. AGTTGAGTCTGTAGATCGTTTCAGTATATCACTGATTAATGTACTACTGT'rATGATGAAATATAGAATCG
2B1. ATATTGGCATTTAACTGTTTTGTTATACTAAGTCTAGATTTTAAATCTTCrAGTAATATG~.~rATTTAATA
351. TAAAAGCTTCCACGTTTTTGTATACATTTCTTTCCATATTAGTAGCTACTACTAAATGATTATCTTCTTT
921 CATATCTTGTAGATAAGATAGACTATCTTTATCITTATTAGTAGAAA.ATACTTtTGGCCATACATCGTTA
BamHl (532) 49:1. AATIT~TGTTGTTGTTAGATATF~ATATTAAATATCTAGAGGATCCTATTATTTGTGGTAAAATGTTTA
561 TAGAGTAAAATGATCTGGCTATTAAACATAGGCCAGTTACC1?TAGAATGCTGCTTCCCGTTACAGTGTTT
63. TACCATFACCATACATCTGCCTGTATTGTTGATACATATAACAGCTGTAAATCC'i'P.AAAAATTCCTATCA

TP.ATTATTP.ATATTAG.~"TAP.TTCATTTCCATGTGAAAGATAGACTAATTTTI?TP.TCCTTTACCTCCAAAT
77. r'~hTTATTTACATCTCTTAAACP.ATCTATZTTAATATC~.TTAACTGGTA2°Pt TATAP~'ATCCAG.AAAG~~TT

911. TTATACCCFTAAGTTP.P~TTTATATTTACfiITCTCATCATCTGACTTAGTTAGTTTGTAATF.AGGTGTGT

CTGAP.AAF~TTAP.F.FGGTAATTCGTTGF~?TCP.F:GCTGTATITGCTGTATC1~TTTTTATCTAATT2TGGAG
1051.
ATTTAGCAGTACTTACTTCAT'TACIeAGAAGAATCTGCCIjGTTCCTGTCTAZTACTGATATTTCGTTTCAT
1121. TATTATATGATTTATATIT'rA_~~TCAATTATATATACTCATTTGACTAGTTAATG~ATAA.AAAGAFT
1191. TTCGACTTAGGGTTTAAGTGGGG:T3T"?TrAAGATTAAATTCTCTGAATTGTACATACATGGTTACACGG
233<ProLysLeuProProAspLysLeuAsnPheG(uArgPheGInVaITyrMetThrValArgl 5tul (1306) ATATTGTAGTCCT_GGTCGTATTTA'TGT'_T'i'TCGAACGCAGCGCCGAGGCGTACGTGGTCCACATTTCC1~G
212< I eAsnTyrAspGl nAspTyrLysSerAsnGl uPheAl aAl aGl yLeuGl yVa I Hi sAspValAsnGl ySe 1331 AGGTTTGTAGTCTCAGCCAAAGCTGATTCCTTTTGTTATTTGGTTGGAAGTAAT~:AATAGTGGAGTCP.AG
189< r Thr GI nLeuArgLeuT rpLeuGl nAsnArgLysAsnAsnProGl nPheTyrAspl I eThr SerAspleu 1401 AACAGGT'~I'GGGTCTGAAGTP_~:CC-GGAGTGGTAGGAGAAGGGTTGGGGGATTGTATGGCGGUAGGAGTAG
166 Va l ProLysProThr PheTyrArgSer Hi sTyrSer PheProGl nProl I eThr Hi sArgSer Ser TyrA
Ndel (1475) 1471 TTTACATATGGGTC~TAGGTTAGGGCTGTGGCCTTTGTTACAAAGTTATCATCrAGAATAACAGCAGTGG
142 ~ snVal Ty rP roAsp T yrThr LeuAl aThr AI aLysThr Va 1 PheAsnAspAspLeu I I
eVa IAI aThr Se EcoRl (1584) 1541 AGCCCACT~CCCCTATCACCCTG~.-GTGATGGGGGAGCAGGGCCAGAATTCAACCTTAACC'~TCTTATTCT
1191 r GI yVa I GI yA rgAspGl yGl nThr I 1 eP roSer CysP ro T rpPheGl uVa I
LysVa I Lys Arg I I eArg Smal (1651) 1611 GTAGTATT~LGGGTATAGAGAT~TTGTTGGTCCCCCCTCCCGGGGGAAChAAGTCGTCAATTTTAAAT
96~TyrTyrGluPheProIleSer IIeLysAsnThrGIyGIyGIyProProVaIPheAspAspl IeLysPheA

168.. CTCATCATGTCCACCGCCC~:GGAGGGCGTTGTGACTGTGGTACGCTTGAC~GTPTATCCGAAGGTGCGGG
71!~r gN'~etMetAspVaIAI aT rpSer ProThrThr VaIThrThrArgLysVaIThrTyrGIyPheThrArgSe 1751. AGAGGCGGGTGTTGAAGATGCCATTTTTCCTTCTCCAACGGTAGCGGTGGCGGGGGTGGACGAGCCAGGG
9~~~ r LeuArg'fhrAsnPhe I I eGl yAsnLysArgArgT rpArgTyrArgHi sArgProHi sVa I
LeuT rpPro 1821.
GCGGCGGCGGAGGATC'I'G~CCAAGATGGw.~TGCGGGGGCGGTGTCTTCTTCTGCGGTAACGCCTCCTTGGA
2~~~A rgArgArgLeu I I eGl nGl yLeuHi sSer A rgP roArgHi sArgArgArgAraTyrArgArgArgProT
Nrul (1921) EcoRV (1917) 1891 TACGTCATTACGATACAAACTTAACGGATATCGCGATAATGAAATAATTTATGP~'TATTTCiCGCTTTCA
~:~y rThrMet 1961 ATTTAACACAACCCTCAAGP.ACCTTTGTATTTATTTTCACTTTTTAAGTATAGAATAAAGP.AGCTGGGGG
203.. ATCA.nTTCCTGCAGCCCTGCAGCTAATTAATTAAGCTACAAATAGTTTCGTTT~CCTTGTCTAATAAC
BamHl (2112) 210.. TAATTr ~TTAAGCATCCCCCA
GCTTCTTTATTCTATACTTAAAAAGTGAAAATP_~FTACAAAGGTTCTTG
EcoRV (2224) Nrul (2220) 2171. AGG:TTGTGTTAAATTGAAAGCGAGAAATAATCATAAATTATTTCATTATCGCGATATCCGTTAAGTTTG
2247.
TATCGTP_n.TGCCCAGCAAGAAGP.ATGGAAGAAGCGGACCCCP.ACCACATAAAAGGTGGGTG?'TCACGCrG
It Met P roSer LysLysAsnGl yA rgSer GI yP roGl nProHi sLysArgT rpVa I PheThr Leu Sacl (2347) 2317. AATAATCCTTCCGAAGACGAGCGCPACAAAATACGGGAGCTCCCAATCTCCCTATTTCATTATTTTATTG
2~~AsnAsnProSer GI uAspGl uArgLysLys I I eArgGl uLeuProl I e5er LeuPheAspTyrPhel I eV
2381 TTGGCGAGGAGGGTAATGAGGAAGGACGP.ACACCTCACCTCCAGGGGTTCGCTAATTTTGTCAAGAAGCA
9_°°:t a I GI yGl uGl uGl yAsnGl uGl uGl yA rgThr P roHi sLeuGl nGl yPheAl aAsnPheVa I LysLysGl l3cll (2514) 2451 AACTTTTT~TAAAGTGAAGTGGTATTTGGGTGCCCGCTGCCACATCGAGAPAGCCA;~AGGAACTGATCAG
68~ nThr PheAsnLysVal LysT rpTyrLeuGl yAl aArgCysi-ii s I I eGl uLysAI aLysGl yThrAspGl n Sacl (2570) 2521 G~GAAT~GAATATTGCr.GTP.AAGAAGGC~ACTTACTTATTGAATGTGGAGCTC'CTCGATCTCAAG:~:C
92 ~ GI nAsnLysGl uTyrCysSer LysGl uGl yAsnLeuLeu I I eGl uCysGl yAl aP
roArgSer GI nGl yG
2591 P.ACGGAGTGACCTGTCTACTGCTGTGAGTACCTTGTTGGAGAGCGGGAGTCTGGTGACCGTZ'GCAGAGCA
115 I nArgSerAspLeuSer Thr AI aVa I Ser Thr LeuLeuGl user GI ySer LeuVa I Thr Va IAI aGl uGl 2661 GCACCCTGTPACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTZ'rGAAAGTGAGCGGGAAAATGCAG
138 nHi sProVal Thr PheVaiA rgAsnPheArgGl yLeuAl aGl uLeuLeuLysVal Ser GI
yLysMelGl n 2731 AAGCGTGATTGGAAGACC~.ATGTACACGTCATTGTGGGGCCACCTCGGTGTGGTAAAAGC~AATGGGCTG
1621' LysArgAspT rpLysThrAsnVa I Hi sVa I I I eVa I GI yP roP roGl yCysGl yLysSer LysT rpAl aA
Ncol (2865 2801 CTAATTTTGCAGACCCGGP.AACCACATACTGGAAACCACCTAGAAACAAGTGGT"~GGATGGTTACCATGG
1851'laAsnPheAlaAspProGluThrThrTyrT rpLysProProArgAsnLysTrpTrpAspGlyTyrHisGl 2871 TGAAGAAGTGGTTGTTATTGATGACTTTTATGGCTGG''~'GCCGTGGGATGATCTACTGAGACTGTGTGAT
2081' yGl uGl uVa I Val Va I I I eAspAspPheT y rGl yT rpLeuP roT
rpAspAspLeuLeuArgLeuCysAsp EcoRV (2942) 2941 CGATATCC~TTGACTGTAGAGACTAnAGGTGGAACTGTACCTTTTTTGGCC~C~CAGTATTC'I~iATTACCA
2321'A rgTyrF roLeu T hr Va I GI uThr LysGl yGl yThr Va I ProPheLeuAl aArgSer I I eLeu I I eThr S

WO 01/16330 .PCT/EP00/08781 F' n a 3017. GCAATCAGACCCCGTTGGAATGGTACTCCTCAACTGCTGTCCCAGCTGTAGAAGCTCTCTATCGGAGGAT
25's~ erAsnGl nThr P roLeuGl uT rpTyrSer Ser Thr AI aVa I ProAl aVal GI uAl aLeuTyrAraArgl I
3081. TACTTCCTTGGTATTTTGGAAGAATGCTACAGAACAATCCACGGAGGAAG~'sGGGCCAGTTCGTCACCCTT
27~:;~ eThr Ser LeuVa 1 PheT rpLysAsnAl aThr GI uGl nSer Thr GI uGl uGl yGl yGl nPheVa I Thr Leu Smal (3199) Ndei (3173) Sall (3193) 3157. TCCCCCCCATGCCCTGAATTTCCATATGAAATAAATTACTGAGTCGACCCCGGGTTTTTATAGCTAATTA
30:::~ Ser ProProCysProGl uPheProTyrGl u1 I eAsnTyr 3291. TAACAAAATCCATTATGTATTATTTATAACTGTP.ATTTCTT'TAGCGTAGTTAGATGTCCAATCTCTCTCA

350.
ATTAATATCATCTACT'T'I'AGAATTGATAGGAAATATGAATACCTTTGTAGTAP.TATCTATACTATCTACA
Sacl (3604) Notl (359fi) 3 571 CCTP.ACTCATTAAGACT'I'I~'GATAG.GCGGCCGCGAGCTC

Th015 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Bublot, Michel Perez, Jennifer M.
Charreyre, Catherine E.
(ii) TITLE OF INVENTION: Porcine Circovirus 2 Recombinant Poxvirus (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS: , (A) ADDRESSEE: Viro~enetics Inc.
(B) STREET: 465 Jordan Road (C) CITY: Troy (D) STATE: NY
(E) COUNTRY: USA
(F) ZIP: 12180 (v) COMPUTER REP.DABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION hTUMBER
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Howe, Timothy R.
(B) REGISTRATION NUMBER: 39,228 (C) REFERENCE/DOCKET IvTUMBER: TH015 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (570) 586-1022 (B) TELEFAX: (570) 895-2702 Page i Th015 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3701 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi.) SEQUENCE DESCRIPTION: SEQ ID NO: l:
AAGCTTCTAT CAAAAGTCTT AATGAGTTAG GTGTAGATAG TATAGATATT ACTACAAAGG
TATTCATATT TCCTATCAAT TCTAAAGTAG ATGATATTAA TAACTCAAAG ATGATGATAG

TAGATAATAG ATACGCTCAT ATF.ATGACTG CAAATTTGGA CGGTTCACAT TTTAATCATC

ACGCGTTCAT P.AGTTTCAAC TGCATAGATC P.AAATCTCAC TAAAAAGATA GCCGATGTAT

TTGAGAGAGA TTGGACATCT AACTACGCTA AAGAAATTAC AGTTATAAAT AATACATAAT

GGATTTTGTT ATCATCAGTT ATATTTAACA TAAGTACAAT P_AA.AAGTATT AAATAA.~iAAT

ACTTACTTAC GP~AAA.AATGT CATTATTACA PAAACTATAT TTTACAGAAC AATCTATAGT

AGAGTCCT""" AAGAGTTATA ATTTAAAAGA TFACCATAAT GTAATATTTA CCACATCAGA

TGATGATACT GTTGTAGTAA TAAATGAAGA TAATGTACTG TTATCTACAA GATTATTA'TC

ATTTGATAAA ATTCTGTTTT TTP~1CTCC'~'T TAATAACGGT TTATCAAAAT ACGAAACTAT

TAGTGATACA ATATTAGATA TAGATACTCA TAATTATTAT ATACCTAGTT CTTCTTCTTT

Th015 GTTAGATATT CTF~AAA.AAA GAGCGTGTGA TTTAGAATTA GAAGATCTAA ATTATGCGTT

AATAGGAGAC AATAGTAACT TATATTATAA AGATATGACT TACATGAATA ATTGGTTATT

TACTAAAGGA TTATTAGATT ACAAGTTTGT ATTATTGCGC GATGTAGATA AATGTTACAA

ACAGTATAAT AAAP.AGAATA CTATAATAGA TATAATACAT CGCGATAACA GACAGTATAA

CATATGGGTT AAAPATGTTA TAGAATACTG TTCTCCTGGC TATATATTAT GGTTACATGA

TCTAAAAGCC GCTGCTGAAG ATGATTGGTT AAGATACGAT AACCGTATAA ACGAATTATC

TGCGGATAAA TTATACACTT TCGAGTTCAT AGTTATATTA GAAAATAATA TAAAACATTT

ACGAGTAGGT ACF.ATAATTG TACATCCAAA CAAGATAATA GCTAATGGTA CATCTAATAA

TATACTTACT GATTTTCTAT CTTACGTAGA AGAACTAATA TATCATCATA ATTCATCTAT

F.ATATTGGCC GGATATTTTT TAGAATTCTT TGAGACCACT ATTTTATCAG AATTTATTTC

TTCATCTTCT GAATGGGTAA TGAATAGTAA CTGTTTAGTA CACCTGAAAA CAGGGTATGA

AGCTATACTC TTTGATGCTA GTTTATTTTT CCAACTCTCT ACTAAAAGCA ATTATGTAAA

ATATTGGACA AAGAAAACTT TGCAGTATAA GAACTTTTTT AAAGACGGTA P.ACAGTTAGC

A.~1AATATATA ATTAAG_~AAG ATAGTCAGGT GATAGATAGA GTATGTTATT TACACGCAGC

Th015 TGTATATAAT CACGTAACTT ACTTAATGGA TACGTTTAAA ATTCCTGGTT TTGATTTTAA

ATTCTCCGGA ATGATAGATA TACTACTGTT TGGAATATTG CATAAGGATA ATGAGAATAT

ATTTTATCCG AAACGTGTTT CTGTAACTAA TATAATATCA GAATCTATCT ATGCAGATTT

TTACTTTATA TCAGATGTTA ATAAATTCAG TAA.AA.AGATA GAATATAAAA CTATGTTTCC

TATACTCGCA GAAAACTACT ATCCAAAAGG AAGGCCCTAT TTTACACATA CATCTAACGA

AGATCTTCTG TCTATCTGTT TATGCGAAGT AACAGTTTGT AAAGATATP.A F.AAATCCATT

ATTATATTCT AA.AAAGGATA TATCAGCAAA ACGATTCATA GGTTTATTTA CATCTGTCGA

TATAAATACG GCTGTTGAGT TAAGAGGATA TAAAATAAGA GTAATAGGAT GTTTAGAATG

GCCTGAA.A.AG ATAAAAATAT TTAATTCTA~ TCCTACATAC ATTAGATTAT TACTAACAGA

AAGACGTTTA GATATTCTAC ATTCCTATCT GCTTAAATTT AATATAACAG AGGATATAGC

TACCAGAGAT GGAGTCAGP.A ATAATTTACC TATAATTTCT TTTATCGTCA GTTATTGTAG

ATCGTATACT TATAAATTAC TAAATTGCCA TATGTACAAT TCGTGTAAGA TP.ACAAAGTG

TAAATATAAT CAGGTAATAT ATAATCCTAT ATAGGAGTAT ATATAATTGA P.AAAGTAAAA

TATAAATCAT AT.~ATAATGr AACGAAATAT CAGTAATAGA CAGGAACTGG CAGATTCTTC

Paae Th015 TTCTAATGAA GTAAGTACTG CTAAATCTCC P.AAATTAGAT AAA.AATGATA CAGCAAATAC

AGCTTCATTC AACGAATTAC CTTTTAATT'~' TTTCAGACAC ACCTTATTAC AAACTAACTA

AGTCAGATGA TGAGAAAGTA AATATAAATT TAACTTATGG GTATAATATA ATAAAGATTC

ATGATATTAA TAATTTACTT AACGATGTTn ATAGACTTAT TCCATCAACC CCTTCAAACC

TTTCTGGATA TTATAAA.ATA CCAGTTAATG ATATTAAAAT AGATTGTTTA AGAGATGTAA

ATAATTATTT GGAGGTAAAG GATATAAP~'=' TAGTCTATCT TTCACATGGA AATGAATTAC

CTAATATTAA TAATTATGAT AGGAATTT'_"-" TAGGATTTAC AGCTGTTATA TGTATCAACA

ATACAGGCAG ATCTATGGTT ATGGTAP.F.AC ACTGTAACGG GP1~GCAGCAT TCTATGGTAA

CTGGCCTATG TTTA_~TAGCC AGATCATT'='T ACTCTATAAA CATTTTACCA CAAATAATAG

GATCCTCTAG ATATTTAATA TTATATC';'_..A CAACAACAAA AAAATTTAAC GATGTATGGC

CAGAAGTAmT TTCTACTAAT AAAGATPL'.G ATAGTCTATC TTATCTACAA GATATGAAAG

AAGATAATCA TTTAGTAGTA GCTACTAFTA TGGAAAGAAA TGTATACAA.~ AACGTGGAAG

CTTTTATATT AAATAGCATA TTACTAGriAG ATTTAAAATC TAGACTTAGT ATF.ACAAAAC

AGTTAAATGC CAATATCGAT TCTATAT'='TC ATCATAACAG TAGTACATTA ATCAGTGATA

TACTGAAACG ATCTACAGAC TCAACTA''GC AAGGAATAAG CAATATGCCA A'='TATGTCTA
gage 5 Th015 ATATTTTAAC TTTAGAACTA AAACGTTCTA CCAATACTAA AAATAGGATA CGTGATAGGC

TGTTAAAAGC TGCAATAAAT AGTAAGGATG TAGAAGAAAT ACTTTGTTCT ATACCTTCGG

AGGAAAGAAC TTTAGAACAA CTTAAGTTTA ATCAAACTTG TATTTATGAA CACTATAAAA

AAATTATGGA AGATACAAGT AAAAGAATGG ATGTTGAATG TCGTAGTTTA GAACATAACT

ATACGGCTAA CTTATATAAA GTGTACGGAC AAAACGAATA TATGATTACT TATATACTAG

CTCTCATP.AG TAGGATTAAT AATATTATAG AAACTTTAAA ATATAATCTG GTGGGGCTAG

ACGAATCTAC AATACGTAAT ATAAATTATA TAATTTCACA AAGAACAP.AA P_AAAATCAAG

TTTCTAATAC CTTATAGATA AACTATATTT TTTACCACTG A

(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
ATCATCGAGC TCGCGGCCGC CTATCAAAAG TCTTAATGAG TT

(2) INFORMATION FOR SEQ ID N0:3:
Pa4e 6 Th015 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GAATTCCTCG AGCTGCAGCC CGGGTTTTTA TAGCTAATTA GTCATTTTTT CGTAAGTAAG
TATTTTTATT TAA

(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CCCGGGCTGC AGCTCGAGGA ATTCTTTTTA TTGATTAACT AGTCAAATGA GTATATATAA
TTGAP.AA.AGT AA

(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Th015 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GATGATGGTA CCTTCATAAA TACAAGTTTG ATTAAACTTA AGTTG
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CATCATCATG ATATCCGTTA AGTTTGTATC GTAATGACGT ATCCAAGGAG GCG

(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TACTACTACG TCGACTTAGG GTTTAAGTGG GGGGTC

(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2520 base pairs (B) TYPE: nucleic acid (C) STRAND~DNESS: single (D) TOPOLOGY: linear Th015 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GGTACCTTCA TAAATACAAG TTTGATTAAA CTTAAGTTGT TCTAAAGTTC TTTCCTCCGA
AGGTATAGAA CAAAGTATTT CTTCTACATC CTTACTATTT ATTGCAGCTT TTAACAGCCT

ATCACGTATC CTATTTTTAG TATTGGTAGA ACGTTTTAGT TCTAAAGTTA AAATATTAGA

CATAATTGGC ATATTGCTTA TTCCTTGCAT AGTTGAGTCT GTAGATCGTT TCAGTATATC

ACTGATTAAT GTACTACTGT TATGATGAAA TATAGAATCG ATATTGGCAT TTAACTGTTT

TGTTATACTA AGTCTAGATT TTAAATCTTC TAGTAATATG CTATTTAATA TAAAAGCTTC

CACGTTTTTG TATACATTTC TTTCCATATT AGTAGCTACT ACTAAATGAT TATCTTCTTT

CATATCTTGT AGATAAGATA GACTATCTTT ATCTTTATTA GTAGAAAATA CTTCTGGCCA

TACATCGTTA AATTTTTTTG TTGTTGTTAG ATATAATATT AAATATCTAG AGGATCCTAT

TATTTGTGGT AAAATGTTTA TAGAGTAAAA TGATCTGGCT ATTAAACATA GGCCAGTTAC

CATAGAATGC TGCTTCCCGT TACAGTGTTT TACCATAACC ATAGATCTGC CTGTATTGTT

GATACATATA ACAGCTGTAA ATCCTAAi~ ATTCCTATCA TAATTATTAA TATTAGGTAA

TTCATTTCCA TGTGAAAGAT AGACTAATTT TATATCCTTT ACCTCCAAAT AATTATTTAC

Th015 ATCTCTTAAA CAATCTATTT TAATATCATT AACTGGTATT TTATAATATC CAGAAAGGTT

TGAAGGGGTT GATGGAATAA GTCTATTAAC ATCGTTAAGT AAATTATTAA TATCATGAAT

CTTTATTATA TTATACCCAT AAGTTAAATT TATATTTACT TTCTCATCAT CTGACTTAGT

TAGTTTGTAA TAAGGTGTGT CTGAAA.AA.AT TAAAAGGTAA TTCGTTGAAT GAAGCTGTAT

TTGCTGTATC ATTTTTATCT AATTTTGGAG ATTTAGCAGT ACTTACTTCA TTAGAAGAAG

AATCTGCCAG TTCCTGTCTA TTACTGATAT TTCGTTTCAT TATTATATGA TTTATATTTT

ACTTTTTCAA TTATATATAC TCATTTGACT AGTTAATCAA TAA.AAAGAAT TCCTGCAGCC

CTGCAGCTAA TTAATTAAGC TACAAATAGT TTCGTTTTCA CCTTGTCTAA TAACTP.ATTA

ATTAAGGATC CCCCAGCTTC TTTATTCTAT ACTTAAAA_AG TGA.AAATAAA TACAAAGGTT

CTTGAGGGTT GTGTTAAATT GAAAGCGAGA AATAATCATA AATTATTTCA TTATCGCGAT

ATCCGTTP.AG TTTGTATCGT AATGACGTAT CCAAGGAGGC GTTACCGCAG AAGAAGACAC

CGCCCCCGCA GCCATCTTGG CCAGATCCTC CGCCGCCGCC CCTGGCTCGT CCACCCCCGC

CACCGCTACC GTTGGAGAAG GAA.AAATGGC ATCTTCAACA CCCGCCTCTC CCGCACCTTC

GGATATACTG TCAAGCGTAC CACAGTCACA ACGCCCTCCT GGGCGGTGGA CATGATGAGA

Th015 TTTAAAATTG ACGACTTTGT TCCCCCGGGA GGGGGGACCA ACAAAATCTC TATACCCTTT

GAATACTACA GAATAAGAAA GGTTAAGGTT GAATTCTGGC CCTGCTCCCC CATCACCCAG

GGTGATAGGG GAGTGGGCTC CACTGCTGTT ATTCTAGATG ATAACTTTGT AACAAAGGCC

ACAGCCCTAA CCTATGACCC ATATGTAAAC TACTCCTCCC GCCATACAAT CCCCCAACCC

TTCTCCTACC ACTCCCGTTA CTTCACACCC AAACCTGTTC TTGACTCCAC TATTGATTAC

TTCCAACCAA ATAACAAAAG GAATCAGCTT TGGCTGAGAC TACAAACCTC TGGAAATGTG

GACCACGTAG GCCTCGGCGC TGCGTTCGAA AACAGTAAAT ACGACCAGGA CTACAATATC

CGTGTAACCA TGTATGTACA ATTCAGAGP.A TTTAATCTTA AAGACCCCCC ACTTAAACCC

TAAGTCGACC CCGGGTTTTT ATAGCTAATT AGTCATTTTT TCGTAAGTAA GTATTTTTAT

TTAATACTTT TTATTGTACT TATGTTAAAT ATAACTGATG ATAACAAAAT CCATTATGTA

TTATTTATAA CTGTAATTTC TTTAGCGTAG TTAGATGTCC P.ATCTCTCTC P..AATACATCG

GCTATCTTTT TAGTGAGATT TTGATCTATG CAGTTGAAAC TTATGAACGC GTGATGATTA

AAATGTGAAC CGTCCAAATT TGCAGTCATT ATATGAGCGT ATCTATTATC TACTATCATC

ATCTTTGAGT TATTAATATC ATCTACTTTA GAATTGATAG GAAATATGAA TACCTTTGTA

GTAATATCTA TACTATCTAC ACCTAACTCA TTAAGACTTT TGATAGGCGG CCGCGAGCTC
Paqe 11 Th015 (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CATCATCATT CGCGATATCC GTTAAGTTTG TATCGTAATG CCCAGCAAGA AGAATGG

(2) INFORMATION FOR SEQ ID NO:10:
( i ) SEQUENCE CHP_R..ACTERISTICS
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TACTACTACG TCGACTCAGT AATTTATTTC ATATGG

(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3609 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Th015 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGTACCTTCA TAAATACAAG TTTGATTAAA CTTAAGTTGT TCTAAAGTTC TTTCCTCCGA
AGGTATAGAA CAAAGTATTT CTTCTACATC CTTACTATTT ATTGCAGCTT TTAACAGCCT

ATCACGTATC CTATTTTTAG TATTGGTAGA ACGTTTTAGT TCTAAAGTTA AAATATTAGA

CATAATTGGC ATATTGCTTA TTCCTTGCAT AGTTGAGTCT GTAGATCGTT TCAGTATATC

ACTGATTAAT GTACTACTGT TATGATGAAA TATAGAATCG ATATTGGCAT TTAACTGTTT

TGTTATACTA AGTCTAGATT TTAAATCTTC TAGTAATATG CTATTTAATA TAAAAGCTTC

CACGTTTTTG TATACATTTC TTTCCATATT AGTAGCTACT ACTAAATGAT TATCTTCTTT

CATATCTTGT AGATAAGATA GACTATCTTT ATCTTTATTA GTAGAAAATA CTTCTGGCCA

TACATCGTTA AATTTTTTTG TTGTTGTTAG ATATAATATT AAATATCTAG AGGATCCTAT

TATTTGTGGT AAAATGTTTA TAGAGTAF~~1 TGATCTGGCT ATTAAACATA GGCCAGTTAC

CATAGAATGC TGCTTCCCGT TACAGTGT'~'T TACCATAACC ATAGATCTGC CTGTATTGTT

GATACATATA ACAGCTGTAA ATCCTAAP~A ATTCCTATCA TAATTATTAA TATTAGGTAA

TTCATTTCCA TGTGAAAGAT AGACTAATTT TATATCCTTT ACCTCCAAAT AATTATTTAC

ATCTCTTP.A.A CAATCTATTT TAATATCFTT AACTGGTATT TTATAATATC CAGAAAGGTT

Th015 TGAAGGGGTT GATGGAATAA GTCTATTP.AC ATCGTTAAGT AAATTATTAA TATCATGAAT

CTTTATTATA TTATACCCAT AAGTTAAATT TATATTTACT TTCTCATCAT CTGACTTAGT

TAGTTTGTAA TAAGGTGTGT CTGAAAAAAT TAAAAGGTAA TTCGTTGAAT GAAGCTGTAT

TTGCTGTATC ATTTTTATCT AATTTTGGAG ATTTAGCAGT ACTTACTTCA TTAGAAGAAG

AATCTGCCAG TTCCTGTCTA TTACTGATAT TTCGTTTCAT TATTATATGA TTTATATTTT

ACTTTTTCAA TTATATATAC TCATTTGACT AGTTAATCAA TAA.A.P~AGAAT TTCGACTTAG

GGTTTAAGTG GGGGGTCTTT AAGATTAAAT TCTCTGAATT GTACATACAT GGTTACACGG

ATATTGTAGT CCTGGTCGTA TTTACTGTTT TCGAACGCAG CGCCGAGGCC TACGTGGTCC

ACATTTCCAG AGGTTTGTAG TCTCAGCCAA AGCTGATTCC TTTTGTTATT TGGTTGGAAG

TAATCAATAG TGGAGTCAAG AACAGGT'='TG GGTGTGAAGT AACGGGAGTG GTAGGAGAAG

GGTTGGGGGA TTGTATGGCG GGAGGAGTAG TTTACATATG GGTCATAGGT TAGGGCTGTG

GCCTTTGTTA CAAAGTTATC ATCTAGAATA ACAGCAGTGG AGCCCACTCC CCTATCACCC

TGGGTGATGG GGGAGCAGGG CCAGAATTCA ACCTTAACCT TTCTTATTCT GTAGTATTCA

AAGGGTATAG AGATTTTGTT GGTCCCCCCT CCCGGGGGAA CAAAGTCGTC AATTTTAAAT

Th015 CTCATCATGT CCACCGCCCA GGAGGGCGTT GTGACTGTGG TACGCTTGAC AGTATATCCG

AAGGTGCGGG AGAGGCGGGT GTTGAAGATG CCATTTTTCC TTCTCCAACG GTAGCGGTGG

CGGGGGTGGA CGAGCCAGGG GCGGCGGCGG AGGATCTGGC CAAGATGGCT GCGGGGGCGG

TGTCTTCTTC TGCGGTAACG CCTCCTTGGA TACGTCATTA CGATACAAAC TTAACGGATA

TCGCGATAAT GAAATAATTT ATGATTATTT CTCGCTTTCA ATTTAACACA ACCCTCAAGA

ACCTTTGTAT TTATTTTCAC TTTTTAAGTA TAGAATAAAG AAGCTGGGGG ATCAATTCCT

GCAGCCCTGC AGCTAATTAA TTAAGCTACA AATAGTTTCG TTTTCACCTT GTCTAATAAC

TAATTAATTA AGGATCCCCC AGCTTCTTTA TTCTATACTT AAAAAGTGAA AATAAATACA

AAGGTTCTTG AGGGTTGTGT TAAATTGAAA GCGAGAAATA ATCATAAATT ATTTCATTAT

CGCGATATCC GTTAAGTTTG TATCGTAATG CCCAGCAAGA AGAATGGAAG AAGCGGACCC

CAACCACATA AAAGGTGGGT GTTCACGCTG AATAATCCTT CCGAAGACGA GCGCAAGAAA

ATACGGGAGC TCCCAATCTC CCTATTTGAT TATTTTATTG TTGGCGAGGA GGGTAATGAG

GAAGGACGAA CACCTCACCT CCAGGGGTTC GCTAATTTTG TGAAGAAGCA AACTTTTAAT

AAAGTGAAGT GGTATTTGGG TGCCCGCTGC CACATCGAGA AAGCCAAAGG AACTGATCAG

CAGAATAAAG AATATTGCAG TAAAGAAGGC AACTTACTTA TTGAATGTGG AGCTCCTCGA

Th015 TGATGATTAA AATGTGAACC GTCCAAATTT GCAGTCATTA TATGAGCGTA TCTATTATCT

ACTATCATCA TCTTTGAGTT ATTAATATCA TCTACTTTAG AATTGATAGG AAATATGAAT

ACCTTTGTAG TAATATCTAT ACTATCTACA CCTAACTCAT TAAGACTTTT GATAGGCGGC

CGCGAGCTC

(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
CATCATCATG ATATCCGTTA AGTTTGTATC GTAATGACGT GGCCAAGGAG GCG

(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Paqe 17 Th015 TACTACTACG TCGACTTATT TATTTAGAGG GTCTTTTAGG

Claims (15)

1. Use of a PCV-2 immunogen to formulate a vaccine composition comprising a pharmaceutically acceptable carrier, excipient or vehicle, to prevent or treat pigs against myocarditis and/or abortion and/or intrauterine infection associated with PCV-

2.

2. The use according to claim 1, characterized in that the immunogen is a live attenuated PCV-2, or an inactivated PCV-2, or PCV-2 subunits or fragments thereof, or a recombinant vector comprising and expressing in vivo a sequence, fragment or epitope of PCV-2.

3. The use according to claim 1 or 2, characterized in that the PCV-2 strain is Imp999, Imp 1010, Imp 1011-48121 or Imp 1011-48285.

4. The use according to claim 1 or 2, characterized in that the PCV-2 strain is strain 1103 or 1121.

5. The use according to any one of claims 1-4, characterized in that the immunogen is formulated with an adjuvant.

6. Culture of strain 1103 as deposited at the ECACC under V00012710.

7. Culture of strain 1121 as deposited at the ECACC under V00012709.

8. Vaccine composition comprising a pharmaceutically acceptable carrier, excipient or vehicle and a PCV-2 immunogen, characterized in that the PCV-2 immunogen is a strain 1103 or strain 1121 immunogen.

9. Vaccine composition according to claim 8, characterized in that the PCV-2 immunogen is a live attenuated PCV-2. or an inactivated PCV-2, or PCV-2 subunits or fragments thereof, or a recombinant vector comprising and expressing in vivo a sequence, fragment or epitope of PCV-2.

10. DNA fragment comprising SEQ ID NO : 6.

11. DNA fragment comprising SEQ ID NO : 7.

12. Use of a PCV-2 immunogen to formulate a vaccine composition comprising a pharmaceutically acceptable carrier, excipient or vehicle, to prevent or treat pigs against myocarditis and/or abortion and/or intrauterine infection associated with PCV-2 and/or post-weaning multisystemic wasting syndrome and other pathologic sequalae associated with PCV-2, wherein the vaccine composition is intended to be administered to female pigs with at least one inoculation before serving, preferably followed by an inoculation during gestation, preferably at about 6-8 weeks of gestation, and/or by an inoculation at the end of gestation, preferably at about 11-13 weeks of gestation.

13. Method of vaccination of-pigs to prevent or treat pigs against myocarditis and/or abortion and/or intrauterine infection associated with PCV-2 and/or post-weaning multisystemic wasting syndrome and other pathologic sequalae associated with PCV-2, wherein a vaccine composition comprising a pharmaceutically acceptable carrier, excipient or vehicle and a PCV-2 immunogen, is administered to female pigs with at least one inoculation before serving, preferably followed by an inoculation during gestation, preferably at about 6-8 weeks of gestation, and/or by an inoculation at the end of gestation, preferably at about 11-13 weeks of gestation.

14. Use of a PCV-2 immunogen to formulate a vaccine composition comprising a pharmaceutically acceptable carrier, excipient or vehicle, to prevent or treat pigs against myocarditis and/or abortion and/or intrauterine infection associated with PCV-2 and/or post-weaning multisystemic wasting syndrome and other pathologic sequalae associated with PCV-2, wherein the vaccine composition is intended to be administered to piglets within the first week of life or within the third week of life, preferably followed by a booster two to four weeks later.

15. Method of vaccination of pigs to prevent or treat pigs against myocarditis and/or abortion and/or intrauterine infection associated with PCV-2 and/or post-weaning multisystemic wasting syndrome and other pathologic sequalae associated with PCV-2, wherein a vaccine composition comprising a pharmaceutically acceptable carrier, excipient or vehicle and a PCV-2 immunogen, is administered to piglets within the first week of life or within the third week of life, preferably followed by a booster two to four weeks later.