Patent Application: US-58354500-A

Abstract:
what is described is a recombinant poxvirus , such as avipox virus , containing 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 composition is administered . also described are methods of treating or preventing disease caused by porcine circovirus 2 by administering the immunological compositions of the invention to an animal in need of treatment or susceptible to infection by porcine circovirus 2 .

Description:
in one aspect , the present invention relates to a recombinant virus , such as a recombinant poxvirus , containing therein a dna sequence from pcv2 , e . g ., 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 “ cocktail ” 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 . pat . 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 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 &# 39 ; 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 maleic 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 . pat . 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 maleic anhydride and alkenyl derivative , the copolymers ema ® ( monsanto ) which are copolymers of maleic 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 , 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 : r 1 and r 2 , which are identical or different , represent h or ch 3 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 nacl , preferably physiological saline ( nacl 9 g / l ) 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 % w / v , 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 . the invention encompasses vectors encoding and expressing equivalent nucleotide sequences , that is to say the sequences which change neither the functionality or the strain specificity ( say strain of type 1 and strain of 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 , included . the pcv - 2 sequences used in the examples are derived from meehan et al . ( strain imp . 1010 ; orf1 nucleotides 398 - 1342 ; orf2 nucleotides 1381 - 314 ; and correspond respectively to orf4 and orf13 in u . s . application ser . no . 09 / 161 , 092 of sep . 25 1998 and to col4 and col13 in wo - a - 9918214 ). other pcv - 2 strains and their sequences have been published in wo - a - 9918214 and are called imp1008 , imp999 , imp1011 - 48285 and imp1011 - 48121 , as well as in a . l . hamel et al . j . virol . june 1998 , vol 72 , 6 : 5262 - 5267 ( genbank af027217 ) and in i . morozov et al . j . clinical microb . september 1998 vol . 36 , 9 : 2535 - 2541 , as well as genbank af086834 , af086835 and af086836 , and give access to equivalent orf sequences . these sequences , or orfs therefrom , or regions thereof encoding an antigen or epitope of interest can also be used in the practice of this invention . the invention also encompasses the equivalent sequences to those used 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 of the whole genome between pcv types 1 and 2 is about 75 %. for orf1 , it is about 86 %, and for orf2 , about 66 %. on the contrary , homologies between genomes and between orfs within type 2 are generally above 95 %. also , equivalent sequences useful in the practice of this present invention , for orf1 , are those sequences having an homology equal or greater than 88 %, advantageously 90 % or greater , preferably 92 % or 95 % or greater with orf1 of strain imp1010 , and for orf2 , are those sequences having an homology equal or greater than 80 %, advantageously 85 % or greater , preferably 90 % or 95 % or greater with orf2 of strain imp1010 . orf1 and orf2 according to meehan 1998 has the potential to encode proteins with predicted molecular weights of 37 . 7 kd and 27 . 8 kd respectively . orf3 and orf4 ( according to meehan et al . 1998 , correspond to orf7 and orf10 respectively in wo - a - 9918214 ) has the potential to encode proteins with predicted molecular weights of 11 . 9 and 6 . 5 kd respectively . the sequence of these orfs is 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 orf1 and / or orf2 . the other orfs 1 - 3 and 5 , 6 , 8 - 9 , 11 - 12 disclosed in u . s . application ser . no . 09 / 161 , 092 of sep . 25 1998 ( cols 1 - 3 and 5 , 6 , 8 - 9 , 11 - 12 in wo - a - 9918214 ), or region ( s ) 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 1 and 2 or region ( s ) thereof encoding antigen ( s ) or epitope ( s ). this invention also encompasses the use of equivalent sequences ; for instance , from orfs of various pcv - 2 strains cited herein . for homology , one can determine that there are equivalent sequences which come from a pcv strain having an orf2 and / or an orf1 which have an homology as defined above with the corresponding orf of strain 1010 . for orf3 according to meehan , 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 orf3 of strain implo10 . for orf4 according to meehan 1998 , advantageously an equivalent sequence has homology that is equal or greater than 86 %, advantageously 90 % or greater , preferably than 95 % or greater with orf4 of strain imp1010 . from the genomic nucleotide sequence , e . g . those disclosed in wo - a - 99 18214 , it is routine art to determine the orfs using a standard software , such as macvector ®. 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 of the genome of another strain ( e . g . other strains disclosed in wo - a - 99 18214 or in other herein cited documents ). using software or making sequence alignment is not undue experimentation and provides direct access to equivalent orfs or nucleic acid molecules . nucleotide sequence homology can be determined using the “ align ” program of myers and miller , (“ optimal alignments in linear space ”, cabios 4 , 11 - 17 , 1988 , incorporated herein by reference ) 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 ( n ref - n dif )* 100n ref , wherein n dif is the total number of non - identical residues in the two sequences when aligned and wherein n ref 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 ( n ref = 8 ; n dif = 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 , incorporated herein by reference ), for instance , using a window size of 20 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 ™ 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 rna 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 , incorporated herein by reference ) and available at ncbi . the following references ( each incorporated herein by reference ) 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 s b and wunsch c d , “ 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 t f and waterman m s , “ comparison of bio - sequences ,” advances in applied mathematics 2 : 482 - 489 ( 1981 ); smith t f , waterman m s and sadler j r , “ statistical characterization of nucleic acid sequence functional domains ,” nucleic acids res ., 11 : 2205 - 2220 ( 1983 ); feng d f and dolittle r f , “ progressive sequence alignment as a prerequisite to correct phylogenetic trees ,” j . of molec . evol ., 25 : 351 - 360 ( 1987 ); higgins d g and sharp p m , “ fast and sensitive multiple sequence alignment on a microcomputer ,” cabios , 5 : 151 - 153 ( 1989 ); thompson j d , higgins d g and gibson t j , “ 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 ). this invention 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 ). preferably , female pigs are inoculated prior to breeding ; and / or prior to serving , and / or during gestation . advantageously , at least one inoculation is done before serving and it is preferably 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 mating and / o serving and a booster inoculation during gestation . thereafter , there can be reinoculation before each serving and / or during gestation at about mid - gestation and / or at the end of gestation . preferably , reinoculations are during gestation . male pigs also can be inoculated , e . g ., prior to mating . 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 . 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 ). advantageously , such piglets are then boosted two to four weeks later . the present invention is additionally described by the following illustrative , non - limiting examples . the invention in a preferred embodiment is directed to recombinant poxviruses 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 ). cell 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 , wis .). the complete nucleotide sequence of the imp . 1010 - stoon pcv2 strain has been determined by meehan et al . ( 1998 ) and is available under the genbank 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 used as the parental virus in in vitro recombination tests . the plaque purified canarypox isolate is designated alvac . alvac was deposited nov . 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 . fig1 ( seq id no : 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 bp pcr fragment was amplified from genomic canarypox dna using oligonucleotide primers c6a1 ( seq id no : 2 ) and c6b1 ( seq id no : 3 ). a 1155 bp pcr fragment was amplified from genomic canarypox dna using oligonucleotide primers c6c1 ( seq id no : 4 ) and c6d1 ( seq id no : 5 ). the 380 bp and 1155 bp fragments were fused together by adding them together as template and amplifying a 1613 bp pcr fragment using oligonucleotide primers c6a1 ( seq id no : 2 ) and c6d1 ( seq id no : 5 ). this fragment was digested with saci and kpni , and ligated into pbluescript sk + ( stratagene , la jolla , calif ., usa ) digested with saci / kpni . the resulting plasmid , pc6l was confirmed by dna sequence analysis . it consists of 370 bp of canarypox dna upstream of c6 (“ c6 left arm ”), vaccinia early termination signal , translation stop codons in six reading frames , an mcs containing smai , psti , xhoi and ecori sites , vaccinia early termination signal , translation stop codons in six reading frames and 1156 bp of downstream canary pox sequence (“ c6 right arm ”). plasmid pjp099 was derived from pc6l by ligating 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 smai / ecori 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 sali site located between the stop codon of the foreign gene and the c6 left arm . the ˜ 4 . 5 kb ecorv / sali or nrui / sali fragment from pjp099 contains therefore the plasmid sequence ( pbluescript sk +; stratagene , la jolla , calif ., usa ), the 2 c6 arms and the 5 ′ end of the h6 promoter until the ecorv or nrui site . plasmid pgem7z - imp1010 - stoon - ecori no . 14 , containing the pcv2 genome as an ecori fragment in plasmid pgem - 7z , was digested with ecori , and a 1768 bp fragment was isolated and ligated . in order to insert pcv2 orf 2 into an appropriate alvac insertion vector : primers jp760 ( seq id no : 6 ) and jp773 ( seq id no : 7 ) were used to amplify pcv2 orf 2 from the 1768 bp ligated ecori fragment ( see above ) resulting in pcr j1304 . primer jp760 ( seq id no : 7 ) contains the 3 ′ end of the h6 promoter from ecorv and the 5 ′ end of pcv2 orf 2 . primer jp773 ( seq id no : 8 ) contains the 3 ′ end of pcv2 orf 2 followed by a sali site . the product of pcr j1304 was then digested with ecorv / sali and cloned as a ˜ 750 bp fragment into a ˜ 4 . 5 kb ecorvisali 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 fig2 and the sequence ( seq id no : 9 ) in fig3 ). 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 ). construction of an alvacc donor plasmid for pcv2 0rf2 and orf1 pcv2 orf1 was amplified by pcr using primers jp774 ( seq id no : 9 ) and jp775 ( seq id no : 10 ) on plasmid pgem7z - imp1010 - stoon - ecori no . 14 resulting in pcr j1311 . primer jp774 ( seq id no : 10 ) contains the 3 ′ end of the h6 promoter from nrui and the 5 ′ end of pcv2 orf1 . primer jp775 ( seq id no : 11 ) contains the 3 ′ end of pcv2 orf1 followed by a sali site . the product of pcr j1311 (˜ 1 kb ) was cloned into pcr2 . 1 ( invitrogen , carlsbad , calif .). 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 bp nruilsali fragment was isolated from pjp104 and cloned into a ˜ 4 . 5 kb nrui / sali fragment from pjp099 ( see example 1 ), resulting in a plasmid which was confirmed by restriction analysis and designated pjp105 ( see fig4 ). the donor plasmid pjp105 could be used in an in vitro recombination test ( described in example 6 ) to generate alvac recombinant expressing the pcv2 orf1 . a ˜ 838 bp bamhi / sali from pjp102 ( see example 2 ) was blunted using the klenow fragment of dna polymerase , and was cloned into the klenow - blunted ecori site of pjp105 . 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 fig5 and the sequence ( seq id no : 11 ) in fig6 ). the donor plasmid pjp107 ( linearized with noti ) was used in an in vitro recombination 5 ( ivr ) test to generate the alvac recombinant vcp1615 ( see example 6 ). plasmid ppcv1 ( b . meehan et al . j . gen . virol . 1997 . 78 . 221 - 227 ), containing the pcv1 genome as a psti fragment in plasmid pgem - 7z , was used as a template to amplify the pcv1 orf2 . in order to insert pcv2 orf 2 into an appropriate alvac insertion vector : primers jp787 ( seq id no : 16 ) and jp788 ( seq id no : 17 ) were used to amplify pcv1 orf 2 from plasmid ppcv1 ( see above ) resulting in pcr j1315 . primer jp787 ( seq id no : 16 ) contains the 3 ′ end of the h6 promoter from ecorv and orf 2 followed by a sali site . the product of pcr j1315 was then digested with ecorv / sali and cloned as a ˜ 750 bp fragment into a ˜ 4 . 5 kb ecorv / sali fragment from pjp099 ( see above in example 1 ). the resulting plasmid was confirmed by sequence analysis and designated pjp113 . the sequence of orf 2 matches sequence available in genbank , accession number u49186 . the donor plasmid pjp113 ( linearized with noti ) was used in an in vitro recombination ( ivr ) test to generate alvac recombinant vcp1621 ( see example 7 ). construction of an alvacc donor plasmid for pcv1 rf2 and orf1 plasmid ppcv1 ( see example 4 above ), containing the pcv1 genome as a psti fragment in plasmid pgem - 7z , was digested with psti , and a 1759 bp fragment was isolated and ligated . primers jp789 ( seq id no : 14 ) and jp790 ( seq id no : 15 ) were used to amplify pcv1 orf1 from the 1759 bp ligated psti fragment ( see above ), resulting in pcr j1316 . primer jp789 ( seq id no : 14 ) contains the 3 ′ end of the h6 promoter from nrui and the 5 ′ end of pcv1 orf1 . primer jp790 ( seq id no : 15 ) contains the 3 ′ end of pcv1 orf1 followed by a sali site . the product of pcr j1316 (˜ 1 kb ) was cloned into pcr2 . 1 ( invitrogen , carlsbad , calif .). 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 bp nrui / sali fragment was isolated from pjp114 and cloned into a ˜ 4 . 5 kb nrui / sali fragment from pjp099 ( see example 1 ), resulting in a plasmid which was confirmed by restriction analysis and designated pjp115 . the donor plasmid pjp115 could be used in an in vitro recombination test ( described in example 7 ) to generate alvac recombinant expressing the pcv1 orf1 . a ˜ 838 bp bamhi / sali from pjp113 ( see example 4 ) was blunted using the klenow fragment of dna polymerase , and was cloned into the klenow - blunted ecori site of pjp115 . 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 pjp117 . the donor plasmid pjp117 ( linearized with noti ) was used in an in vitro recombination ( ivr ) test to generate the alvac recombinant vcp1622 ( see example 7 ). plasmids pjp102 ( see example 2 and fig2 ) and pjp107 ( see example 3 and fig5 ) were linearized 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 pjp102 , 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 pjp107 , and it contains the pcv2 orf2 and orf1 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 & amp ; pcv2 210 7g5gd ) or orf2 ( pcv2 190 4c7cf , pcv2 190 2b1bc & amp ; 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 . wo - a - 99 18214 , 1998 , french applications nos . 97 / 12382 , 98 / 00873 , 98 / 03707 , filed oct . 3 , 1997 , jan . 22 , 1998 , and mar . 20 , 1998 , and wo99 / 29717 , 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 vcp1614 and cells infected with vcp1615 . pcv2 specific immunofluorescence with the two orf1 - specific antibodies could be detected in cells infected with vcp1615 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 . plasmids pjp113 ( see example 4 ) and pjp117 ( see example 5 ) were linearized 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 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 between 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 pjp117 , 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 pcv1 orf1 can be generated using the donor plasmid pjp115 described in example 5 . immunofluorescence . in order to determine if the pcv1 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 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 ). pcv1 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 vcp1621 and in cells infected with vcp1622 . no fluorescence was detected in parental alvac infected vero cells , nor in uninfected vero cells . 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 carbopol ™ 974p manufactured by the company bf goodrich ( molecular weight of # 3 , 000 , 000 ). a 1 . 5 % carbopol ™ 974p stock solution is first prepared in distilled water containing 1 g / l of sodium chloride . this stock solution is then used for manufacturing a 4 mg / ml carbopol ™ 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 carbopol ™ 974p 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 e 8 pfu per dose of 2 ml , one can dilute 0 , 1 ml of a 10 e 9 pfu / ml stock solution into 1 , 9 ml of the above carbopol ™ 974p 4 mg / ml ready - to - use solution . in the same fashion , carbopol ™ 974p 2 mg / ml ready - to - use solutions can also be prepared . 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 ( nacl 0 . 9 %). suitable ranges for viral suspensions can be determined empiracally , but will generally range from 10 6 to 10 10 , and preferably about 10 10 , pfu / dose . vaccine solutions can also be prepared by mixing the recombinant virus suspension with a solution of carbopol ™ 974p , as described in example 8 . 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 . after 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 pwms macroscopic lesions . tissue samples are prepared from liver , lymph nodes , spleen , kidneys and thymus in order to look for specific histological lesions . 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 ( nacl 0 . 9 %). vaccine solutions can also be prepared by mixing the recombinant virus suspension with a solution of carbopol ™ 974p , as described in example 8 . 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 . necropsies 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 . groups of 3 or 4 piglets , caesarian - delivered day oare placed into isolators . day 2 the piglets are vaccinated with 10 8 pfu of vcp1614 , vcp1615 or parental alvac vector 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 alvac recombinant is well tolerated by piglets and no evidence of 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 . 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 vcp 1614 and vcp1615 ( 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 . 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 . hassard , e . clark , j . harding , 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 . winslow , 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 . harding 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 . 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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 ., virologyl87 , 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 ). acttacttac gaaaaa atg tca tta tta caa aaa cta tat ttt aca gaa caa 412 tct ata gta gag tcc ttt aag agt tat aat tta aaa gat aac cat aat 460 ser ile val glu ser phe lys ser tyr asn leu lys asp asn his asn gta ata ttt acc aca tca gat gat gat act gtt gta gta ata aat gaa 508 gat aat gta ctg tta tct aca aga tta tta tca ttt gat aaa att ctg 556 ttt ttt aac tcc ttt aat aac ggt tta tca aaa tac gaa act att agt 604 gat aca ata tta gat ata gat act cat aat tat tat ata cct agt tct 652 tct tct ttg tta gat att cta aaa aaa aga gcg tgt gat tta gaa tta 700 gaa gat cta aat tat gcg tta ata gga gac aat agt aac tta tat tat 748 aaa gat atg act tac atg aat aat tgg tta ttt act aaa gga tta tta 796 gat tac aag ttt gta tta ttg cgc gat gta gat aaa tgt tac aaa cag 844 tat aat aaa aag aat act ata ata gat ata ata cat cgc gat aac aga 892 cag tat aac ata tgg gtt aaa aat gtt ata gaa tac tgt tct cct ggc 940 gln tyr asn ile trp val lys asn val ile glu tyr cys ser pro gly tat ata tta tgg tta cat gat cta aaa gcc gct gct gaa gat gat tgg 988 tta aga tac gat aac cgt ata aac gaa tta tct gcg gat aaa tta tac 1036 act ttc gag ttc ata gtt ata tta gaa aat aat ata aaa cat tta cga 1084 gta ggt aca ata att gta cat cca aac aag ata ata gct aat ggt aca 1132 tct aat aat ata ctt act gat ttt cta tct tac gta gaa gaa cta ata 1180 tat cat cat aat tca tct ata ata ttg gcc gga tat ttt tta gaa ttc 1228 ttt gag acc act att tta tca gaa ttt att tct tca tct tct gaa tgg 1276 gta atg aat agt aac tgt tta gta cac ctg aaa aca ggg tat gaa gct 1324 val met asn ser asn cys leu val his leu lys thr gly tyr glu ala ata ctc ttt gat gct agt tta ttt ttc caa ctc tct act aaa agc aat 1372 tat gta aaa tat tgg aca aag aaa act ttg cag tat aag aac ttt ttt 1420 aaa gac ggt aaa cag tta gca aaa tat ata att aag aaa gat agt cag 1468 gtg ata gat aga gta tgt tat tta cac gca gct gta tat aat cac gta 1516 act tac tta atg gat acg ttt aaa att cct ggt ttt gat ttt aaa ttc 1564 tcc gga atg ata gat ata cta ctg ttt gga ata ttg cat aag gat aat 1612 gag aat ata ttt tat ccg aaa cgt gtt tct gta act aat ata ata tca 1660 glu asn ile phe tyr pro lys arg val ser val thr asn ile ile ser gaa tct atc tat gca gat ttt tac ttt ata tca gat gtt aat aaa ttc 1708 agt aaa aag ata gaa tat aaa act atg ttt cct ata ctc gca gaa aac 1756 ser lys lys ile glu tyr lys thr met phe pro ile leu ala glu asn tac tat cca aaa gga agg ccc tat ttt aca cat aca tct aac gaa gat 1804 tyr tyr pro lys gly arg pro tyr phe thr his thr ser asn glu asp ctt ctg tct atc tgt tta tgc gaa gta aca gtt tgt aaa gat ata aaa 1852 aat cca tta tta tat tct aaa aag gat ata tca gca aaa cga ttc ata 1900 asn pro leu leu tyr ser lys lys asp ile ser ala lys arg phe ile ggt tta ttt aca tct gtc gat ata aat acg gct gtt gag tta aga gga 1948 gly leu phe thr ser val asp ile asn thr ala val glu leu arg gly tat aaa ata aga gta ata gga tgt tta gaa tgg cct gaa aag ata aaa 1996 tyr lys ile arg val ile gly cys leu glu trp pro glu lys ile lys ata ttt aat tct aat cct aca tac att aga tta tta cta aca gaa aga 2044 cgt tta gat att cta cat tcc tat ctg ctt aaa ttt aat ata aca gag 2092 arg leu asp ile leu his ser tyr leu leu lys phe asn ile thr glu gat ata gct acc aga gat gga gtc aga aat aat tta cct ata att tct 2140 asp ile ala thr arg asp gly val arg asn asn leu pro ile ile ser ttt atc gtc agt tat tgt aga tcg tat act tat aaa tta cta aat tgc 2188 phe ile val ser tyr cys arg ser tyr thr tyr lys leu leu asn cys cat atg tac aat tcg tgt aag ata aca aag tgt aaa tat aat cag gta 2236 his met tyr asn ser cys lys ile thr lys cys lys tyr asn gln val met ser leu leu gln lys leu tyr phe thr glu gln ser ile val glu ser phe lys ser tyr asn leu lys asp asn his asn val ile phe thr phe asn asn gly leu ser lys tyr glu thr ile ser asp thr ile leu tyr ala leu ile gly asp asn ser asn leu tyr tyr lys asp met thr trp val lys asn val ile glu tyr cys ser pro gly tyr ile leu trp asn arg ile asn glu leu ser ala asp lys leu tyr thr phe glu phe leu thr asp phe leu ser tyr val glu glu leu ile tyr his his asn asn cys leu val his leu lys thr gly tyr glu ala ile leu phe asp glu tyr lys thr met phe pro ile leu ala glu asn tyr tyr pro lys gly arg pro tyr phe thr his thr ser asn glu asp leu leu ser ile tyr ser lys lys asp ile ser ala lys arg phe ile gly leu phe thr ser val asp ile asn thr ala val glu leu arg gly tyr lys ile arg val ile gly cys leu glu trp pro glu lys ile lys ile phe asn ser leu his ser tyr leu leu lys phe asn ile thr glu asp ile ala thr atccgttaag tttgtatcgt a atg acg tat cca agg agg cgt tac cgc aga 1431 aga aga cac cgc ccc cgc agc cat ctt ggc cag atc ctc cgc cgc cgc 1479 ccc tgg ctc gtc cac ccc cgc cac cgc tac cgt tgg aga agg aaa aat 1527 ggc atc ttc aac acc cgc ctc tcc cgc acc ttc gga tat act gtc aag 1575 gly ile phe asn thr arg leu ser arg thr phe gly tyr thr val lys cgt acc aca gtc aca acg ccc tcc tgg gcg gtg gac atg atg aga ttt 1623 aaa att gac gac ttt gtt ccc ccg gga ggg ggg acc aac aaa atc tct 1671 ata ccc ttt gaa tac tac aga ata aga aag gtt aag gtt gaa ttc tgg 1719 ccc tgc tcc ccc atc acc cag ggt gat agg gga gtg ggc tcc act gct 1767 pro cys ser pro ile thr gln gly asp arg gly val gly ser thr ala gtt att cta gat gat aac ttt gta aca aag gcc aca gcc cta acc tat 1815 gac cca tat gta aac tac tcc tcc cgc cat aca atc ccc caa ccc ttc 1863 asp pro tyr val asn tyr ser ser arg his thr ile pro gln pro phe tcc tac cac tcc cgt tac ttc aca ccc aaa cct gtt ctt gac tcc act 1911 ser tyr his ser arg tyr phe thr pro lys pro val leu asp ser thr att gat tac ttc caa cca aat aac aaa agg aat cag ctt tgg ctg aga 1959 ile asp tyr phe gln pro asn asn lys arg asn gln leu trp leu arg cta caa acc tct gga aat gtg gac cac gta ggc ctc ggc gct gcg ttc 2007 leu gln thr ser gly asn val asp his val gly leu gly ala ala phe gaa aac agt aaa tac gac cag gac tac aat atc cgt gta acc atg tat 2055 glu asn ser lys tyr asp gln asp tyr asn ile arg val thr met tyr gta caa ttc aga gaa ttt aat ctt aaa gac ccc cca ctt aaa ccc 2100 pro ser trp ala val asp met met arg phe lys ile asp asp phe val arg ile arg lys val lys val glu phe trp pro cys ser pro ile thr gln gly asp arg gly val gly ser thr ala val ile leu asp asp asn phe thr pro lys pro val leu asp ser thr ile asp tyr phe gln pro val asp his val gly leu gly ala ala phe glu asn ser lys tyr asp gln asp tyr asn ile arg val thr met tyr val gln phe arg glu phe pro lys leu pro pro asp lys leu asn phe glu arg phe gln val tyr met thr val arg ile asn tyr asp gln asp tyr lys ser asn glu phe asp ile thr ser asp leu val pro lys pro thr phe tyr arg ser his ile lys phe arg met met asp val ala trp ser pro thr thr val thr ile gly asn lys arg arg trp arg tyr arg his arg pro his val leu trp val phe thr leu asn asn pro ser glu asp glu arg lys lys ile arg glu leu pro ile ser leu phe asp tyr phe ile val gly glu glu gly asn glu glu gly arg thr pro his leu gln gly phe ala asn phe val lys lys gln thr phe asn lys val lys arg tyr leu gly ala arg cys his ile glu lys ala lys gly thr asp gln gln asn lys glu tyr cys ser lys glu gly asn leu leu ile glu cys gly ala pro arg ser ser gly ser leu val thr val ala glu gln his pro val thr phe val arg asn phe arg gly leu ala glu leu leu lys val ser gly lys met gln lys arg asp trp lys thr asn val his val ile val gly pro pro gly cys gly lys ser lys trp ala ala asn phe ala asp pro glu thr glu glu val val val ile asp asp phe tyr gly trp leu pro trp asn gly gly thr val pro phe leu ala arg ser ile leu ile thr ser asn ala leu tyr arg arg ile thr ser leu val phe trp lys asn ala thr