(12) United States Patent
`Ellis et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006517843Bl
`US 6,517,843 Bl
`Feb.11,2003
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) REDUCTION OF PORCINE CIRCOVIRUS-2
`VIRAL LOAD WITH INACTIVATED PCV-2
`
`(75)
`
`Inventors: John Albert Ellis, Saskatoon (CA);
`Gordon Allan Moore, Belfast (GB);
`Brian Meehan, Belfast (GB); Edward
`Clark, Saskatoon (CA); Deborah
`Haines, Saskatoon (CA); Lori Hassard,
`Saskatoon (CA); John Harding,
`Humboldt (CA); Catherine Elisabeth
`Charreyre, Saint-Laurent de Mure
`(FR); Gilles Emile Chappuis, Lyons
`(FR); George Steve Krakowka,
`Columbus, OH (US); Jean-Christophe
`Francis Audonnet, Lyons (FR);
`Francis McNeilly, Newtownards (GB)
`
`(73) Assignees: Merial, Lyons (FR); University of
`Saskatchewan, Saskatoon (CA); The
`Queen's University of Belfast, Belfast
`(GB)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 23 days.
`
`(21) Appl. No.: 09/583,350
`
`(22) Filed:
`
`May 31,2000
`
`Related U.S. Application Data
`( 60) Provisional application No. 60/151,564, filed on Aug. 31,
`1999.
`
`(51)
`
`Int. Cl? ........................ A61K 39/12; AOlK 63/00;
`C12N 7/04
`(52) U.S. Cl. ..................... 424/204.1; 424/93.6; 514/44;
`435/236
`(58) Field of Search ........................... 424/184.1, 199.1,
`424/202.1, 204.1, 96.3, 93.6; 514/44; 435/236
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`6,391,314 B1 * 5!2002 Allan eta!. .............. 424/204.1
`
`OTHER PUBLICATIONS
`
`Cruse et al. 1995. Illustrated Dictionary of Immunology.
`CRC Press, Inc., p. 156.*
`Allan et al. 2000. 1 Vet Diagn Invest. 12: 3-14. *
`West et al. 1999. 1 Vet Diagn Invest. 11: 530-532.*
`Allan et al. 2000. Veterinary Record. 147 (6): 170-171.*
`Krakowka et al. 2001. Vet Pathology. 38: 31--42.*
`
`* cited by examiner
`
`Primary Examiner-James Housel
`Assistant Examiner---Shanon Foley
`(74) Attorney, Agent, or Firm---Frommer Lawrence &
`Haug, LLP; WilliamS. Frommer; Thomas J. Kowalski
`
`(57)
`
`ABSTRACT
`
`Porcine circovirus-2 (PCV-2) is a recently identified agent
`wherein the potential spectrum of PCV-2-associated disease
`has been expanded by evidence of vertical and sexual
`transmission and associated reproductive failure in swine
`populations. 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. Inoculation of female pigs with a compo(cid:173)
`sition including an immunogen from PCV-2 or an epitope of
`interest from such an immunogen or with a vector express(cid:173)
`ing such an immunogen or epitope of interest prior to
`breeding, such as within the first five weeks of life, or prior
`to the perinatal period, or repeatedly over a lifetime, or
`during pregnancy, such as between the 6'h and 8'h and/or the
`lO'h and 13'h weeks of gestation, can prevent myocarditis,
`abortion and intrauterine infection associated with porcine
`circovirus-2. In addition, innoculation of male and/or female
`pigs with the aforementioned compositions can be carried
`out to prevent transmission of PCV-2 from male to female
`(or vice versa) during mating. Thus, the invention involves
`methods and compositions for preventing myocarditis, abor(cid:173)
`tion and intrauterine infection associated with porcine
`circovirus-2.
`
`6 Claims, 8 Drawing Sheets
`
`CEV Exhibit 1001_001
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 1 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 1
`
`Figure 1
`
`aattcaacct taacctttct tattctgtag tattcaaagg gcacagagcg ggggtttgag 60
`ccccctcctg ggggaagaaa gtcattaata ttgaatctca tcatgtccac cgcccaggag 120
`ggcgttctga ctgtggttcg cttgacagta tatccgaagg tgcgggagag gcgggtgttg 180
`aagatgccat ttttccttct ccagcggtaa cggtggcggg ggtggacgag ccaggggcgg 240
`cggcggagga tctggccaag atggctgcgg gggcggtgtc ttcttctccg gtaacgcctc 300
`cttggatacg tcatatctga aaacgaaaga agtgcgctgt aagtattacc agcgcacttc 360
`ggcagcggca gcacctcggc agcacctcag cagcaacatg ccgagcaaga agaatggaag 420
`aagcggaccc caaccccata aaaggtgggt gttcactctg aataatcctt ccgaagacga 480
`gcgcaagaaa atacgggatc ttccaatatc cctatttgat tattttattg ttggcgagga 540
`gggtaatgag gaaggacgaa cacctcacct ccaggggttc gctaattttg tgaagaagca 600
`gacttttaat aaagtgaagt ggtatttggg tgcccgctgc cacatcgaga aagcgaaagg 660
`aacagatcag cagaataaag aatactgcag taaagaaggc aacttactga tggagtgtgg 720
`agctcctaga tctcagggac aacggagtga cctgtctact gctgtgagta ccttgttgga 780
`gagcgggagt ctggtgaccg ttgcagagca gcaccctgta acgtttgtca gaaatttccg 840
`cgggctggct gaacttttga aagtgagcgg gaaaatgcag aagcgtgatt ggaagactaa 900
`tgtacacgtc attgtggggc cacctgggtg tggtaaaagc aaatgggctg ctaattttgc 960
`agacccggaa accacatact ggaaaccacc tagaaacaag tggtgggatg gttaccatgg 1020
`tgaagaagtg gttgttattg atgactttta tggctggctg ccctgggatg atctactgag 1080
`actgtgtgat cgatatccat tgactgtaga gactaaaggt ggaactgtac cttttttggc 1140
`ccgcagtatt ctgattacca gcaatcagac cccgttggaa tggtactcct caactgctgt 1200
`cccagctgta gaagctcttt atcggaggat tacttccttg gtattttgga agaatgctac 1260
`agaacaatcc acggaggaag ggggccagtt cgtcaccctt tcccccccat gccctgaatt 1320
`tccatatgaa ataaattact gagtcttttt tatcacttcg taatggtttt tattattcat 1380
`taagggttaa gtggggggtc tttaagatta aattctctga attgtacata catggttaca 1440
`cggatattgt attcctggtc gtatatactg ttttcgaacg cagtgccgag gcctacgtgg 1500
`tctacatttc cagcagtttg tagtctcagc cacagctggt ttcttttgtt gtttggttgg 1560
`aagtaatcaa tagtggaatc taggacaggt ttgggggtaa agtagcggga gtggtaggag 1620
`aagggctggg ttatggtatg gcgggaggag tagtttacat aggggtcata ggtgagggct 1680
`gtggcctttg ttacaaagtt atcatctaga ataacagcac tggagcccac tcccctgtca 1740
`ccctgggtga tcggggagca gggccag
`1767
`
`CEV Exhibit 1001_002
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 2 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 2
`
`Figure 2
`
`aattcaacct taacctttct tattctgtag tattcaaagg gcacagagcg ggggtttgag 60
`ccccctcctg ggggaagaaa gtcattaata ttgaatctca tcatgtccac cgcccaggag 120
`ggcgttttga ctgtggttcg cttgacagta tatccgaagg tgcgggagag gcgggtgttg 180
`aagatgccat ttttccttct ccagcggtaa cggtggcggg ggtggacgag ccaggggcgg 240
`cggcggagga tctggccaag atggctgcgg gggcggtgtc ttcttctccg gtaacgcctc 300
`cttggatacg tcatatctga aaacgaaaga agtgcgctgt aagtattacc agcgcacttc 360
`ggcagcggca gcacctcggc agcacctcag cagcaacatg cccagcaaga agaatggaag 420
`aagcggaccc caaccccata aaaggtgggt gttcactctg aataatcctt ccgaagacga 480
`gcgcaagaaa atacgggatc ttccaatatc cctatttgat tattttattg ttggcgagga 540
`gggtaatgag gaaggacgaa cacctcacct ccaggggttc gctaattttg tgaagaagca 600
`gacttttaat aaagtgaagt ggtatttggg tgcccgctgc cacatcgaga aagcgaaagg 660
`aacagatcag cagaataaag aatactgcag taaagaaggc aacttactga tggagtgtgg 720
`agctcctaga tctcagggac aacggagtga cctgtctact gctgtgagta ccttgttgga 780
`gagcgggagt ctggtgaccg ttgcagagca gcaccctgta acgtttgtca gaaatttccg 840
`cgggctggct gaacttttga aagtgagcgg gaaaatgcag aagcgtgatt ggaagactaa 900
`tgtacacgtc attgtggggc cacctgggtg tggtaaaagc aaatgggctg ctaattttgc 960
`agacccggaa accacatact ggaaaccacc tagaaacaag tggtgggatg gttaccatgg 1020
`tgaagaagtg gttgttattg atgactttta tggctggctg ccctgggatg atctactgag 1080
`actgtgtgat cgatatccat tgactgtaga gactaaaggt ggaactgtac cttttttggc 1140
`ccgcagtatt ctgattacca gcaatcagac cccgttggaa tggtactcct caactgctgt 1200
`cccagctgta gaagctcttt atcggaggat tacttccttg gtattttgga agaatgctac 1260
`agaacaatcc acggaggaag ggggccagtt cgtcaccctt tcccccccat gccctgaatt 1320
`tccatatgaa ataaattact gagtcttttt tatcacttcg taatggtttt tattattcat 1380
`taagggttaa gtggggggtc tttaagatta aattctctga attgtacata catggttaca 1440
`cggatattgt attcctggtc gtatatactg ttttcgaacg cagtgccgag gcctacgtgg 1500
`tctacatttc cagtagtttg tagtctcagc cacagctgat ttcttttgtt gtttggttgg 1560
`aagtaatcaa tagtggaatc taggacaggt ttgggggtaa agtagcggga gtggtaggag 1620
`aagggctggg ttatggtatg gcgggaggag tagtttacat aggggtcata ggtgagggct 1680
`gtggcctttg ttacaaagtt atcatctaga ataacagcac tggagcccac tcccctgtca 1740
`ccctgggtga tcggggagca gggccag
`1767
`
`CEV Exhibit 1001_003
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 3 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 3
`
`Figure 3
`
`aattcaacct taaccttttt tattctgtag tattcaaagg gtatagagat tttgttggtc 60
`ccccctcccg ggggaacaaa gtcgtcaata ttaaatctca tcatgtccac cgcccaggag 120
`ggcgttctga ctgtggtagc cttgacagta tatccgaagg tgcgggagag gcgggtgttg 180
`aagatgccat ttttccttct ccaacggtag cggtggcggg ggtggacgag ccaggggcgg 240
`cggcggagga tctggccaag atggctgcgg gggcggtgtc ttcttctgcg gtaacgcctc 300
`cttggatacg tcatagctga aaacgaaaga agtgcgctgt aagtattacc agcgcacttc 360
`ggcagcggca gcacctcggc agcacctcag cagcaacatg cccagcaaga agaatggaag 420
`aagcggaccc caaccacata aaaggtgggt gttcacgctg aataatcctt ccgaagacga 480
`gcgcaagaaa atacgggagc tcccaatctc cctatttgat tattttattg ttggcgagga 540
`gggtaatgag gaaggacga:a cacctcacct ccaggggttc gctaattttg tgaagaagca 600
`aacttttaat aaagtgaagt ggtatttggg tgcccgctgc cacatcgaga aagccaaagg 660
`aactgatcag cagaataaag aatattgcag taaagaaggc aacttactta ttgaatgtgg 720
`agctcctcga tctcaaggac aacggagtga cctgtctact gctgtgagta ccttgttgga 780
`gagcgggagt ctggtgaccg ttgcagagca gcaccctgta acgtttgtca gaaatttccg 840
`cgggctggct gaacttttga aagtgagcgg gaaaatgcag aagcgtgatt ggaagaccaa 900
`tgtacacgtc attgtggggc cacctgggtg tggtaaaagc aaatgggctg ctaattttgc 960
`agacccggaa accacatact ggaaaccacc tagaaacaag tggtgggatg gttaccatgg 1020
`tgaagaagtg gttgttattg atgactttta tggctggctg ccgtgggatg atctactgag 1080
`actgtgtgat cgatatccat tgactgtaga gactaaaggt ggaactgtac cttttttggc 1140
`ccgcagtatt ctgattacca gcaatcagac cccgttggaa tggtactcct caactgctgt 1200
`cccagctgta gaagctctct atcggaggat tacttccttg gtattttgga agaatgctac 1260
`agaacaatcc acggaggaag ggggccagtt cgtcaccctt tcccccccat gccctgaatt 1320
`tccatatgaa ataaattact gagtcttttt tatcacttcg taatggtttt tat tat teat 1380
`ttagggttta agtggggggt ctttaagatt aaattctctg aattgtacat acatggttac 1440
`acggatattg tagtcctggt cgtatatact gttttcgaac gcagtgccga ggcctacgtg 1500
`gtccacattt ctagaggttt gtagcctcag ccaaagctga ttccttttgt tatttggttg 1560
`gaagtaatca atagtggagt caagaacagg tttgggtgtg aagtaacggg agtggtagga 1620
`gaagggttgg gggattgtat ggcgggagga gtagtttaca tatgggtcat aggttagggc 1680
`tgtggccttt gttacaaagt tatcatctag aataacagca gtggagccca ctcccctatc 1740
`accctgggtg atgggggagc agggccag
`1768
`
`CEV Exhibit 1001_004
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 4 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 4
`
`Figure 4
`
`aattcaacct taacctttct tattctgtag tattcaaagg gtatagagat tttgttggtc 60
`ccccctcccg ggggaacaaa gtcgtcaatt ttaaatctca tcatgtccac cgcccaggag 120
`ggcgttgtga ctgtggtacg cttgacagta tatccgaagg tgcgggagag gcgggtgttg 180
`aagatgccat ttttccttct ccaacggtag cggtggcggg ggtggacgag ccaggggcgg 240
`cggcggagga tctggccaag atggctgcgg gggcggtgtc ttcttctgcg gtaacgcctc 300
`cttggatacg tcatagctga aaacgaaaga agtgcgctgt aagtattacc agcgcacttc 360
`ggcagcggca gcacctcggc agcacctcag cagcaacatg cccagcaaga agaatggaag 420
`aagcggaccc caaccacata aaaggtgggt gttcacgctg aataatcctt ccgaagacga 480
`gcgcaagaaa atacgggagc tcccaatctc cctatttgat tattttattg ttggcgagga 540
`gggtaatgag gaaggacgaa cacctcacct ccaggggttc gctaattttg tgaagaagca 600
`aacttttaat aaagtgaagt ggtatttggg tgcccgctgc cacatcgaga aagccaaagg 660
`aactgatcag cagaataaag aatattgcag taaagaaggc aacttactta ttgaatgtgg 720
`agctcctcga tctcaaggac aacggagtga cctgtctact gctgtgagta ccttgttgga 780
`gagcgggagt ctggtgaccg ttgcagagca gcaccctgta acgtttgtca gaaatttccg 840
`cgggctggct gaacttttga aagtgagcgg gaaaatgcag aagcgtgatt ggaagaccaa 900
`tgtacacgtc attgtggggc cacctgggtg tggtaaaagc aaatgggctg ctaattttgc 960
`agacccggaa accacatact ggaaaccacc tagaaacaag tggtgggatg gttaccatgg 1020
`tgaagaagtg gttgttattg atgactttta tggctggctg ccgtgggatg atctactgag 1080
`actgtgtgat cgatatccat tgactgtaga gactaaaggt ggaactgtac cttttttggc 1140
`ccgcagtatt ctgattacca gcaatcagac cccgttggaa tggtactcct caactgctgt 1200
`cccagctgta gaagctctct atcggaggat tacttccttg gtattttgga agaatgctac 1260
`agaacaatcc acggaggaag ggggccagtt cgtcaccctt tcccccccat gccctgaatt 1320
`tccatatgaa ataaattact gagtcttttt tatcacttcg taatggtttt tattattcat 1380
`ttagggttta agtggggggt ctttaagatt aaattctctg aattgtacat acatggttac 1440
`acggatattg tagtcctggt cgtatttact gttttcgaac gcagcgccga ggcctacgtg 1500
`gtccacattt ccagaggttt gtagtctcag ccaaagctga ttccttttgt tatttggttg 1560
`gaagtaatca atagtggagt caagaacagg tttgggtgtg aagtaacggg agtggtagga 1620
`gaagggttgg gggattgtat ggcgggagga gtagtttaca tatgggtcat aggttagggc 1680
`tgtggccttt gttacaaagt tatcatctag aataacagca gtggagccca ctcccctatc 1740
`accctgggtg atgggggagc agggccag
`1768
`
`CEV Exhibit 1001_005
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 5 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 5
`
`Figure 5
`
`aattcatatt tagcctttct aatacggtag tattggaaag gtaggggtag ggggttggtg 60
`ccgcctgagg gggggaggaa ctggccgatg ttgaatttga ggtagttaac attccaagat 120
`ggctgcgagt atcctccttt tatggtgagt acaaattctg tagaaaggcg ggaattgaag 180
`atacccgtct ttcggcgcca tctgtaacgg tttctgaagg cggggtgtgc caaatatggt 240
`cttctccgga ggatgtttcc aagatggctg cgggggcggg tccttcttct gcggtaacgc 300
`ctccttggcc acgtcatcct ataaaagtga aagaagtgcg ctgctgtagt attaccagcg 360
`cacttcggca gcggcagcac ctcggcagcg tcagtgaaaa tgccaagcaa gaaaagcggc 420
`ccgcaacccc ataagaggtg ggtgttcacc cttaataatc cttccgagga ggagaaaaac 480
`aaaatacggg agcttccaat ctcccttttt gattattttg tttgcggaga ggaaggtttg 540
`gaagagggta gaactcctca cctccagggg tttgcgaatt ttgctaagaa gcagactttt 600
`aacaaggtga agtggtattt tggtgcccgc tgccacatcg agaaagcgaa aggaaccgac 660
`cagcagaata aagaatactg cagtaaagaa ggccacatac ttatcgagtg tggagctccg 720
`cggaaccagg ggaagcgcag cgacctgtct actgctgtga gtaccctttt ggagacgggg 780
`tctttggtga ctgtagccga gcagttccct gtaacgtatg tgagaaattt ccgcgggctg 840
`gctgaacttt tgaaagtgag cgggaagatg cagcagcgtg attggaagac agctgtacac 900
`gtcatagtgg gcccgcccgg ttgtgggaag agccagtggg cccgtaattt tgctgagcct 960
`agggacacct actggaagcc tagtagaaat aagtggtggg atggatatca tggagaagaa 1020
`gttgttgttt tggatgattt ttatggctgg ttaccttggg atgatctact gagactgtgt 1080
`gaccggtatc cattgactgt agagactaaa gggggtactg ttcctttttt ggcccgcagt 1140
`attttgatta ccagcaatca ggccccccag gaatggtact cctcaactgc tgtcccagct 1200
`gtagaagctc tctatcggag gattactact ttgcaatttt ggaagactgc tggagaacaa 1260
`tccacggagg tacccgaagg ccgatttgaa gcagtggacc caccctgtgc ccttttccca 1320
`tataaaataa attactgagt cttttttgtt atcacatcgt aatggttttt atttttattt 1380
`atttagaggg tcttttagga taaattctct gaattgtaca taaatagtca gccttaccac 1440
`ataattttgg gctgtggctg cattttggag cgcatagccg aggcctgtgt gctcgacatt 1500
`ggtgtgggta tttaaatgga gccacagctg gtttctttta ttatttgggt ggaaccaatc 1560
`aattgtttgg tccagctcag gtttgggggt gaagtacctg gagtggtagg taaagggctg 1620
`ccttatggtg tggcgggagg agtagttaat ataggggtca taggccaagt tggtggaggg 1680
`ggttacaaag ttggcatcca agataacaac agtggaccca acacctcttt gattagaggt 1740
`gatggggtct ctggggtaa
`1759
`
`CEV Exhibit 1001_006
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 6 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 6
`
`Figure 6
`
`gaattcaacc ttaacctttt ttattctgta gtattcaaag ggtataaaga ttttgttggt 60
`cccccctccc gggggaacaa agtcgtcaat attaaatctc atcatgtcca ccgcccagga 120
`gggcgttctg actgtggtag ccttgacagt atatccgaag gtgcgggaga rgcgggtgtt 180
`gaaaatgcca tttttccttc tccaacggta gcggtggcgg gggtggacma nccacgggcg 240
`gcggcggawg atctggccaa gatggctgcg ggggcggtgt cttcttctgc ggtaacgcct 300
`ccttggatac gtcatagctg aaaacgaaag aagtgcgctg taagtattac cagcgcactt 360
`cggcagcggc agcacctcgg cagcacctca gcagcaacat gcccagcaag aagaatggaa 420
`gaagcggacc ccaaccacat aaaaggtggg tgttcacgct gaataatcct tccgaagacg 480
`agcgcaagaa aatacgggag ctcccaatct ccctatttga ttattttatt gttggcgagg 540
`agggtwwtga ggaangacga acacctcacc tccaggggtt cgctaatttt gtgaagaagc 600
`aaacttttaa taaagtgaag tggtatttgg gtgcccgctg ccacatcgag aaagccaaag 660
`gaactgatca gcagaataaa gaatattgca gtaaagaagg caacttactt attgaatgtg 720
`gagctcctcg atctcaagga caacggagtg acctgtctac tgctgtgagt accttgttgg 780
`agagcgggag tctggtgacc gttgcagagc agcaccctgt aacgtttgtc agaaatttcc 840
`gcgggctggc tgaacttttg aaagtgagcg ggaaaatgca gaagcgtgat tggaagacca 900
`atgtacacgt cattgtgggg ccacctgggt gtggtaaaag caaatgggct gctaattttg 960
`cagacccgga aaccacatac tggaaaccac ctagaaacaa gtggtgggat ggttaccatg 1020
`gtgaagaagt ggttgttatt gatgactttt atggctggct gccgtgggat gatctactga 1080
`gactgtgtga tcgatatcca ttgactgtag agactaaagg tggaactgta cnnnnnnngg 1140
`cccgcagtat tctgattacc agcaatcaga ccccgttgga atggtactcc tcaactgctg 1200
`tcccagctgt agaagctctc tatcggagga ttacttcctt ggtattttgg aagaatgcta 1260
`cagaacaatc cacggaggaa gggggccagt tngtcaccct ttccccccca tgccctgaat 1320
`ttccatatga aataaattac tgagtctttt ttatcacttc gtaatggttt ttattattca 1380
`tttagggttt aagtgggggg tctttaagat taaattctct gaattgtaca tacatggtta 1440
`cacggatatt gtagtcctgg tcgtatatac tgttttcgaa cgcagtgccg aggcctacgt 1500
`ggtccacatt tctagaggtt tgtagcctca gccaaagctg attccttttg ttatttggtt 1560
`ggaagtaatc aatagtggag tcaagaacag gtttgggtgt gaagtaacgg gagtggtagg 1620
`agaagggttg ggggattgta tggcgggagg agtagtttac atatgggtca taggttaggg 1680
`ctgtggcctt tgttacaaag ttatcatcta gaataacagc agtggagccc actcccctat 1740
`caccctgggt gatgggggag cagggcca
`1768
`
`CEV Exhibit 1001_007
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 7 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 7
`
`Figure 7
`
`accagcgcac ttcggcagcg gcagcacctc ggcagcacct cagcagcaac atgcccagca 60
`agaagaatgg aagaagcgga ccccaaccac ataaaaggtg ggtgttcacg ctgaataatc 120
`cttccgaaga cgagcgcaag aaaatacggg agctcccaat ctccctattt gattatttta 180
`ttgttggcga ggagggtaat gaggaaggac gaacacctca cctccagggg ttcgctaatt 240
`ttgtgaagaa kcaaactttt aataaagtga agtggtattt gggtgcccgc tgccacatcg 300
`agaaagccaa aggaactgat cagcagaata aagaatattg cagtaaagaa ggcaacttac 360
`ttattgaatg tggagctcct cgatctcaag gacaacggag tgacctgtct actgctgtga 420
`gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagcaccct gtaacgtttg 480
`tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg cagaagcgtg 540
`attggaagac caatgtacac gtcattgtgg ggccacctgg gtgtggtaaa agcaaatggg 600
`ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac aagtggtggg 660
`atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg ctgccgtggg 720
`atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaa ggtggaactg 780
`tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttg gaatggtact 840
`cctcaactgc tgtcccagct gtagaagctc tctatcggag gat tact tee ttggtatttt 900
`ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcacc ctttcccccc 960
`catgccctga atttccatat gaaataaatt actgagtcyt ttttatcact tcgtaatggt 1020
`ttttattatt catttagggg ttaagtgggg ggtctttaag attaaattcc ctgaattgta 1080
`catacagggt tacacggata ttgtagtcct ggtcgtattt actgttttcg aacgcagtgc 1140
`cgaggcctac gtggtccaca tttctagagg tttgtagcct cagccaaagc tgattccttt 1200
`tgttatttgg ttggaagtaa tcaatagtgg agtcaagaac aggtttgggt gtgaagtaac 1260
`gggagtggta ggagaagggt tgggggattg tatggcggga ggagtagttt acatatgggt 1320
`catatgtttg ggctgtggcc tttggtacaa agttatcatc tagaataaca gcagtggagc 1380
`ccactcccct atcaccctgg gtgatggggg agcagggcca gaattcaacc ttaacctttc 1440
`ttattctgta gtattcaaag ggtatagaga ttttgttggt cccccctccc gggggaacaa 1500
`agtcgtcaat tttaaatctc atcatgtcca ccgcccagga gggcgttgtg actgtggtac 1560
`gcttgacagt atatccgaag gtgcgggaga ggcgggtgtt gaagatgcca tttttccttc 1620
`tccaacggta gcggtggcgg gggtggacga gccaggggcg gcggcggagg atctggccaa 1680
`gatggctgcg ggggcggtgt cttcttctgc ggtaacgcct ccttggatat gtcatagctg 1740
`aaaacgaaag aagtgcgctg taagtatt
`1768
`
`CEV Exhibit 1001_008
`
`

`

`U.S. Patent
`
`Feb.11,2003
`
`Sheet 8 of 8
`
`US 6,517,843 Bl
`
`SEQ ID No. 8
`
`Figure 8
`
`accagcgcac ttcggcagcg gcagcacctc ggcagcacct cagcagcaac atgcccagca 60
`agaagaatgg aagaagcgga ccccaaccac ataaaaggtg ggtgttcacg ctgaataatc 120
`cttccgaaga cgagcgcaag aaaatacggg agctcccaat ctccctattt gattatttta 180
`ttgttggcga ggagggtaat gaggaaggac gaacacctca cctccagggg ttcgctaatt 240
`ttgtgaagaa gcaaactttt aataaagtga agtggtattt gggtgcccgc tgccacatcg 300
`agaaagccaa aggaactgat cagcagaata aagaatattg cagtaaagaa ggcaacttac 360
`ttattgaatg tggagctcct cgatctcaag gacaacggag tgacctgtct actgctgtga 420
`gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagcaccct gtaacgtttg 480
`tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg cagaagcgtg 540
`attggaagac caatgtacac gtcattgtgg ggccacctgg gtgtggtaaa agcaaatggg 600
`ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac aagtggtggg 660
`atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg ctgccgtggg 720
`atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaa ggtggaactg 780
`tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttg gaatggtact 840
`cctcaactgc tgtcccagct gtagaagctc tctatcggag gat tact tee ttggtatttt 900
`ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcacc ctttcccccc 960
`catgccctga atttccatat gaaataaatt actgagtctt ttttatcact tcgtaatggt 1020
`ttttattatt catttagggg ttaagtgggg ggtctttaag attaaattct ctgaattgta 1080
`catacatggt tacacggata ttgtagtcct ggtcgtattt actgttttcg aacgcagtgc 1140
`cgaggcctac gtggtccaca tttctagagg tttgtagcct cagccaaagc tgattccttt 1200
`tgttatttgg ttggaagtaa tcaatagtgg agtcaagaac aggtttgggt gtgaagtaac 1260
`gggagtggta ggagaagggt tgggggattg tatggcggga ggagtagttt acatatgggt 1320
`cataggttag ggctgtggcc tttggtacaa agttatcatc tagaataaca gcagtggagc 1380
`ccactcccct atcaccctgg gtgatggggg agcagggcca gaattcaacc ttaacctttt 1440
`ttattctgta gtattcaaag ggtatagaga ttttgttggt cccccctccc gggggaacaa 1500
`agtcgtcaat tttaaatctc atcatgtcca ccgcccagga gggcgttgtg actgtagtac 1560
`gcttgacagt atatccgaag gtgcgggaga ggcgggtgtt gaagatgcca tttttccttc 1620
`tccaacggta gcggtggcgg gggtggacga gccaggggcg gcggcggagg atctggccaa 1680
`gatggctgcg ggggcggtgt cttcttctgc ggtaacgcct ccttggatac gtcatagctg 1740
`aaaacgaaag aagtgcgctg taagtatt
`1768
`
`CEV Exhibit 1001_009
`
`

`

`1
`REDUCTION OF PORCINE CIRCOVIRUS-2
`VIRAL LOAD WITH INACTIVATED PCV-2
`
`2
`cited or referenced in herein cited documents are hereby
`incorporated herein by reference.
`
`US 6,517,843 Bl
`
`BACKGROUND OF THE INVENTION
`
`Porcine circovirus-2 (PCV-2) was recently identified as an
`agent that has been consistently associated with post(cid:173)
`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
`10 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 noncyto(cid:173)
`pathic contaminant of porcine kidney (PK/15) cell line
`(Meehan et al. 1998; Tischer et al. 1974). Pigs with naturally
`15 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,
`20 lymphadenopathy, interstitial pneumonia, hepatitis and
`nephritis (Allan et al. 1998; Allan et al. 1999; Ellis et al.
`1998; Ellis et al. 1999). 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
`25 PCV-2-caused myocarditis, and/or abortion and/or intrau(cid:173)
`terine infection.
`
`RELATED APPLICATIONS
`This application claims priority from and is hased upon 5
`U.S application Ser. No. 60/151,564, filed Aug. 31, 1999.
`Reference is made to: WO-A-01409, published from PCT/
`EP99/04698, filed Jun. 28, 1999 and U.S. application Ser.
`No. 09/347,594, filed Jul. 1, 1999, both claiming priority
`from French application No. 98 08777, filed Jul. 6, 1998;
`U.S. application Ser. No. 09/161,092, filed Sep. 25, 1998 as
`a continuation-in-part of U.S. application Ser. No. 09/082,
`558, filed May 21, 1998, claiming priority from French
`applications Nos. 97 12382, 98 00873 and 98 03707, filed
`Oct. 3, 1997, Jan. 22, 1998 and Mar. 20, 1998, respectively;
`WO-A-99 18214; and the U.S. applications of Audonnet et
`al. and Bublot et al., Ser. Nos. 60/138,352 and 60/138,478,
`respectively, both filed Jun. 10, 1999 ("DNA VACCINE(cid:173)
`PCV", and "PORCINE CIRCOVIRUS RECOMBINANT
`POXVIRUS VACCINE"). Reference is additionally made to
`each of the documents cited in the text and in the record or
`prosecution of each of the aforementioned U.S. and French
`applications, including without limitation WO 98/03658,
`published Jan. 29, 1998 from PCT/FR97/01313, filed Jul.
`15, 1997 and designating the U.S. and claiming priority from
`French application 96 09338, filed Jul. 19, 1996 (the U.S.
`continuation-in-part of PCT/FR97/01313 being U.S. appli(cid:173)
`cation Ser. No. 09/232,468, filed Jan. 15, 1999 for "POLY(cid:173)
`NUCLEOTIDE VACCINE FORMULA AGAINST POR(cid:173)
`CINE REPRODUCTIVE AND RESPIRATORY 30
`PATHOLOGIES"). Mention is also made of PCT W099/
`29717. Each of the aforementioned U.S., PCT and French
`applications (including parenthetically), and each document
`cited in the text and the record or prosecution of each of the
`aforementioned U.S., PCT and French applications
`(including parenthetically) ("application cited documents")
`and each document cited or referenced in each of the
`application cited documents, is hereby incorporated herein
`by reference; and, technology in each of the aforementioned
`U.S., PCT and French applications (including
`parenthetically), and each document cited in the text and the
`record or prosecution of each of the aforementioned U.S.,
`PCT and French applications (including parenthetically) can
`be used in the practice of this invention.
`
`OBJECTS AND SUMMARY OF THE
`INVENTION
`
`45
`
`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 encom(cid:173)
`pass live attenuated or inactivated PCV-2, or subunit(s) from
`35 PCV-2 obtained by in vitro expression or by extraction, or
`fragment(s) comprising at least one epitope of interest which
`can be obtained by chemical synthesis or by in vitro recom(cid:173)
`binant expression, as well as recombinant vector(s) com(cid:173)
`prising and expressing in vivo sequence(s) or fragment(s) or
`40 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.
`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 mul(cid:173)
`tisystemic wasting syndrome and/or pathologic sequelae
`50 including but not limited to post-weaning multisystemic
`wasting syndrome; and, methods for formulating such com(cid:173)
`positions and uses of a PCV-2 immunogen (which compo(cid:173)
`sitions can also include a porcine parvovirus (PPV)
`immunogen, wherein when recombinant vector expression
`55 is used, the vector can co-express both the PPV and the
`PCV-2 immunogens, inter alia) for formulating such com(cid:173)
`positions.
`Another object of the invention is the isolation and
`characterisation of new PCV-2 strains identified 1103
`60 (1103/1 P.2) and 1121 (1121!1 P.l), 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
`65 pathologic sequelae associated therewith.
`The invention provides also for inoculation of female pigs
`(e.g., sows, gilts) with a composition comprising a (at least
`
`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 pre(cid:173)
`paring such compositions or for performing such methods,
`inter alia.
`Various documents are cited in this text. Citations in the
`text can be by way of a citation to a document in the
`reference list, e.g., by way of an author(s) and document
`year citation to a document listed in the reference list, or by
`full citation in the text to a document that may or may not
`also be listed in the reference list.
`There is no admission that any of the various documents
`cited in this text are prior art as to the present invention. Any
`document having as an author or inventor person or persons
`named as an inventor herein is a document that is not by
`another as to the inventive entity herein. All documents cited
`in this text ("herein cited documents") and all documents
`
`CEV Exhibit 1001_010
`
`

`

`US 6,517,843 Bl
`
`3
`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 multisys(cid:173)
`temic 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(cid:173)
`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 inocu(cid:173)
`lated 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 also comprehends immunogenic or
`vaccine compositions 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. An immunogenic (or immunological) compo(cid:173)
`sition elicits an immunological response-local or systemic.
`A vaccine composition elicits a local or systemic protective
`response. The terms "immunological composition" and
`"immunogenic composition" include a "vaccine composi(cid:173)
`tion" (as the two former terms can be protective
`compositions). The composition can comprise a PCV-2
`immunogen (which composition can. also include a PPV
`immunogen).
`And, the invention further comprehends uses of a PCV-2
`immunogen (which composition can also include a PPV
`immunogen) to formulate an immunogenic or vaccine com(cid:173)
`position for preventing or treating myocarditis and/or abor(cid:173)
`tion 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 intrau(cid:173)
`terine 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
`
`4
`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
`5 pleuropneumoniae, E. coli, Bordetella bronchiseptica, Pas(cid:173)
`teurella multocida, Erysipelothrix rhusiopathiae,
`Pseudorabies, Hog cholera, Swine Influenza, and Porcine
`Parvovirus (PPV).