WO2007094893A2 - Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs - Google Patents

Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs Download PDF

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Publication number
WO2007094893A2
WO2007094893A2 PCT/US2006/062662 US2006062662W WO2007094893A2 WO 2007094893 A2 WO2007094893 A2 WO 2007094893A2 US 2006062662 W US2006062662 W US 2006062662W WO 2007094893 A2 WO2007094893 A2 WO 2007094893A2
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WO
WIPO (PCT)
Prior art keywords
group
antigen
pcv2
pigs
porcine
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Application number
PCT/US2006/062662
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French (fr)
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WO2007094893A3 (en
WO2007094893A9 (en
Inventor
Michael B. Roof
Phillip Wayne Hayes
Marc Eichmeyer
Greg Nitzel
Merrill Schaeffer
Original Assignee
Boehringer Ingelheim Vetmedica, Inc.
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Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=38371964&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2007094893(A2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to AU2006338182A priority Critical patent/AU2006338182B2/en
Priority to CA002635430A priority patent/CA2635430A1/en
Priority to MX2016013804A priority patent/MX356667B/en
Priority to PL16185663T priority patent/PL3127551T3/en
Priority to ES06850366.3T priority patent/ES2625903T3/en
Priority to JP2008548847A priority patent/JP5739602B2/en
Priority to MX2008008311A priority patent/MX2008008311A/en
Priority to EP16185663.8A priority patent/EP3127551B1/en
Priority to EP20184598.9A priority patent/EP3766518A1/en
Priority to KR1020147001544A priority patent/KR20140019479A/en
Priority to DK06850366.3T priority patent/DK1976558T3/en
Priority to EP06850366.3A priority patent/EP1976558B1/en
Priority to MX2012007937A priority patent/MX343001B/en
Priority to CN200680053578.7A priority patent/CN101426522B/en
Priority to MX2013012700A priority patent/MX338626B/en
Priority to BRPI0620823A priority patent/BRPI0620823B1/en
Application filed by Boehringer Ingelheim Vetmedica, Inc. filed Critical Boehringer Ingelheim Vetmedica, Inc.
Publication of WO2007094893A2 publication Critical patent/WO2007094893A2/en
Publication of WO2007094893A3 publication Critical patent/WO2007094893A3/en
Publication of WO2007094893A9 publication Critical patent/WO2007094893A9/en

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Definitions

  • the present invention relates to the use of an immunogenic composition comprising a porcine circ ⁇ vints type 2 (PCV2) antigen for treatment of several clinical manifestations (diseases).
  • PCV2 porcine circ ⁇ vints type 2
  • the present invention is concerned with an immunological composition effective for providing 3ii im ⁇ me response that reduces, or lessens the severity, of the clinical symptoms associated with PCV2 infection.
  • the immunological composition comprises a recombinant!)- produced antigen of PCV2.
  • the PCV2 antigen is a recombinant!)' produced protein encoded by one of the open reading frames (ORFs) in the PCV2 genome.
  • the antigen is PCV 2 ORF2 protein.
  • Ae present invention is concerned with an immunological composition effective for treatment of clinical sypmt ⁇ ms associated with PCV2 infections in swine receiving ⁇ he immunological composition, and wherein the composition comprises the prolan expressed by ORF2 of PCV2.
  • Another aspect of the present invention is the use of any of the compositions provided herewith as a medicament, preferably as a veterinary medicament, even more preferably as a vaccine.
  • the present invention also relates to the use of any of the compositions described herein, for the preparation of a medicament for reducing or lessening the severity of clinical symptoms associated with PCV2 infection.
  • the medicament is for the invention of a PC V2 infection, even more preferably in swine.
  • a further aspect of the present invention relates So a process for the production of a medicament, comprising an immunogenic composition of PCV2 for the treatment of several clinical manifestations.
  • PCV2j Porcine circovir ⁇ s type 2
  • PCV2j is a small ( 17 -22 rim in diameter), icosahedral, non-enveloped DMA virus, which contains a single-stranded circular genome.
  • PCV2 shares approximately 80% sequence identity with porcine circovirus type S. (PCVI).
  • PCVI porcine circovirus type S.
  • PCV K which is generally non-virulent, swine infecied w ith PCV 2 exhibit a syndrome commonly referred to as Post-weaning Muitisysteinic Wasting Syndrome (PMWS).
  • PMWS is clinically characterized by wasting, paleness of the skin, anth.iiftj.ness, respiratory distress, diarrhea, icterus, and jaundice.
  • Cw tent appioaches to treat PCV2 infections include DW-ba ⁇ ed vaccines such as thobc described in I S Patent ⁇ o 6 7» ⁇ 02 ⁇ iio«e ⁇ ei bitch uieu ⁇ cs kne beui nieffcctiw st confcii ing pioleciivt, innnunits against PCV 2 infection ot reducing lessening tin.
  • the immunogenic compositions used herein appear to be safe w hen administered to voting pigs prefesabh to pigs, not oldei than H weeks of age more piefetabh not oidei than ⁇ > weeks of age c ⁇ en more preferabh not older tlia ⁇ i ⁇ weeks most prefcrabh not oldei than 2 weeks ⁇ Jtematt ⁇ eh it is psefened that the admmistiation of the immunogenic compositions of the present inv ention occ ur w ithin at feast 2 and prefer ⁇ bh within at lcaM " * ⁇ Kccks of c ⁇ os>urc to ⁇ ⁇ iniluit PCV According to a futthcr embodiment the immunogenic compositions used herein for
  • Huis one asjKct of the pte ⁇ cni nnctitioo relates to she use of an immunogenic composition comprising POV2 antigen prefesabh recombinant PCV2 antigen and nioic prcfcrabh PC ⁇ r 2 ORF2 protein as pio ⁇ idcd herewith for the preparation of a medicament foi the prevention lessening ⁇ m ⁇ or reduction of h tnphadenopaUix .
  • said medicament is cffcctn e for the prex ention, lessening and/or reduction of h itiphadcnopath ⁇ h mphoid depletion and or tnultmucJcatcd gtanl hssUocx tes, associated wtth PC V2 infections, in sw ine SuH more piefeiabh & ⁇ d mcdicarncnt is.
  • ciTccti ⁇ c for the prev ention leshimg and or reditction of i ⁇ inphadenopatln , hmphoid depiction and/or mulU ⁇ uclealed/giant hist ⁇ oe>tes> associated w ith PC V 2 mfecuons m pigs when administered to pigs not oSdes than J 5 weeks of age mote picferabh not older than t> weeks of see, e ⁇ en more prefcrabK not older than 3 ⁇ ecks mid most piefciabh uot oldei than 2 ⁇ eeks.
  • Anothei aspect of the present intention relates Io a method fot the Ueatmcnt of i ⁇ mpruidc ⁇ opalhv ly mphoid depiction and/or muH ⁇ nucJe ⁇ itcd''giant histiocy tes in swi ⁇ c computing the administration of an immunogenic composition as prm idcd herew ith to a pig said immunogenic composition comprising a PC ⁇ '2 antigen preferabK a icc-omfamant PCV2 antigen and ⁇ ioieptcfciabh PCV2 0RF2 protein
  • ⁇ ct anotl ⁇ i aspect the present n ⁇ ention pio ⁇ jclcs ⁇ inelliod fot the Ueatmcm of Is niphadeiiopathv K mphoid depletion and/ot mtiitiiHJcicatvd gtsnt h ⁇ s
  • one aspect of the present invention relates, to the use of an immunogenic composition c-oinpti&ing PCV2 antigen, prefctabh a recombinant PCV2 antigen and more prcfetablv PCV2 ORF2 ptoicm as provided herew ith foi the prepaiation of a medicament fot the prevention lessening and'Or icdu ⁇ ion of h niphadcnopathv in combination w ith one or a multiple of the following sy mptoms m pigs (1 > intcisliUal pneumonia with mte: lobular edema, (2) cutaneous pallor or tctcr ⁇ s 0) mottled ati opine hveis.
  • said medicament is effect ⁇ c foi the prevention, lessening and/ot reduction of h mphadenopatln in combination with one or a multiple of the folSou sng sy mptoms associated w ith PCV2 infection in pigs ( 1 ) interstitial pneumonia w ith interlobular edem ⁇ i (2) cutsiieou*; ps ⁇ Jor or ictcras (3 i mottled atropine lncrs (4 ⁇ gastric aleeis ( ⁇ > ncph ⁇ t ⁇ s and (( ⁇ rcpioduct ⁇ c diwtder ⁇ e g abortion ⁇ tillbmks mummies etc Arcoidmg to a further aspect said medicament is effective for the
  • ⁇ t is preferred that the ⁇ idinmis.tiation of the immunogenic compositions of the present invention occtn w ithin at least 2 and ps efcrabh w tll ⁇ n at least 3 weeks of e ⁇ pos ⁇ ire to ⁇ ti ulent PC Y
  • the picseni mvcnuon also ielatcs to a method fot the treatment of l ⁇ mphadcuop ⁇ iliv in combination with one oi a multiple of the following symptoms, in pigs O) interstitial pneumonia with interlobular edema, (2) cutaneous pallor or icterus. (3) mottled atrophic livers. (4) gastric ulcers . (5) nephritis and ⁇ 6 ⁇ reproductive disorders, e.g. abortion.
  • the present invention also relates to a method for the treatment of iyniphadenopathy in combination with one or a multiple of ⁇ he following symptoms associated with PCV 2 infection in pigs: (1 ) interstitial pneumonia wish interlobular edema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers. (4) gastric ulcers. (51 nephritis and ⁇ >) reproductive disorders, e.g. abortion, stillbirths.
  • said method comprising ⁇ he administration of an immunogenic composition comprising PCV2 antigen, prefereably recombinant PCV2 antigen and more preferably PCV2 0R.F2 protein, as provided herewith, to a pig.
  • said treatment results in the lessening or reduction of the lymphadenopathy, and one or multiple of the following symptoms associated wish PCV2 infection in pigs: Cl) interstitial pneumonia with interlobular edema.
  • nephritis and (6) reproductive disorders e.g. abortion, stillbirths, mummies, etc.
  • said methods for treatment further comprise administration of the immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen and snore preferably PCV2 ORF2 protein, as provided herein, to pigs not older than 15 weeks of age, more preferably not older than 6 weeks of age. even more preferably not older than 3 weeks, and most preferably not older than 2 weeks, Alternatively, it is preferred that the administration of the immunogenic- compositions of the present invention occur within at least 2 and preferably within at least 3 weeks of exposure to virulent PC: V.
  • an immunogenic composition comprising PCV antigen, preferably recombinant PCV2 antigen and moie prcierabh PC V2 OR.P2 ptotem as prov ided heicwnh can aho reduce or lessen Pta ⁇ ikc lesions notmaih known to be associated with Law soma mtraccllula ⁇ s in j ections (Ileitis)
  • one aspect of the present imcniion relates, to tlic use of an immunogenic t (imposition comprising PCV2 antigen prtfeidbh itcoinbinanl PC ⁇ 2 antigen and more preferabK PC ⁇ 2 ORF2 piotcm as pro ⁇ ided herew ith fbt the ptepaiatton of a medicament for the pio cntton lessening tlic ⁇ ,e ⁇ c ⁇ t ⁇ of and ot icd ⁇ ctton o!
  • Pia like lestons iiotinslh known (o K, d ⁇ soc i ⁇ icd w ith L ⁇ w ⁇ oma intiaccihilaiis infections in w uic According (o a fin lher js>pt,ci said medicament is Uftxih c fot the p ⁇ c ⁇ cniion lessening of the ⁇ e ⁇ citt ⁇ of an ⁇ ot iedu ⁇ tion of Pi ⁇ liie lesions normaih known to be a ⁇ o*, ⁇ atcd w ith Law soma intraceiiulans lufc ⁇ ions when ⁇ dnjimstcjcd to pigs not oido jlisn 1 *> weeks of age mote prefeiabh not oldci than 6 weeks of sgc e ⁇ en more pfcferabh not older than 3 weeks and most prdcrnbh not
  • nho relates to a method for the treatment of Pta iikc iesiom noin ⁇ ih know n Io be d ⁇ ooiaied nith Law soma mtu ⁇ e!iu!d ⁇ »> mfeuions said method L-ompi tsing the admiiustratiosi of an immiisiogenit composition ctwnpming PC ⁇ 2 antigen picicrabh iccombinant PCV2 antigen and snore preferabh PCV 2 ORF2 piotcm as proMdcd hetein Io ⁇ i pig Pidenibh wn
  • the immunogenic composition is an immunogenic composition
  • the immunogenic composition as used herein is effective for inducing an immune response against PCV2 and prexenting, reducing and/or lessening the sexerity of the clinical symptoms associated with PCV2 infection.
  • the composition generally comprises at ieast one PCV2 antigen. Unless defined otherwise, ail technical and scientific terms used herein have the same meanings as common!) understood by one of ordinary skill in She art to which this invention belongs.
  • the terns "immunogenic composition” as used herein refers to any pharmaceutical composition containing a PCV2 antigen, which composition can be used to prevent or treat a PCV2 infection-associated disease or condition in a subject.
  • a preferred immunogenic composition can induce, stimulate or enhance the immune response against PCV2. The term thus encompasses both summit immunogenic compositions, as described below, as well as compositions containing whole killed or attenuated and/or inactivated PCV 2.
  • sidnmit immunogenic composition refers to a composition containing at least one immunogenic polypeptide or antigen, but not all antigens, derived front or homologous to an antigen from PCV2. Such a composition is substantially free of intact PCV2.
  • a "subunit immunogenic composition” is prepared from at least partially purified or fractionated (preferably substantially purified) immunogenic poly peptides from PCV2. or recombinant analogs thereof.
  • a subutrit immunogenic composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from FC ⁇ 2 or m fractionated from ⁇ prefei rcd immunogenic subunjt composition comprises thec PC V2 ORF2 protein as described below
  • composition ot ⁇ acemc is the development m the hos>t of a celhilat and'' 01 antitxxh -mediated immune response to the compos it ion ot utc ⁇ ne of iiiicicsl Csualh , an "immune response" inc ludes but is not limned to one 01 more of the follow ing effects the pioduciion ot activ ation of antibodies.
  • helper T edls helper T edls, siippicssor T cells, and ni c ⁇ totn ⁇ tc T cells and/oi j d T cells, dnected spec if ⁇ calK to an antigen or antigens included m the composition ot ⁇ ace me of tntetcsi Ptef ⁇ obh , the host w ili dispisn etthct a jiierapcutic ot piotcctiv e uumunol ⁇ gtcal response such thai iesutance to new infection will be enhanced and/oi the ciimcai se ⁇ et ⁇ n of the disease reduced
  • Such piotcouoti w ill be dctRonstraicd b ⁇ eithei a icduciton in immbei ot sc ⁇ ettt ⁇ of, or Kick of one Oi moi
  • immunogenic protein ot pol ⁇ peptide ot refers to an ammo a ⁇ d sequence which elicits a « immunological response as described abo ⁇ c ⁇ .n
  • uniminogcme piotcsn oi poh peptide, as used herein, includes the fuii-iength sequence of an ⁇ P( ⁇ ' 2 proteins, analogs thereof, or immunogenic fragments thereof
  • the terra "immunogenic fragment” iefcis to a fiagmetit of a piotcm winch includes one or more epitopes, and thus choits the jiumunoJogical response described abo ⁇ e
  • Such fragments can be sdenufsed using am sntmbei of epitope mapping techniques, well known in the att See e g , Epitope Mapping Piotocols in Methods in Molecular Bioiog> , Voi
  • conformational epitopes ate readily identified by determining spatial conformation of amino acids such as by. e.g., x-ray crystallography and 2-diniensional nuclear magnetic resonance. See, e.g.. Epitope Mapping Protocols, supra.
  • Synthetic antigens are also included within the definition, for example, polycpiiopes, flanking epitopes, and other recombinant or synthetically derived antigens. See. e.g.. Bergmann et ai. (!993 ) Eur. J. Immunol. 23:2777-27S l ; Bergmann et al. (! 996). J. Immunol 157:3242-3249: Suhrbier. A. (1997). Immunol, and Cell Biol. 75 402-408; Gardner et al . ( 1998) 12th Worid AIDS Conference, Geneva. Switzerland. June 28-Juiy 3. 1998
  • an immunogenic composition thai induces an immune response and. more preferably, confers protective immunity against the clinical signs of PC.V2 infection, is provided.
  • the composition .most preferably comprises the poly peptide, or a fragment thereof, expressed by ORF 2 of FCVZ as the antigenic component of the composition.
  • PCV2 ORF2 DNA and protein, used herein for the preparation of the compositions and within the processes provided herein is a highly conserved domain within PCV2 isolates thereby, am PCV 2 ORF2 would be effective as the source of the PCV ORF2 DNA and/or polypeptide as used herein.
  • a preferred PCV2 ORF2 protein is that of SEQ ID NO. U .
  • a preferred PCV ORF2 polypeptide is provided herein as SEQ ID NO. 5, but it is understood by those of skill in the art that this sequence could vary by as imtch as 6-10% in sequence homology and still retain the antigenic characteristics that render it useful in immunogenic compositions.
  • the antigenic characteristics of an immunological composition can be, for example, estimated by the challenge experiment as provided by Example 4. Moreov er, the antigenic characteristic of a modified antigen is still retained, vUseis the modified ant J gen argue at least 7 ⁇ >%, prefesabh 80%.
  • a furthe rreferred PC ⁇ r 2 ORF2 poly peptide prov ided heret ⁇ is encoded b ⁇ the nucleotide sequences of SEQ ID NO 3 or SLQ IO NO 4
  • A is understood b ⁇ those of skill m the art that lhis> sequence could ⁇ ar> bj as much as b-20% m secjitence homolog) and still retam the antigenic chaiactc ⁇ siics that render it useful m immunogenic compositions In some forms a truncated ot substituted form ot fiag ⁇ nent of this.
  • P ⁇ 'C2 ORF2 poly peptide is used as the antigenic component in the composition Prcfeiabix such truncated or substituted forms, or fragments will comprise at least IS contiguous nucleotides from the full-length ORF2 nucleotide sequence, e.g. of SEQ 1I> NO: 3 or SEQ ID NO: 4. More preferably, the truncated or substituted formj. or fragments, vv ⁇ J! have at ieasi 30, more preferably at least 45, and still more preferably at least 57 contiguous nucleotides of the AiH- length ORF2 nucleotide sequence, e.g. SEQ ID NO: 3 or SEQ ⁇ D NO, 4.
  • Sequence Identity refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence Similarity, as determined by the match between sitings of such sequences. Upon such alignment, sequence identify is ascertained on a posilion-by-position basis, e.g., the sequences are " ⁇ identical" at a particular position if st that position, the nucleotides or amino acid residues are identical.
  • Sequence identity can be readily calculated by known methods, including but not limited to. those described in Computational Molecular Biology. Lesk. A. N., ed.. Oxford University Press. New York ( 1988), Bioeomputing: Informatics and Genome Projects. Smith. D.W.. ed.. Academic Press, New York (1993): Computer Analysis of Sequence Data. Fart I, Griffin. A.M. and Griffin. H. G.. eds.. Humana Press. New Jersey (1994); Sequence Analysis in Molecular Biology, von Meinge. G.. Academic Press (1987); Sequence Analysis Primer.
  • BLASTX ptogram is publicly available from NCBl and other sources (BLAST Manual, Ahschul, S. et al., NiCV! NLM NTH Scihesda. MD 20894, Altschul, S, F. et aL J. Molec. Biol., 2 15:403-41 U (1990), (he teachings of which are incorporated herein by -reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences.
  • nucleotide sequence having ai least, for example. 85%, preferably 90%, even more preferably 95% "sequence identity" to a reference nucleotide sequence it is intended ⁇ hat the nucleotide sequence of " the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 13, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • nucleotide sequence having at least 85%, preferably 90% in a polynucleotide having at least 85%, preferably 90%.
  • a polypeptide having a given amino acid sequence having at least, for example, 85V preferably 90%, e ⁇ en more prefcrabh 95% sequence identity to a reference ammo acid sequence it is intended that the gh en amino acid sequence of the pol y peptide is identical to the reference -sequence except that the gh cn poh peptide sequence nia ⁇ include up to 15. prefcrabh up Io 10, even more ⁇ referabh up Io 5 amino ac ⁇ l alterations per each 100 amino acids of the reference amino acid sequence In other words, to obtain a ghen p ⁇ K peptide sequence ha ⁇ ing at least 85%.
  • preferabK WV even more preferabK 95% sequence identi t yth a reference amino acid sequence, up to 15°b. preferabK up to 10%. even more preferabK up to 5% of the amino acid residues in the reference sequence ma> be deleted or substituted with another amino acid, or a number of ammo acids up to 15%, prcicrabh up to i ⁇ %. even more preicrablj up to 5% of the total number of ammo acid residues in the reference sequence ma> be inserted into the reference sequence.
  • residue positions w hich are not identical differ b> conserv ative ammo acid substitutions lkwe ⁇ er, eonsenatne substitutions arc not included as a match w lien determining sequence identit ⁇ ,
  • Sequence homology refers to a method of determining the rel ⁇ ledncss of wo sequences. " Jo determine sequence homologj , wo or more sequences arc optima Uj aligned, and gaps arc introduced if neccssan HOWC ⁇ C ⁇ .
  • sequence ideotit ⁇ ' ⁇ consen atnc amino acid substitutions are counted as a match when detcmiimng sequence homologj in other words, to obtain a polypeptide or poh nucleotide inn ing 95% sequence homologj n ith a reference sequence, K5')o, preferably 90%, e ⁇ e ⁇ more preJcrabl) 95% of (lie amino acid residues or nucleotides in the reference sequence must match or comprise a conscn am c substitution % ⁇ ith another amino acid oi nucleotide, or a number of ammo acids ot nucleotides up to i *% preferabl yp to J0% c ⁇ en more prefer ⁇ bh up Io ">% oi the total ammo aud residues 01 nucleotides not meludmg c-onsen am e substitutions in the reference.
  • the immunogenic composition as used herein also icicrs to a composition thai comprises PC V2 ORI 2 protein wheiem said PCV 2 OR! 2 piotcm is am one of those desL- ⁇ bed abo ⁇ e Prefeiabh said ?C ⁇ 2 DlU 2 protetn is
  • SLQ ID NO 9 SLQ lD NO 10 OJ SLQ i D NO 1 1 ii ⁇ a ⁇ poK peptide that is at least 80° ⁇ • liomologoits to the pohpepttde of 0 i «) am immunogenic poition of tiie poh peptides oi 0 and oi ii) i ⁇ i the immunogenic portion of HI) comprising at lu&t 10 contiguous ammo acsds included in the sequences of SLQ ⁇ D NO * SLQ ID NO 6 SLQ ID ⁇ O Q SFQ lD KO 10 or SFQ ID XO I l v) a poh peptide thai is encoded b ⁇ a DNA comprising the sequence of SBO
  • PCV2 ORF2 piotem is pimided in the immunological composition at an antigen inclusion IC ⁇ CI effective for inducing the dciucd uninunc response, nameh ted ⁇ cmg the incidence of lessening the se ⁇ erit ⁇ of ot prev enting one ot more cluneal stgns ics ⁇ ltmg from PCV2 infection
  • Prefesabh ⁇ he
  • PC V2 OR ⁇ -2 prolejn mcluston ic ⁇ cl ii at least ⁇ > 2 ⁇ g antigen mi of the final immunogenic composition (ug mJi more prefeiabh from abotit O 2 to abotit 400 ⁇ g ml, still moie preferabh from about O " * to about 200 ⁇ g ml, e ⁇ en inoic prefer abh from about O 35 to about 100 ⁇ g ml, still mose prefesabh
  • the OR.F2 antigen inclusion level is at least 0.2 ⁇ g/ PCV2 0RF2 protein as described above per dose of the final antigenic composition ( ⁇ g/dosc). More preferably from about 0.2 to about 400 ⁇ g/dose, still more preferably from about 0.3 to about 200 ⁇ g/dose.
  • PCV2 O.RF2 polypeptide used in the immunogenic composition in accordance with the present invention can be derived in. any fashion including isolation, and purification, of PCV2 O.RF2. standard protein synthesis, and recombinant methodology. Preferred methods for obtaining PCV2 OR.F2 polypeptide are provided in U S. Patent Application Serial No. 1 1/034,797, the teachings and content of which are hereby incorporated by reference. Briefly, susceptible cells are infected with a recombinant viral vector containing PCV2 O.RF2 DNA coding sequences.
  • PCV2 OR.F2 polypeptide is expressed by the recombinant virus, and the expressed PCV2 OR.F2 polypeptide is recovered from the supernate by filtration and inactivated by any conventional method, preferably using binary ethyjcnimine, which is then neutralized to stop the inactivatbn process.
  • the immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 protein described above, preferably in concentrations described above, and ii) at least a portion of the viral vector expressing said PCV2 ORF2 protein, preferably of a recombinant baculovirus.
  • the immunogenic composition can comprise i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ii ⁇ at least a portion of the viral vector expressing said PCV2 ORF2 protein, preferably of a recombinant bacuiov tr ⁇ s. and iii) a portion of the DCi culture supemate.
  • the immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ii) at least a portion of She viral vector expressing said PCV2 OR.F2 protein, preferably of a recombinant baculov ⁇ rus. and Hi) a portion of the cell culture; wherein about
  • the immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ti) at least a portion of the viral vector expressing said PCV2 ORF2 protein, iii) a portion of the cell culture, i ⁇ ) and inactivating agent to inactivate the recombinant viral vector preferably BEl, wherein about 90% of the components i) to iii) have a size smaller than 1 ⁇ ro.
  • BEl is present in concentrations effective to inactivate the baculovinis. Effective concentrations are described above.
  • the immunogenic composition as used herein also refers to a composition that comprises t) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, it) at least a portion of the vira! vector expressing said PCV2 ORF2 protein, iii) a portion of the ceil culture, iv) an inactivating agent to inactivate the recombinant viral vector preierabiy BET. and v) an neutralization agent to stop the inactivalion mediated by the inactivating agent, wherein about 90% of the coinpo.ne.nis i) to iii) have a size smaller than J.
  • the inactivating agent is BEL said composition comprises sodium thiosulfate in equivalent amounts to BEl.
  • the polypeptide ss incorporated into a composition thai can be administered to an animal susceptible to PCV 2 infection in preferred forms.
  • the composition inaj also include additional components known io those of skill in the art (sec also Remington's Pharmaceutical Sciences ( 1990), 18th cd.
  • the composition ma ⁇ include one or more ⁇ eterinarj -acceptable earners ⁇ s used herein, "a ⁇ etc ⁇ nar> -acceptable earner” includes an ⁇ and all solvents, dispersion media, coatings, adjin anls, stabilizing agents, dduenfe. piemen ath es. anubactei ial and antifungal agents, isotonic ageins, adsotption delaung agents, and the like
  • the immunogenic composition conjpns.es PC V2 ORF2 protein as piovjded herewith, preferabh m concentrations described above, which is mixed with an adjm ant. picferabh CarbopoL and pin siological salme
  • compositions used herein. can include known injectable, physiologically acceptable sterile sohitton*; For preparing a rcad> -Jo-usc solution for parenteral injection or mfusion.
  • aqueous isotonic solutions such as e g saline or corresponding plasma protein solutions, are readih available
  • the immunogenic and utccuie compositions of the present can include diluents, isotonic agents, stabilizers, or adjuvants.
  • Diluents can include nater. saline, dextrose. efhan ⁇ l.
  • Isotonic agents can include sodtuni chloride, dextrose, manmtoi. sorbitol, and lactose, among others.
  • Stabsls/ers include albumin and alkali salts of elirj lei ⁇ diamintetracetic acid, among others
  • ⁇ dju ⁇ anis can include aluminum hulrovule and aluminum phosphate, saponins e- g , Quil A. QS-21 (Cambridge Biotech Inc , Cambridge M ⁇ >. GPl- OjOO (Galer ⁇ ca Pharmaceuticals. Inc.. Birmingham. ⁇ Lh water-m-oil emulsion.
  • emulsion can be based in particular on light liquid paraffin oil (Euiopcan Pliatmacopca ij pe), isoprenoid oil such as &quaiaue oi squalene oil resulting from theoligoraerization of alke ⁇ es, in particular of isobuiene or decene; esters of acids or of alcohols containing a linear slkyi group, more particularly plant oils, ethyl oleate.
  • light liquid paraffin oil Euiopcan Pliatmacopca ij pe
  • isoprenoid oil such as &quaiaue oi squalene oil resulting from theoligoraerization of alke ⁇ es, in particular of isobuiene or decene
  • esters of acids or of alcohols containing a linear slkyi group more particularly plant oils, ethyl oleate.
  • propylene glycol dKcaprylate/caprate propylene glycol dKcaprylate/caprate
  • glyceryl trj-(capryiate/caprate) or propylene glycol dioleate esters of branched laity acids or alcohols, in particular isostearic acid esters.
  • the oil is used in combination with emulsifierss to form the emulsion.
  • the emuSsifiers are preferably noniotiic sinfaciatits, in particular esters of sorbitan, of niatinide (e.g.
  • anlrydro ⁇ iatnsit ⁇ l oleate of glycol, of poiyglycerol, of propylene glycol and of oleic, isostearic, ricinoieic or hydroxystearic acid, ⁇ vhich are optionally ethoxylsted. and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L 121.
  • an adjuvant is a compound chosen from the polymers of acrylic or Hiethacrylic acid and the copolymers of inaleic anhydride and aikenvl derivative.
  • Advantageous adjuvant compounds are the polymers of acrylic or r ⁇ ethacrylic acid which are cross-linked, especially with polyaikeny! ethers of sugars or polyalcohols. These compouads are known by the term carbomer (Phameisropa 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 hydroxy! groups. preferably not more than 8.
  • the hydrogen atoms of at least three hydroxy Is being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms.
  • the preferred radicals are those containing fromn 2 to 4 carbon atoms c g ⁇ m ⁇ ls. aSh ls and othei etln lerucaJh unsaturated gjoups I he misahnated radicals ma ⁇ tiiemseh es contain other subsUHients.
  • sun ⁇ blc ⁇ djii ⁇ anis include but aic not intuted to ⁇ he R ⁇ B1 ad]u ⁇ anl s ⁇ stera (Ribi iiic ), Biock co-poh tnci (,(MR ⁇ Atlanta GA), SAF-M (Ch ⁇ on, Fmowilie C A) mo ⁇ ophcsphoi ⁇ !
  • Prcf ⁇ tabh the adjuv ant is added m an amount of about 1Ou ⁇ g to about H) mg pei dose Hcsi more prefeiabh ⁇ he adpj ⁇ ant is added in an amount of about 100 ug to about 10 mg pei dose Ih en more prcferabh the adjmant is added in an amount of about 500 ⁇ g to about * mg per dose ⁇ en more preferabh .
  • ihss imtiuinogctnc composition further unnp ⁇ ses s phanwseeui ical acceptabSe sail prcierabh a phosphate salt m phv sioiogicalh acceptable concentrattons Picicrabh the pH of said immunogenic composition is adjusted to a pin M ⁇ iogical pli mediimg between about 6 5 and
  • the immunogenic composition as used hetein also refei s to a composition thai comprises pes one mi S) at least 1 O ug of PC V2 ORi 2 piotcm desc ⁇ lx'd abt ⁇ e ii) at least a [X)HiOt) of bacuioMrus c ⁇ ptess)t)g said PC V 2 0RF2 protein iii) a pouion of the cell uiltute iv ) tilxnft 2 So 8 m ⁇ i BFT - ⁇ ) sodmni thiosulfate in cqti ⁇ aknl amounts, to BFl and ⁇ i) about !
  • the immunogenic compositions can further include one or more other immunoniothilatory agents such as. e.g.. interieiikiiis. interferons, or other cytokines.
  • the immunogenic coin positions can also include G ⁇ ntaniiein and Merthioiate.
  • compositions comprising from about 50 ⁇ g to about 20(H) ⁇ g of adjuvant and preferably about 250 ⁇ g/ ml dose of the vaccine composition.
  • the immunogenic composition as iised herein also refers to a composition that comprises from about l ug/ml to aboui 60 ⁇ g/nil of antibiotics, and more preferably less than about 30 ⁇ g/nil of antibiotics.
  • the immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ti) at least a portion of the viral vector expressing said PCV2 ORF2 protein, iii) a portion of the cell culture, iv) an inactivating agent to inactiv ate the recombinant viral vector preferably BEl. and v) an neutralization agent to stop the mactivation mediated by the inactivating agent, preferably sodium thiosulfate in equivalent amounts to BEi; vi) a suitable adjuvant, preferably Carbopol 971 in.
  • PCV2 ORF2 protein was highly stable over a period of 24 months, ⁇ t has also been found the immunogenic compositions are very effective in reducing She clinical symptoms associated with PCV2 infections. Tt was also discovered, that the immunogenic compositions comprising the recombinant baculovirus expressed PCV2 ORF2 protein as described above, are surprisingly more effective ⁇ ian an immunogenic composition comprising the whole PCV2 virus in an inactivated font), or isolated viral PCV 2 ORF2 antigen. In particular, it has been surprisingly found, tiiat the recombinant baciil ⁇ virus expressed PCV2 ORf ; 2 protein is effective in very low concentrations., which means in concentrations up to 0.25 ⁇ g/dose. This unexpected high immunogenic potential of the PCV2 ORF2 protein is increased by CarbopoL Examples 1 to 3 disclose in detail the production of PCV2 GR F2 comprising immunogenic compositions.
  • the immunogenic composition as used herein also refers to Ingelvac i. CireoFLEX" * 1 . ⁇ Bochringer Tnge ⁇ heira Vcimcdica. Inc., St Joseph, MO, USA), CircoVaeOt- (Medal SAS, Lyon, France! CircoVenl (intervet inc.. Miiisboro. DE. USA), or Sitvaxyn PCV-2 One Dose,f (Fort Dodge Animal Health, Kansas City . RA, USA.).
  • the composition according to the invention may be applied intradenrially, intratrachealh", or iniravagmalh'.
  • the composition preferably may be applied i ⁇ ramnscularly or intranasally. most preferably intramuscular! Iy, ⁇ an ani ⁇ ia! body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous or by direct injection into target tissues.
  • intravenous, intravasc ⁇ iar, intramuscular, intranasal, intraarterial intraperitoneal, oral or intrathecal ro ⁇ tci are preferred.
  • a more local application can be effected subcutaneouily, intradermals, intracutaneous! ⁇ ?, intracardtally, ijrtralobaHy.
  • compositions according to the invention may be administered once or several times, also iittentuttcnth fos instance on a dash basis, for several d ⁇ s necks or months and in different dosages.
  • Prdcrabh at least one dose of the immunogenic compositions as described abov e is intramuscula administered to the subjec t need ⁇ heieof ⁇ t cording to a Au the: ⁇ i>>pcc l ⁇ the PC ⁇ -2 antigen ot the immunogenic composition contpt isi ⁇ g an ⁇ such PCV-2 antigen as desc ribed abmc is formulated and administered in one ( I I ml pet dose
  • pjcferablv in combmauon with C'atbopol can be lnrihcr confiimcd In the sdrainistr ⁇ uoii of the lngc! ⁇ ⁇ k, PRRS ⁇ L ⁇ • ⁇ i(,C)t ⁇ ⁇ i.ec Fxampic >) PC V 2 cluneal signs and disease manifesiations atv greajK magnified when PRRS wfcctton is ptescnt Ho ⁇ e ⁇ ei the immunogenic compositions *; and ⁇ a «.cjnaiio» sitategies ss prm tded herew ith Serened th « cfleet great h and more than expected In other voids an unexpected sujcrgistic eftect was observed when animals pjdenibh piglets were treated w nh am of the PC ⁇ 2 ORF2 immunogenic, compositions as pro ⁇ idc
  • I-igtiic 1 is> a schematic flow diagram of a piefcncd construction of PC ⁇ r 2 ORP2 recombinant KIUSICA iru& and
  • Ligs 2a and 2b are each schematic How diagrams of how to produce one of the compositions os «.d in accordance v ⁇ ith the present invention DE TAILED DESCRIP T ION OF THE PREF ERRED EMBODIMENT S
  • the following examples set forth psefcned materials and pioocduies in accoi dance vwih the ⁇ icscnt im cmion
  • am methods and materials similar or equivalent to those described herein can be uv,d in the piactice or testing of (ho present iincnjinn the prcfct tcd inelhixk dev ices and materials aic now desc ribed
  • liowev ci that these examples arc provided b ⁇ % ⁇ a ⁇ of illusitaiion onK and noil ⁇ riji ilierem should be deemed a limitation upon (he otcwll bcopc of the intention
  • the p ⁇ 1 1392 pln ⁇ tntd ootitsitijug the PCV 2 ORF2 gene was designated N4? ⁇ 064 ⁇ and then co-iroiisfcoicd with BaculoGoldJC (BD Bioscicnces Phomiiugcn) baciiim iius D ⁇ ⁇ into Sf- inscci ecus.
  • PC ⁇ r 2 ORf 2 antigen as. dcteoied b ⁇ poh clonal serum or monoclonal antsbodses m an mdu ca Ouorcsceiit anUbtxh assa ⁇ Addiuonalh the tdenittj of liic PC ⁇ r 2 ORF2 ba ⁇ tkn inis ⁇ a ⁇ > confmncd b ⁇ ⁇ -temimal amino nct ⁇ sequencing
  • the PCX 2 0RF2 bauiio ⁇ uui MSX was also tested for pu ⁇ ft in accoidance -v ⁇ «h 9 C f R 1 P 27 (e) 1 13 28 and 3 13 5 1 ⁇ Lach iccombu ⁇ tn bae ⁇ kn jrus seeded into lite spinner flasks, had van ing multip ⁇ femes of infection (MOIs) Flask 1 was seeded w ith 7 ⁇ 2m ⁇ of 08H
  • the flasks After being seeded with (he baculo virus, the flasks were then incubated at 2? & 2 X for 7 days and were also agitated at l ⁇ l)rpm (hiring that time. The flasks used ventilated caps to allow fot air flow. Samples from each flask were taken every 24 hours for the next 7 days.
  • each sample was eentrifuged. and both the pellet: and the supernatant were separated and then mieroftiiered through a 0.45-1 ,0 ⁇ m pore size membrane.
  • the resulting samples then had the amount of OR.F2 present within them quantified via an ELTSA assay.
  • the ELlSA assay was conducted with capture antibody Swine anti- PCV2 Pab IgG Pr ⁇ t G purified (diiuted 5 :250 in PBS) diluted to 1 :6000 m 0 05 M Carbonate buffer (p K 9.6). 100 ⁇ L of the antibody was then placed in ⁇ lie wells of ⁇ he inictrotiter plate, sealed, and incubated overnight at 37 ⁇ C. The plate was then washed three times with a wash solution which comprised 0.5ml.. of Tween 20 (Sigma, Si Louis. MO).
  • the sample was then diluted at a 1:240 ratio and a 1 :480 ratio, and 100 ⁇ L of each of these diluted samples was then added to one of the top welis on the dilution plate (i.e. one top well received 100 ⁇ L of the 1:240 dilution and the other received 100 ⁇ L of the 1 :480 dilution).
  • Serial dilutions were then done for the remainder of the plate by removing K ) O ⁇ L form each successive well and transferring it to the nest well on the plate. Each well was mixed prior to doing the next transfer.
  • the test plate washing included w ashmg the plaic three tmses with the wash buffer.
  • the plate was then sealed and incubated for an hour al 37X before being washed three more times w ith the wash buffer.
  • the detection antibod> used was monoclonal amibod ⁇ to PCV ORF2 St w as diluted to ! 300 in diluent solution, and 100 ⁇ L of the diluted detection aiitiboch w as then added to the wells
  • the plate was then scaled and incubated dor an hour at 3 T 5 C before being washed three times w ith the wash buffer.
  • Conjugate diluent was then prepared b> adding normal rabbit scr ⁇ m (Jackson Immuuorc search. West Gtwe, PA) to the diluent solution to ⁇ % concentration.
  • Conjugate antibod> Goat anti-mouse (H + ] )-HRP (Jackson imnninorescareh) was diluted in the conjugate diluent to L K)J)Oo. l oo ⁇ L of the diluted conjugate antibod> was then added to each of the veils The plate was. then sealed and incubated for 45 mmutes at 3? ⁇ C before being washed three times with the wash buffer. 100 ⁇ L. of substrate (TMB Peroxidase Substrate, Kirkgaard and Pern. Laboratories (KPLs. Gaithcrsbcrg.
  • the iiask was then incubated at 27"C for a total of 6 das s After incubation, the fiask was then eentrifiiged and three samples of the resulting supernatant were harvested and inactivated. The supernatant was inactnated b> bringing its temperature to 37 -*- 2 0 C.
  • EXAMPLE 3 This example demonstrates that the present invention is scalable from small scale production of recombinant PCV2 OR.F2 to large scale production of recombinant PCV2 ORF2.
  • 5,0 x Mf cells/ml of SF> cells/m! in 700OmL of ExCeIl 420 media was planted in a 2000OmL ⁇ pplikon Bioreactor. The media and cells were then incubated at 27°C and agitated at !00RPM for the nesX 68 hours.
  • 4i,3mL of PCV2 ORF2 Baculovi ⁇ is MSV+3 was added to 7000ml.. of ExCeIl 420 medium.
  • vORF2 isolated vira lRF2: rORF2 ::: iecombiniint h ⁇ ctilov ⁇ us ex])resse_! ORP2: ktllec! whole cet! virus ⁇ PCV2 virus grown in suitable cell culture
  • Groups i through 7 were treated with assigned vaccines. Piglets in. Group 7 were given a booster treatment on Day 14. Piglets were observed for adverse events and injection site reactions following vaccination and on Day 19, piglets were moved to the second study site. At the second study site. Groups S -S were group housed in one buiiding vhiie Group 9 was housed in a separate building. All pigs -received keyhole limpet hemoevaniti (KLH (/incomplete Freund ⁇ s adjuvant (ICFA) on Days 21 and 27 and on Day 24. Groups 1-8 were challenged wish a virulent PCV2.
  • KLH keyhole limpet hemoevaniti
  • Pre- and post-challenge blood samples were collected for PCV2 serology. Post- challenge, body weight data for determination of average daily weight gain (ADWG). and clinical symptoms, as well as nasal swab samples to determine nasal shedding of PCV2, were collected. Oft Day 49, all surviving pigs were necropsied, kings were scored for lesions, and selected tissues were preserved in formalin for imtmmoMstotliemtstsy (IHC) testing at a lster date.
  • IHC imtmmoMstotliemtstsy
  • CDCD pigs 9 to 14 days of age on Day 0.
  • PCV2 ⁇ FA titers of sows were ⁇ 1 : 1000.
  • serologic status of sows were from a known PRRS- negative herd.
  • Twenty-eight (28) sows were tested tor PCV2 serological status.
  • sows had a PCV2 titer of ⁇ 1000 and were transferred to the first study site.
  • One hundred ten ( 1 10) piglets were delivered by cesarean section sisrgeries and were available for this study on Day -4.
  • On Day -3, J 08 CDCD pigs at the first study site were weighed, identified with ear tags, blocked by weight and. randomly assigned to 1 of 9 groups, as set forth above in table 4.
  • foliows Group 1 was designed to administer ImI of Mtal ORF2 ⁇ 1)RF2) containing 16 ⁇ g ORF2/m ⁇ This was done b ⁇ miving 10 24 ml of urai ORF2 (256 ug/25 ⁇ g/ml - 10 24 ml ⁇ ORF2> w sth 3 2 m ⁇ of 0 5"i> Catbopol and 2 ⁇ 6 ml of phosphate buflered saline at a pH of 7 4 This produced l t> ml of formulation for group 1 Group 2 was designed to ddmimstcr Im!
  • Group 5 was designed to administer ImS of rORF2 containing S ⁇ g rORF2/ml, This was clone by mixing 1.11 ml of rORF2 (256 ⁇ g/230 ⁇ g/ml ⁇ i. i l ml rORF2) with 6.4 ml of 0.5% Carbopol and 24.49 ml of phosphate-buffered saline at & pH of 7.4. Tlits produced 32 mi of formulation for group 5.
  • Group 6 was designed to administer Im! of rORF2 containing S ⁇ g rORF2/ml. This ⁇ va& done by mixing 0.56 ml of iORF2 ( !
  • Animals were housed at the first study site m individual cages in five rooms from birth to approximately 5 1 days of age (approximately Day D of the study). Each room was identical in layout and consisted of stacked individual stainless steel cages with heated and filtered air supplied separately to each isolation unit. Each room had separate heat and ventilation, thereby preventing cross-contamination of air between rooms. Animals were housed in two different buildings at ⁇ lie second study site. Group 9 (The Strict negative control group) was housed separately in a converted finisher building and Groups 1 -8 were housed in converted nursery building. Each group was housed s.u a separate pen (1 .1-12 pigs per pen) and each pen provided approximately 3.0 square feet per pig. Each pen was on an elevated deck with plastic slatted doors. A pit below the pens served as a holding tank for excrement and waste. Each building had its own separate heating and ventilation systems, with little likelihood of cross-contamination of air between buildings.
  • pigs assigned to Group 7 received 2.0 mL of PCY2 Vaccine No. 7 IM in the right neck region using a sterile 3.0 us L Luer-Joek suinge and a sterile 2Og x ' ⁇ " needle.
  • Body weights were recorded on the Body Weight Fonn Day -3 body weights were utilised to Mock pigs prior to randomization Da> 24 and Day 49 weight data nas uti ⁇ i/cd to determine the average daih weight gain (ADWCi) for each pig during these time potnts For pigs that died after challenge and before Day 4' ⁇ the ADWG was adjusted to represent the ADWG from Da> 24 to the day of death,
  • a sterile dacron swab was insetted intra nasally into either the left or right nostril of each pig (one swab per pig) as aseptically as possible. swished around for a few seconds and then removed. Each swab was then placed info a single sterile snap-cap tube containing 1.0 mi, of EMEM media with 2% IFBS. 500 imils/mL of PenicJliin. 500 ⁇ g/mL of Streptomycin and 2.5 ⁇ g/mL of Fungizone.
  • Table 8 The results from the post-challenge clinical observations are presented below in Table 8. This summary of results includes observations for Abnormal Behavior, Abnormal Respiration. Cough and Diarrhea. Table 9 includes the results from the Summary of Group Overall incidence of Clinical Symptoms and Table 10 includes results from the Summary of Group Mortality Rates Post -challenge.
  • Pigs receiving the 2 lower doses of vORF2 pigs receiving 16 ⁇ g of rORF2 and pigs receiving 2 doses of KV vaccine had incidence rates of > 27.3%.
  • Pigs receiving 8 ⁇ g of rORF2 and (he strict negative control group had no abnormal behavior. None of the pigs in ⁇ his study demonstrated any abnormal respiration. Coughing was noted frequently ⁇ i all groups (0 to 25% ⁇ , as w as diarrhea (0-20%). None of the clinical symptoms noted were pa ⁇ hognomic for PMWS,
  • vOR F2 :: isolated viral OK F2; i()RF2 ::: recombinant baeiitovirns expressed ORF2. killed, whole eel] s'jrus - PCV2 virus armvn in suitable cell culture
  • PCV2 nasal shedding results are presented below in Table S.1. Following challenge on Day 24. 1 pig in Group 7 began shedding PCV2 on Da ⁇ 2?. None of ⁇ he other groups experienced shedding nntii Day 33. The bulk of nasal shedding was noted from Day 35 to Day 45. Groups receiving any of the three vORF2 vaccines and groups receiving either 4 or 8 ⁇ g of rORF2 had the lowest incidence of nasal shedding of PCV2 ⁇ 9, 1%). The challenge control group (Group 8) had the highest shedding rate (80%). followed by the strict negative control group (Group 9), which had a» incidence rate of 63.6%.
  • Group 5 which received one dose of H ⁇ g of rORF2 antigen, outperformed the other 6 vaccine groups.
  • Group 5 had tiic highest ADWG (0.94 * 0.22 lbs/clay X the lowest incidence of abnormal behavior (0%). the second lowest incidence of cough (8.3%), the lowest incidence of overall clinical symptoms (8,3%).
  • Group 4 which received a higher dose of rORF2 antigen than Group 5, did not perform as well or better than.
  • Group 5 Group 4 had a slightly lower ADWG, a higher incidence of abnormal behavior, a higher incidence of overall clinical symptoms, a higher rate of nasai shedding of FCV2, a higher mean % lung lesions, and a higher rate for positive ⁇ 1TC tissues than Group 5.
  • Statistical analysis which may have indicated that the differences between these two groups were not statistically significant, was not conducted on these data, but there was an observed trend that Group 4 did not perform as well as Group 5.
  • gtoup had the highest mean 1 O lung lesion scoic ⁇ 9 88 -r 29 2%) Jn all foui gio ⁇ ps the mean ⁇ l ⁇ lung lesion scores were clc ⁇ a ⁇ ed due to one pig m each of these gjoups that had x en high lung legion scores Most of the lung lessons warn described as red purple and consolidated ⁇ j ptcalh .
  • lung lesions associated ⁇ MUI PMWS mo dc&cnbed as> tan and non -collapsible vuth interlobular edema The lung lesions noted in this, stud ⁇ were either not associated w ith PG ⁇ 2 infection ot a second pulnso ⁇ an infectious agent maj ha ⁇ e been ptesenl W tthsn the conteM of this studs the "o iung lesion scores ptobabh do not reflect a tiue nieasuic of the amount of lung mfecuoa due to PC V 2 Other researchers have demonstrated a direct correlation between the presence of
  • the Minimum Portcctive Dosage (MPD) of a I ml/1 dose recombinant product with extracted PCV2 ORF2 (rORF2) antigen in the CDCD pig mode! in the face of a PCV2 challenge was determined.
  • rORF2 PCV2 ORF2
  • Group 5 (8 ⁇ g of rOR.F2 antigen) clearly had the highest level of protection.
  • Group 5 either had the best results or was tied for the most favorable results with regard to all of the parameters examined.
  • Group 5 was compared with die oiher six vaccine groups post-challenge.
  • Group 1 (16 ⁇ g of vORF2) had the highest level of protection.
  • Group I outperformed Groups 2 and 3 with respect to ADWG, mean % lung lesions, and THC.
  • Groups 1 and 2 (8 ⁇ g of vORF2 antigen) performed equally with respect to overall incidence of clsntcal s ⁇ mptoms.
  • Group 3 (4 ug of ⁇ ORF2 antigen; hu ⁇ the lowest mortal itj rate and al I three gioups perfoinied equalh w ith respect to nasal shedding Oscrsll.
  • ⁇ ORF saccules did not pet form as well as tORF > accincs
  • This example tests the efficacy of eight PCV2 candidate vaccines and reconfirms PCV2 challenge parameters TtOHi earlier challenge studies following exposure to a virulent strain of PCV2.
  • PCV2 Vaccine No. 1 administered at 1 x 2 ml dose to Group 1 > w as a high dose ( 16 ug/2 ml dose) of inactivated recombinant ORF2 anligen adjuvanted with IMS 13 ! 4 ( 16 iig rORF2 - IMS 13 ! 4).
  • PCV2 Vaccine No. 1 administered at 1 x 2 ml dose to Group 1 > w as a high dose ( 16 ug/2 ml dose) of inactivated recombinant ORF2 anligen adjuvanted with IMS 13 ! 4 ( 16 iig rORF2 - IMS 13 ! 4).
  • ml dose Io Group 4. was a high dose ⁇ 16 ug / 1 nil dose) of a partially purified VlDO R- 1 generated PC V 2 ORF2 antigen adjuvanted with Carbopoi (16 ug ⁇ ORF2 - Carbopoi).
  • Vaccine No. 5. administered at 1 x 2 nil dose to Group 5. was a 4 ug/2 ml dose of an inactivated recombinant 0RF2 antigen adjuvanted with Carbopoi ⁇ 4 ug rORF2 - Carbopoi).
  • PCV2 Vaccine ⁇ No. 6.
  • PCV2 Vaccine No. 7 administered at I x 2 ml dose to Group 7, was a low dose ( " 0.25 ug/2 ml dose) of inactivated recombinant ORF2 antigen adjm anted with Carbopol (0.25 ug rORF2 ••• Carbopol).
  • Group 8 administered at 1 x 2 nil dose to Group 8, was a high dose ⁇ pre-inaciivaikm titer > S.0 log/2 ml dose) inactivated Conventional Killed VlDO R-I generated PCV2 Strove antigen adjuva ⁇ ted with Carbopol (>S.u log KV - Carbopol).
  • Groups 1-8 were treated with their assigned vaccines.
  • Groups 1-3 and 5-8 received boosters of their respective vaccines again on Day 14.
  • the effectiveness of a single dose of 16 ⁇ g of vOKF2 - Carbopol % ⁇ as tested on Group 4 which did not receive a booster on Day 14. Piglets v ere observed for adverse events and injection site reactions following both vaccinations.
  • mice were housed at the first study site in individual cages in seven rooms from birth to approximately 11 days of age (approximately Day 0 of the study ) Each room was identical in layout and consisted of stacked individual stainless steel cages with heated and filtered air supplied separately to each isolation unit. Each room had separate heat and ventilation, thereby preventing cross-contamination of air between rooms. Animals were housed in two different buildings at the second study site. Group 10 (The Strict negative control group) was housed separately m a converted nursery building and Groups 1 -9 were housed in a converted farrowing building. Each group was housed in a separate pen (14- 15 pigs per pen) and each pen provided approximately 2.3 square feet per pig. Groups 2.
  • pigs assigned to Groups 1 -9 received 1.0 nil. of PCV2 ISUVDL challenge material (3.98 iogi-> TCIDw ' ml.) IM in ⁇ lie right neck region using a sterile 3.0 ml. Luer-l ⁇ ck syringe and a sterile 2ug x I " needle. An additional 1.0 nil. of the same mate ⁇ al was administered IN to each pig (0.5 mL per nostril) using a sterile 3.0 ml. Luer-lock s> ringe and nasal csnula.
  • Test pigs were observed daily for overall health and adverse events on Da ⁇ -3 and from Day 0 to Day 21. Each of the pigs were scored for norma! or abnormal behavior, respiration, or cough. Observations were recorded on the Clinical Observation Record. All tesf pigs were observed from Day o £o Day 7. and Group 7 was further observed from Day 14 lo 21. for injection site reactions. A ⁇ erage daiK weight gain was determined by weighing each pig on a calibrated scale on Days -3, 25 and .50. or on the day that a pig w as found dead after challenge Body weights were recorded oti the Body Weight Form. Day -3 body weights v. ere utilized to block pigs prior to randomization.
  • ADWG average daily weight gain
  • venous whole blotxl was collected from each piglet from the orbital venous sinus on Days -3 and S 4.
  • blood was collected from the orbital venous sinus by inserting a sterile capillary tube into the medial eanthus of one of the eyes and draining approximately 3.0 niL of whole blood into a 4,0 mL Serum Separator Tube (SST).
  • SST Serum Separator Tube
  • venous whole blood from each pig was collected from the anterior vena cava using a sterile 20g x 1 W Vacutainer& needle (Beclon Dickinson and Company.
  • Pigs were observed once daily from Day 22 to Day 50 for clinical symptoms and scored for normal or abnormal behavior, respiration or cough. Clinical observations were recorded on the Clinical Observation Record.
  • Pigs Nos. 46 (Group 1 ) and 98 (Groups 9) died at the first study site. Both of these deaths were categorized as bleeding deaths and necropsies were not conducted on these two pigs.
  • pigs that died after challenge and prior to Day 50, and pigs euthanized on Day 5i ⁇ were necropsied. Any gross lesions were noted and the percentages of lung lobes with lesions were recorded on the Necropsy- Report Font).
  • tissue sample of tonsil, lung, heart, and mesenteric lymph node was placed into a single container with buffeied 10% fourtaSm. while anothes tissue sample from lhc same aforementioned organs was placed into a Whu ⁇ -pak ft. ⁇ M-fech Diagnostics Ltd . rhelwail t ; K.) and each Whiri-psk# was placed on iec Each container was.
  • Group 4 which received one dose of 16 ug vORF2 •• Carbopol had the highest ADWG- (1.16 * 0.26 lbs/clay). followed by Groups 1, 2, 3, 5, 6, and 10 which had ADWGs that ranged from 1.07 ⁇ 0,23 lbs/day to U l ⁇ 0.26 lbs/day. Group 9 had the lowest ADWG (0.88 ⁇ 0.29 lbs/day >, followed by Groups S and 7, which had ADWGs of 0.93 ⁇ 0.33 lbs/day and 0.V9 ⁇ 0,44 lbs/day . respectively ,
  • Group 3 which received two doses of 16 ug rORF2 - CarbopoL had higher antibody tilers than Group I. which received two doses of .16 ug rORF2 ⁇ TMS 1314.
  • Group 2. which received two doses of 16 ug vORF2. had higher titers than Group 4, which received only one does of the same vaccine.
  • Groups 3, 5, 6, 7. which received decreasing levels of rORF2 - Carbopol, of 5.6, 4, 1. and 0 25 ug respectively, demonstrated correspondingly decreasing antibody titers on Days 25 and 32.
  • vORF2 - isolated viral OE.P2; rOE£P2 ::: recombinant haculovirus exptessed ORF2: KV or killed whole ceil virus ⁇ PCV2 virus arown in suitable ceil culture
  • a ⁇ I OO !FA titer was designated as a titer of "50"; a >640ft IFA tiler was designated as a ⁇ ter of " i 2.8(H)”. **Day of Challenge ***Day of " Necropsy
  • Tabic 20 includes observations for Abnormal Behavior. Abnormal Respiration. Cough and Diarrhea.
  • Table 21 includes (he results from the Summary of Group Overall Incidence of Clinical Symptoms and Table 22 includes results from She Summary of Group Mort ⁇ lily Rates Post-challenge, Hie incidence of abnormal behavior, respiration and cough post- challenge were low in. pigs receiving 16 ug r ⁇ RF2TMiMS 1314 (Group S.), S.6 ug rORF2 ⁇ Carbopol (Group 3). S. ug rORF2-Carbopo ⁇ (Group 6). 0 25 ug rORF2 ⁇ Carbopol (Group 7).
  • pigs receiving 4 ug rORF2-Carbopo3 (Group 5), 0.25 og tORF2— Carbopol ⁇ Group 7), and >8 log KV vaccine had incidence rates of 13.3%; and pigs in the Challenge Control Group (Group 9 ⁇ bad an incidence rate of 14.3%.
  • Group 3 which received two doses of 16 ug rORF2 ⁇ Carbopoi, had the lowest tentative diagnosis of " pneumonia, with 13.3%.
  • Group 9 the challenge control group, had 50% of die group tentatively diagnosed with pneumonia, followed by Group 10. the strict negative control group and Group 2. which received two doses of lf> ug vORF2-Carbopol, with 46 TYa and 40% respectively, tentatively diagnosed with pneumonia.
  • Groups 1. 2, 3. 5, 9. and 10 had 0% of the group tentatively diagnosed as PCV2 infected; while Group 8, which received two doses if KV vaccine, had the highest group rate of tentative diagnosis of PCV2 infection, with 20%.
  • vORF2 :: isolated viriil fJH.P2; ) ⁇ >RP2 ::: rectMiibitiam bitciilo ' vin.t-5 expressed
  • ORF2 KV or kilted whole ceil virus ⁇ PCV2 vints arown in suitable ceil culture
  • PCV2 vaccines were evaluated in this example, which included a high dose (16 ⁇ g) of rORF2 antigen adjuvanted with IMS 1314 administered twice, a high dose ⁇ 16 ⁇ g) of vORF2 antigen adjuvanted with Carbopol adminislered once to one group of pigs and twice to a second group of pigs, a high dose ( 16 ⁇ ,g) of rORF2 antigen adjuvanted with Carbopol administered twice, a 4 ⁇ .g dose of ⁇ ORF2 antigen adjuvanted with Carbopol adra mistered twice, a 1 ⁇ g dose of rORF2 antigen adjuvanted witli Carbopol administered twice, a low dose (0.25 ⁇ g) of rORF2 antigen adjuvanted with Carbopol administered iwice. and a high dose ⁇ > 8 log) of killed whole cell PCV2 vaccine adjuvanted with Carbopol.
  • Groups 2 through ? which received vaccines containing various levels of cither vORF2 or rORF2 antigen adjuvanted wish Carbopol and much belter than Group 8, which received two doses of killed whole ceil PCV2 vaccine.
  • Group 1 had the third highest ADWG ⁇ 1.KO ⁇ 0.3 « lbs/day ), the lowest incidence of abnormal behavior (0%). the lowest incidence of abnormal respiration (0%). a low incidence of cough (7 1%). a low incidence of overall clinical symptoms (7.1%). was tied with three other groups for the lowest mortality rate (0%), the second lowest rate for mean % hsig lesions (0.1.5 & 0.34%). the second lowest rate for pneumonia (21.4%) and the lowest incidence rate for positive MC tissues (0%) Group 1. was. however, the only group s.n which injection site reactions were noted, which included 50% of the vaccinates 1 day after the second vaccination. The other vaccines administered to Groups 2 through 7 performed better than the killed vaccine and nearly as well as the vaccine administered to Group 1.
  • Group 8 which received two doses of killed PCV2 vaccine adjuvanted with Carbopol. had the worst set of results for any vaccine group.
  • Group 8 had the lowest ADWG (0.93 ⁇ 0.33 lbs/day), the second highest rate of abnormal behavior (6.7%), the highest rate of abnormal respiration ⁇ 6.7%), was tied with three other groups for the highest overall incidence fate of clinical symptoms (13.3%), had the highest mortality rate of all groups (20%). and had the highest positive UiO rate (80%) of any vaccine group.
  • a gastric ulcer was noicd in one Group 7 pig, bui lymphad ⁇ nopaihy was not specifically examined for. Based on the presence of lesions thai were consistent with PCV2 infection, three groups had at least one pig tentatively diagnosed with PCV2 (FMWS). Group 8, which received rwo doses of killed vaccine, had 20% tentatively diagnosed with PCV2, while Group ? and Group 4 had 13.3% and 6.7%. respectively, tentatively diagnosed with PCV2. The tnean % lung lesion scotes varied between groups at necropsy . Groups 1. 2, 3, 4. 6 and 10 bad Sow % lung lesion scores that ranged Prom 0.1 1 ⁇ 0.38% to 0.V0 ⁇ 0.1.5%. As expected. Group 9.
  • the challenge control group had the highest mean % lung lesion score ( 10,81 i 23.27%), In four groups, the mean % lung lesion scores were elevated due to one to three pigs in each of these groups having very high lung lesion scores.
  • the lung lesions were red/purple and consolidated. Typically, lung lesions associated with PMVVS arc described as tan. »on ⁇ eollapsible. and with interlobular edema.
  • the lung lesions noted in this study were cither not associated with PCV ' 2 infection or a second pulmonary infectious agent may have been present. Within the context of this study, the % lung lesion scores probably do no reflect a true measure of the amount of lung infection due to PCV2.
  • Group IHC results showed the largest differences between groups.
  • Group I (16 ⁇ g rORF2 - IMS 1334) had the lowest positive IHC results for PCV2 antigen (0%); while Groups 9 and 10 had the highest positive IHC results with incidence rates of 100% and 93.3% respectively.
  • the Minimum Protective Dosage (MPD) of PCV2 rORF2 antigen adj ⁇ varUc ⁇ with Carbopol in the CDCD pig model in the face of a PCV2 challenge is determined.
  • ⁇ he antigenicity of recombinant (rORF2) and VlDO R-I (vURF2) PCV2 antigens were assessed.
  • Group 2 received two doses of 16 ⁇ g vORF2 and Group 3 received two doses of 16 ⁇ g rORP2, Both vaccines were adjuvanted with CarbopoS. Both vaccines were ftmnd io be safe and both had 0% mortality rate.
  • Group 2 had a PCV2 antibody titer of 2507 on Day 25, while Group 3 had a PCV2 antibody titer of 1503.
  • Group 3 had a lower mean % hmg lesion score than Group 2 (0, 1 1 * 0.38% vs. 0.90 ⁇ ( 115%), but Group 2 had a lower IMC positive incidence rate that Group 3 (6.7% vs. 20%).
  • both vaccines had similar antigenicity, but vORF2 was associated with slightly better UiO results.
  • the suitability of two different adjuvants was determined.
  • Groups 1 and 3 both received two doses of vaccine containing 16 ug of rOR.F2 antigen, but Group 3 received the antigen adjuvanted witli IMS 1314 while Group 3 received the antigen adjavanted with Carbopoi.
  • Both groups had essentially the same ADWG, essentially the same incidence of clinical signs post-challenge, (he same mortality rate, and essentially the same mean % King lesions; but Group 1 had an SHC positive rate of 0% while Group 3 had an !HC positive rale of 20%.
  • Group 3 which received She vaccine adj ⁇ vanted wish Carbopoi, had higher IFAT PCV2 liters on Days 23, 32, and 50 than Group 3 , which received the vaccine adjuvanted with IMS 33 14,
  • the PCV2 vaccine adjuvanted with IMS 1314 did provide belter WC results, it did not provide overwhelmingly tetter protection from PCV 2 infection and did induce injection site reaction.
  • ⁇ lie PCV2 vaccine adjuvanted with Carbopoi performed nearly as well as the IMS 1314 adjuvanted vaccine, but was not associated with any adverse events.

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Abstract

The present invention relates to the use of an immunogenic composition that comprises a porcine circovirus type 2 (PCV2) antigen for treatment of several clinical manifestations (diseases). Preferably, the clinical manifestations are associated with a PCV2 infection. Preferably, they include lymphadenopathy, lymphoid depletion and/or multinucleated/giant histiocytes. Moreover, the clinical symptoms include lymphadenopalhy in combination with one or a multiple of the following symptoms in pigs: (1) interstitial pneumonia with interlobular edema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4) gastric ulcers, (51 nephritis and (6) reproductive disorders, e.g. abortion, stillbirths. mummies, etc. Furthermore the clinical symptoms include Pia like lesions, normally known to be associated with Lawsonia intracellularis infections.

Description

USE OF A PCV2 IMMUNOGENIC COMPOSITION FOR LESSENING CLINICAL
SYMPTOMS IN PiGS
SEQUENCE LISTING
This application contains a sequence listing in paper format and in computer readable format, the teachings and content of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the use of an immunogenic composition comprising a porcine circøvints type 2 (PCV2) antigen for treatment of several clinical manifestations (diseases). Preferably, those clinical manifestations are associated with a PCV2 infection. More particularly, the present invention is concerned with an immunological composition effective for providing 3ii imππme response that reduces, or lessens the severity, of the clinical symptoms associated with PCV2 infection. Preferably, the immunological composition comprises a recombinant!)- produced antigen of PCV2. More preferably, the PCV2 antigen is a recombinant!)' produced protein encoded by one of the open reading frames (ORFs) in the PCV2 genome. Still more preferably, the antigen is PCV 2 ORF2 protein. Most particularly. Ae present invention is concerned with an immunological composition effective for treatment of clinical sypmtøms associated with PCV2 infections in swine receiving {he immunological composition, and wherein the composition comprises the prolan expressed by ORF2 of PCV2. Another aspect of the present invention is the use of any of the compositions provided herewith as a medicament, preferably as a veterinary medicament, even more preferably as a vaccine. Moreover, the present invention also relates to the use of any of the compositions described herein, for the preparation of a medicament for reducing or lessening the severity of clinical symptoms associated with PCV2 infection. Preferably, the medicament is for the invention of a PC V2 infection, even more preferably in swine. A further aspect of the present invention relates So a process for the production of a medicament, comprising an immunogenic composition of PCV2 for the treatment of several clinical manifestations.
Description of the Prior Art
Porcine circovirυs type 2 (PCV2j is a small ( 17 -22 rim in diameter), icosahedral, non-enveloped DMA virus, which contains a single-stranded circular genome. PCV2 shares approximately 80% sequence identity with porcine circovirus type S. (PCVI). However, in. contrast with PCV K which is generally non-virulent, swine infecied w ith PCV 2 exhibit a syndrome commonly referred to as Post-weaning Muitisysteinic Wasting Syndrome (PMWS). PMWS is clinically characterized by wasting, paleness of the skin, anth.iiftj.ness, respiratory distress, diarrhea, icterus, and jaundice. In some affected swine, a combination of all symptoms will be apparent while other affected swine will only have one or two of these symptoms. During necropsy, microscopic and macroscopic lesions also appear on multiple tissues and organs, with lymphoid organs being the most common sue for lesions. A strong correlation has been observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. Mortality rates for swine infected with PC V2 can approach 80%. In addition to PMWS. PCV2 has been associated with several other infections including pseudorabics. porcine reproductive and respiratory syndrome < PRR S). Glasser's disease, streptococcal meningitis, salmonellosis, postweøning coiibacillosis, dietetic hepatosis, and suppurative bronchopneumonia. However, research thus far has not confirmed whether any of these clinical symptoms ate in fact, the direct result of a PCV2 infection Moreover u ss not \et knoun w hether an\ of these cluneal s\ mpιoms can be dXecmeh reduced or cured In an acuvc agent duected agamst PC V2
Cw tent appioaches to treat PCV2 infections include DW-ba^ed vaccines such as thobc described in I S Patent λo 6 7»^ 02^ iio«e\ei bitch uieuπcs kne beui nieffcctiw st confcii ing pioleciivt, innnunits against PCV 2 infection ot reducing lessening tin. se\erm of or cui ing am c hmcai sy mptoms assoc iated therewith Moremci > aecnie<- (icsuibed in the prsoi ml «ei<, focused soϋclv on the pit'\enUon of PCX 2 infections m S\MIK- btit did not consider αn\ fiitthcr incdtea! os«.
Λccoidπiglv what )& needed m the art is an immunogenic composilioii foi the tteatmcCT of scleral chmcal manifesiations Fimliet what JS needed in \hc ait )& an immunological composition which contets protcetne immuiitn agaiswi PCV 2 tofecnoo but \\ hid) can also be used to tieat existing cimieai sv mptoms associated Λ\ nil PCV2 infection
DISCL OSU RE OF T HE INV E N T ION The present m\enUo« o^eicomcs the problems inherent m (he prior ait and provides a distinct adv ance in the state of the art The present iinention piov idcs a racdicinal use(»>) of immunogenic composition^) compπsmg PC V 2 antigen
In general no ad^eisc events or mjecttoti site reactions ncic noted for an\ oi the PC V2 antigen immunogenic oomposiuons as used herein ϊ hus the immunogenic compositions used herein appear to be safe w hen administered to voting pigs prefesabh to pigs, not oldei than H weeks of age more piefetabh not oidei than ι> weeks of age c\en more preferabh not older tliaϊi ^ weeks most prefcrabh not oldei than 2 weeks ΛJtematt\ eh it is psefened that the admmistiation of the immunogenic compositions of the present inv ention occ ur w ithin at feast 2 and preferαbh within at lcaM "* ΛKccks of c\μos>urc to ^ \iniluit PCV According to a futthcr embodiment the immunogenic compositions used herein for am medicina lse described herein, is, adtnmslered to pigs, of 3 weeks of age or older piefeiabh of 2 weeks, of age oi older. mo&t ptcfciabiv but not older than 15 weeks of age
I'nexpeetcdh , it was found that the theiapciftte use of the immunogenic compositions described below is effective for lessening the sc> eπt\ of unions* ciimcal sMtiμtoras in sw um In particular, it was disccncicd that the tliciapcutic UAC of the immunogenic compositions of (he pjres.cn! lm entioii, diid speufitaϊh ςnmposj&oiis comptibing PCV2 ORF2 antigϋn ts> e(ϊccu\c for redu-cnig or lessening h mphadcnopaths K mphoid depletion aud ot inuitinuckated/giαni histiocy tes m sπ me iiifeclcd w ith PCV 2 \foico\ei {he thcjapculic use of an antigenic composition, as prαx ided heicwjth <md thai compr ises PC\ 2 antigen prcfεrabh ORF2 antigen reduces the overall citcm it us load and its tmmunosupprc^n c impact jlierctn tesulting m a higher level of genera! disease icsisjancc and a (educed incidence of PO V-2 associated diseases and s% mptoms
Huis one asjKct of the pte^cni nnctitioo relates to she use of an immunogenic composition comprising POV2 antigen prefesabh recombinant PCV2 antigen and nioic prcfcrabh PC \r2 ORF2 protein as pio\ idcd herewith for the preparation of a medicament foi the prevention lessening <mά or reduction of h tnphadenopaUix . h raphotd depletion and'Or nniltinuclcated giant histiocy tes m swmc Frefeiabh , said medicament is cffcctn e for the prex ention, lessening and/or reduction of h itiphadcnopath} h mphoid depletion and or tnultmucJcatcd gtanl hssUocx tes, associated wtth PC V2 infections, in sw ine SuH more piefeiabh &<πd mcdicarncnt is. ciTccti\c for the prev ention lessemng and or reditction of i\ inphadenopatln , hmphoid depiction and/or mulUπuclealed/giant histιoe>tes> associated w ith PC V 2 mfecuons m pigs when administered to pigs not oSdes than J 5 weeks of age mote picferabh not older than t> weeks of see, e\en more prefcrabK not older than 3 Λ\ecks mid most piefciabh uot oldei than 2 \\eeks. Aite»iati\eh , it is prefeπed that tlie administrati on the immunogenic compositions of the present indention ocα« w ithtπ at least 2 and pi efcrabh w illiin al least 3 weeks, of exposure to \ uulenl PC V
Anothei aspect of the present intention relates Io a method fot the Ueatmcnt of i\ mpruidcπopalhv ly mphoid depiction and/or muHιnucJe<itcd''giant histiocy tes in swiπc computing the administration of an immunogenic composition as prm idcd herew ith to a pig said immunogenic composition comprising a PC\'2 antigen preferabK a icc-omfamant PCV2 antigen and πioieptcfciabh PCV2 0RF2 protein In \ct anotlκi aspect the present nπention pio\ jclcs ά inelliod fot the Ueatmcm of Is niphadeiiopathv K mphoid depletion and/ot mtiitiiHJcicatvd gtsnt hιstto-c\ {cs. associated w ith a PC\'2 mlccUon in swine compmmg the αdnjtnistrøuon of an unituiuogenic composition as pio\ idtd hctew ith JO i pig sstd imtnυnogcmc composition compt issiig a PC \ 2 amigen prefeiabh a recombinant PC' V 2 antigen and mote pi cfcrabh PCV2 ORF2 protein Prcfcrablv said ticatmcnt iesidts m the lessening reduction pic\cntton and/oi case of the h inpbsdeiiopatln h mphoid dcpJeJion and'Or multimiclcated giant bisitoo lεs in swtoe fccesuog said immunogenic composition Λccoidmg to a furthej aspect, said methods foi ticatiwent litnhci comprise the admmisuαtion of said jmtniaiogentc composition to pigs not older than 15 weeks of age more picierabi\ not older than 6 weeks of age c\en more piefcrabh not older than 3 weeks and most psefciabh «of oldei than 2 weeks AJtewaUx eh it is preferred {hat the administration of the immunogenic compositions of the present (m ention occur w ithin at least 2 and prcferabK wnhui at least 3 weeks of exposure to Mntlent PCV
It was fimhei discov ered that the therapαsUe itse of an immunogenic compowlion composing PC\'2 antigen, prcfeiabh a rcconunant PCV2 antigen and most preiereabh PC V2 ORI 2 psotesn as pjoudcd herew ith can seduce oi lessen h mphade-nopaths m combination with one oi a multiple of the follow ing sv niptonis in affected sw ine ( 1 ) interstitiai pnetunonis with mieilobulsi edeωa (2) cutaneous pallet or ictei ns (3) mottled atrophic In ess, (-4) gastnc ulcers (?) nephritis and ((A seproduαne disordei s, c g abortion stillbirths, mummies etc
Thus one aspect of the present invention relates, to the use of an immunogenic composition c-oinpti&ing PCV2 antigen, prefctabh a recombinant PCV2 antigen and more prcfetablv PCV2 ORF2 ptoicm as provided herew ith foi the prepaiation of a medicament fot the prevention lessening and'Or icduαion of h niphadcnopathv in combination w ith one or a multiple of the following sy mptoms m pigs (1 > intcisliUal pneumonia with mte: lobular edema, (2) cutaneous pallor or tctcrαs 0) mottled ati opine hveis. (4) gasttie uleers p> nephutis and (6) icpiθductι\c disordos, e g abortion stillbirths mummies, etc , m pigs Picferablv said medicament is effectι\c foi the prevention, lessening and/ot reduction of h mphadenopatln in combination with one or a multiple of the folSou sng sy mptoms associated w ith PCV2 infection in pigs ( 1 ) interstitial pneumonia w ith interlobular edem<i (2) cutsiieou*; psϊJor or ictcras (3 i mottled atropine lncrs (4~\ gastric aleeis (^ > ncphπtϊs and ((Λ rcpioductπc diwtder^ e g abortion ^tillbmks mummies etc Arcoidmg to a further aspect said medicament is effective for the ptcvention, lessening and'Or reduottoti of h mphadcnopath> m combination w ith one or a multiple of the following sj nipioms m pigs (1) mtersnnal pneumonia with mtei lobular edema (2) cutaneous pallor or icterus f "5) mottled atrophic In ers* {4) gastϋc ideas ( ^ ncplirttts and (6) reprodtictne disoideis e g abortion stillbirths mummies, etc , m pigs when administered to pigs, not oides than 15 weeks of age more ptefeiabh not older than <> weeks of age, e\ en snore prefesabh not oldei than 3 weeks, and most prefeπibh not older than 2 \secK& Λϊtematn ei\ . ϊt is preferred that the ϊidinmis.tiation of the immunogenic compositions of the present invention occtn w ithin at least 2 and ps efcrabh w tllπn at least 3 weeks of e\posιire to \ ti ulent PC Y
Moreov er, the picseni mvcnuon also ielatcs to a method fot the treatment of l\ mphadcuopαiliv in combination with one oi a multiple of the following symptoms, in pigs O) interstitial pneumonia with interlobular edema, (2) cutaneous pallor or icterus. (3) mottled atrophic livers. (4) gastric ulcers . (5) nephritis and {6} reproductive disorders, e.g. abortion. stillbirths, mummies, etc., said method comprising the administration of an immunogenic composition comprising PCV2 antigen, preferably a recombinant: PCV2 antigen, and more preferably PCV2 OR.F2 protein as provided herewith. Preferably, the present invention also relates to a method for the treatment of iyniphadenopathy in combination with one or a multiple of ϊhe following symptoms associated with PCV 2 infection in pigs: (1 ) interstitial pneumonia wish interlobular edema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers. (4) gastric ulcers. (51 nephritis and {<>) reproductive disorders, e.g. abortion, stillbirths. mummies, etc., said method comprising {he administration of an immunogenic composition comprising PCV2 antigen, prefereably recombinant PCV2 antigen and more preferably PCV2 0R.F2 protein, as provided herewith, to a pig. Preferably, said treatment results in the lessening or reduction of the lymphadenopathy, and one or multiple of the following symptoms associated wish PCV2 infection in pigs: Cl) interstitial pneumonia with interlobular edema. (2) cutaneous pallor or icterus. {3} mottled atrophic livers. (4) gastric ulcers . (5) nephritis and (6) reproductive disorders, e.g. abortion, stillbirths, mummies, etc. According to a further aspect, said methods for treatment further comprise administration of the immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen and snore preferably PCV2 ORF2 protein, as provided herein, to pigs not older than 15 weeks of age, more preferably not older than 6 weeks of age. even more preferably not older than 3 weeks, and most preferably not older than 2 weeks, Alternatively, it is preferred that the administration of the immunogenic- compositions of the present invention occur within at least 2 and preferably within at least 3 weeks of exposure to virulent PC: V.
It was also unexpectedly found that the therapeutic use of an immunogenic composition comprising PCV antigen, preferably recombinant PCV2 antigen and moie prcierabh PC V2 OR.P2 ptotem as prov ided heicwnh can aho reduce or lessen Pta ϊikc lesions notmaih known to be associated with Law soma mtraccllulaπs injections (Ileitis)
Thus one aspect of the present imcniion relates, to tlic use of an immunogenic t (imposition comprising PCV2 antigen prtfeidbh itcoinbinanl PCΛ 2 antigen and more preferabK PCΛ 2 ORF2 piotcm as pro\ ided herew ith fbt the ptepaiatton of a medicament for the pio cntton lessening tlic <,e\cπt\ of and ot icdαctton o! Pia like lestons iiotinslh known (o K, d^soc iαicd w ith Lαw^oma intiaccihilaiis infections in w uic According (o a fin lher js>pt,ci said medicament is Uftxih c fot the pιc\cniion lessening of the ^e\citt\ of anά ot iedu^tion of Piα liie lesions normaih known to be a^o*,ιatcd w ith Law soma intraceiiulans lufcαions when αdnjimstcjcd to pigs not oido jlisn 1 *> weeks of age mote prefeiabh not oldci than 6 weeks of sgc e\en more pfcferabh not older than 3 weeks and most prdcrnbh not older than 2 weeks \ltci nau\ch it li. preferred thai the αdmimstrauon of the immunogenic compositions of the ptescut im entjon occiu within at least 2 and prefcrabh w ithin at least ^ weeks of exposure Io Λ sruSent VC \ Moreover the present inventio nho relates to a method for the treatment of Pta iikc iesiom noinωih know n Io be d^ooiaied nith Law soma mtuκe!iu!dπ»> mfeuions said method L-ompi tsing the admiiustratiosi of an immiisiogenit composition ctwnpming PC \ 2 antigen picicrabh iccombinant PCV2 antigen and snore preferabh PCV 2 ORF2 piotcm as proMdcd hetein Io <i pig Pidenibh wnϋ treatment iesuUs m the Jesscnuig or seduction of the Pta like lesions itoπnalK known to be associated vuth Law soma mu acel luJaris infections Λccoidmg to a forlhci aspect the methods for treatment dcscπbcd atxnc further comprise the administration of the muntinogcπic composition comprising PC\ 2 antigen ptefeiabh recombsant PC V 2 anugcn and mote prefetabb PC\ 2 ORI" 2 protcm as psosided heron to pigs not older than H weeks of age more ptUeiabh not oldei than 6 γ\ccks of age even more μιcierabl\ not older tlian > v\ceks and most prefci αbh not older than 2 weeks Alternatively, it is preferred that the administration of the immunogenic compositions of the present invention occur within, at least 2 and preferably within at least 3 weeks of exposure to virulent PCV.
The immunogenic composition
The immunogenic composition as used herein is effective for inducing an immune response against PCV2 and prexenting, reducing and/or lessening the sexerity of the clinical symptoms associated with PCV2 infection. The composition generally comprises at ieast one PCV2 antigen. Unless defined otherwise, ail technical and scientific terms used herein have the same meanings as common!) understood by one of ordinary skill in She art to which this invention belongs. The terns "immunogenic composition" as used herein refers to any pharmaceutical composition containing a PCV2 antigen, which composition can be used to prevent or treat a PCV2 infection-associated disease or condition in a subject. A preferred immunogenic composition can induce, stimulate or enhance the immune response against PCV2. The term thus encompasses both summit immunogenic compositions, as described below, as well as compositions containing whole killed or attenuated and/or inactivated PCV 2.
The term "sidnmit immunogenic composition" as used herein refers to a composition containing at least one immunogenic polypeptide or antigen, but not all antigens, derived front or homologous to an antigen from PCV2. Such a composition is substantially free of intact PCV2. Thus, a "subunit immunogenic composition" is prepared from at least partially purified or fractionated (preferably substantially purified) immunogenic poly peptides from PCV2. or recombinant analogs thereof. A subutrit immunogenic composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from FC \ 2 or m fractionated from \ prefei rcd immunogenic subunjt composition comprises thec PC V2 ORF2 protein as described below
\n "immunological or immune response" lo a composition ot \acemc is the development m the hos>t of a celhilat and'' 01 antitxxh -mediated immune response to the compos it ion ot utcαne of iiiicicsl Csualh , an "immune response" inc ludes but is not limned to one 01 more of the follow ing effects the pioduciion ot activ ation of antibodies. B cells, helper T edls, siippicssor T cells, and ni c\ totn\tc T cells and/oi j d T cells, dnected spec ifϊcalK to an antigen or antigens included m the composition ot \ ace me of tntetcsi Ptefαobh , the host w ili dispisn etthct a jiierapcutic ot piotcctiv e uumunolαgtcal response such thai iesutance to new infection will be enhanced and/oi the ciimcai se\et ιn of the disease reduced Such piotcouoti w ill be dctRonstraicd b\ eithei a icduciton in immbei ot sc\etttΛ of, or Kick of one Oi moie of the sunptoms associated \\ ι\h PC\ 2 infections as. described abo^e fhe terms "immunogenic" protein ot pol\ peptide ot "antigen as used beiesn refer to an ammo aαd sequence which elicits a« immunological response as described abo\c Λ.n "uniminogcme" piotcsn oi poh peptide, as used herein, includes the fuii-iength sequence of an\ P( \' 2 proteins, analogs thereof, or immunogenic fragments thereof The terra "immunogenic fragment" iefcis to a fiagmetit of a piotcm winch includes one or more epitopes, and thus choits the jiumunoJogical response described abo\e Such fragments can be sdenufsed using am sntmbei of epitope mapping techniques, well known in the att See e g , Epitope Mapping Piotocols in Methods in Molecular Bioiog> , Voi 06 {Glenn R MoJiis Ed 19%) IIomana Prc&i. Totowa. Nevv Jcrse> For example linear epitopes roaj be detcimmcd b\ e g concurrcnth s\ nthesi/mg large numbess of peptides on solid supports the peptides coπcsponding to poruons of lhc protein molecule and leading the peptides Λ\ tth antibodies while tlie peptides me still attached to the supports Such tcclmic[ucs aic knovn in the art and described in, e.g., U.S. Patent No. 4.708,871 : Gey sen et ai ( J 984) Proc. Nail. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec, Immunol, 23:709-715. Similarly, conformational epitopes ate readily identified by determining spatial conformation of amino acids such as by. e.g., x-ray crystallography and 2-diniensional nuclear magnetic resonance. See, e.g.. Epitope Mapping Protocols, supra.
Synthetic antigens are also included within the definition, for example, polycpiiopes, flanking epitopes, and other recombinant or synthetically derived antigens. See. e.g.. Bergmann et ai. (!993 ) Eur. J. Immunol. 23:2777-27S l ; Bergmann et al. (! 996). J. Immunol 157:3242-3249: Suhrbier. A. (1997). Immunol, and Cell Biol. 75 402-408; Gardner et al . ( 1998) 12th Worid AIDS Conference, Geneva. Switzerland. June 28-Juiy 3. 1998
In a preferred embodiment of the present invention, an immunogenic composition thai induces an immune response and. more preferably, confers protective immunity against the clinical signs of PC.V2 infection, is provided. The composition .most preferably comprises the poly peptide, or a fragment thereof, expressed by ORF 2 of FCVZ as the antigenic component of the composition. PCV2 ORF2 DNA and protein, used herein for the preparation of the compositions and within the processes provided herein is a highly conserved domain within PCV2 isolates
Figure imgf000012_0001
thereby, am PCV 2 ORF2 would be effective as the source of the PCV ORF2 DNA and/or polypeptide as used herein. A preferred PCV2 ORF2 protein is that of SEQ ID NO. U . A preferred PCV ORF2 polypeptide is provided herein as SEQ ID NO. 5, but it is understood by those of skill in the art that this sequence could vary by as imtch as 6-10% in sequence homology and still retain the antigenic characteristics that render it useful in immunogenic compositions. The antigenic characteristics of an immunological composition can be, for example, estimated by the challenge experiment as provided by Example 4. Moreov er, the antigenic characteristic of a modified antigen is still retained, vUseis the modified ant J gen confess at least 7<>%, prefesabh 80%. more preferably 90 of the protcctn c imπnimtj as compared to tiic PCV2 ORi" 2 protein, encoded b\ the poK nucleotide sequence of SFQ ID NO 3 or SEQ ID NO 4 AJJ "immunogenic composition" as used heiein, means a PCV 2 ORF2 prnlein w hich elicits an 'immunological tespoiisc" HΪ the host of a cellular anchor antttaixh -mediated immune response to PCV2 ORF2 protein Prcferablj , this mimunojicinc composition is capable of eliciting Ot enhancing an immune response against PC\ 2 ϊheiebj confcπng protect i\ e lDiinami) again &ι PCV2 infection atid a reduction m the incidence of, se\cπt\ of, ot prev ention, of one ot more, and pjcfcrabh all of tbe clinical signs assocuited thcteu ilb In some forms, immunogenic portions, of PCY2 ORF2 piotcm are ixs.ed as the antigenic component to the coinpositioo The leπn "immunogenic poittoti" as used bαesti refets to tnaicatcd and/or substituted forms, ot fragments of PCV 2 0RF2 piotem and or IMK nucleotide respecti^ch Piefcrabfy such trancated and/oi lυbsttrutcd forms, or fragments w ill comprtsc at least 6 contiguous stmno acsd^ fiom the fulS-lcngth ORF2 ]X>h peptide More preferabh . the truncated or subsfihited forms, or fragments w ill hs\e a{ least 10. more preferabh at least 15. and sui! more preferabh at least i'> contiguous, ammo acids from the full-length 0RF2 rκ)h pepttde Two pieferred sequences m this respect arc prouded bercin as SEQ ID NOs 9 anά 10 U is litrthci understood that such sequences ma> hi a part of larger fragments ot truncated forms. A furthe rreferred PC\r2 ORF2 poly peptide prov ided heretπ is encoded b\ the nucleotide sequences of SEQ ID NO 3 or SLQ IO NO 4 However, A is understood b\ those of skill m the art that lhis> sequence could \ar> bj as much as b-20% m secjitence homolog) and still retam the antigenic chaiactcπsiics that render it useful m immunogenic compositions In some forms a truncated ot substituted form ot fiagϊnent of this. P\'C2 ORF2 poly peptide is used as the antigenic component in the composition Prcfeiabix such truncated or substituted forms, or fragments will comprise at least IS contiguous nucleotides from the full-length ORF2 nucleotide sequence, e.g. of SEQ 1I> NO: 3 or SEQ ID NO: 4. More preferably, the truncated or substituted formj. or fragments, vvϊJ! have at ieasi 30, more preferably at least 45, and still more preferably at least 57 contiguous nucleotides of the AiH- length ORF2 nucleotide sequence, e.g. SEQ ID NO: 3 or SEQ ΪD NO, 4.
"Sequence Identity" as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence Similarity, as determined by the match between sitings of such sequences. Upon such alignment, sequence identify is ascertained on a posilion-by-position basis, e.g., the sequences are "■identical" at a particular position if st that position, the nucleotides or amino acid residues are identical. The total number, of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to. those described in Computational Molecular Biology. Lesk. A. N., ed.. Oxford University Press. New York ( 1988), Bioeomputing: Informatics and Genome Projects. Smith. D.W.. ed.. Academic Press, New York (1993): Computer Analysis of Sequence Data. Fart I, Griffin. A.M. and Griffin. H. G.. eds.. Humana Press. New Jersey (1994); Sequence Analysis in Molecular Biology, von Meinge. G.. Academic Press (1987); Sequence Analysis Primer. Gribskov, M. and Devereux, J.. eds., M. Stockton Press. New York (1991); and Cariiio, H., and Upmaru D., SlAM J. Applied Math.. 48: 1073 ( 1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity arc codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, bui are not limited to, the GCG program package (Devereux. J.. et al.. Nucleic Acids Research, 12( 0:387 (1984», BLASTP. BLASTN and FASTA (Altschul S. F. et al.. J. Moke. Biol., 215:403-410 { 199(1). The BLASTX ptogram is publicly available from NCBl and other sources (BLAST Manual, Ahschul, S. et al., NiCV! NLM NTH Scihesda. MD 20894, Altschul, S, F. et aL J. Molec. Biol., 2 15:403-41 U (1990), (he teachings of which are incorporated herein by -reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having ai least, for example. 85%, preferably 90%, even more preferably 95% "sequence identity " to a reference nucleotide sequence, it is intended {hat the nucleotide sequence of" the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 13, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%. even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%. preferably 10%. even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%. preferably ] 0%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in lite reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85V preferably 90%, e\ en more prefcrabh 95% sequence identity to a reference ammo acid sequence, it is intended that the gh en amino acid sequence of the poly peptide is identical to the reference -sequence except that the gh cn poh peptide sequence nia\ include up to 15. prefcrabh up Io 10, even more μreferabh up Io 5 amino acκl alterations per each 100 amino acids of the reference amino acid sequence In other words, to obtain a ghen pøK peptide sequence ha\ ing at least 85%. preferabK WV even more preferabK 95% sequence identi t yth a reference amino acid sequence, up to 15°b. preferabK up to 10%. even more preferabK up to 5% of the amino acid residues in the reference sequence ma> be deleted or substituted with another amino acid, or a number of ammo acids up to 15%, prcicrabh up to i θ%. even more preicrablj up to 5% of the total number of ammo acid residues in the reference sequence ma> be inserted into the reference sequence. These alterations, of the reference sequence ma> occur at the amino or the carbo\> terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed etiher itidmdualh among residues in the reference sequence or to the one or more contiguous groups w ithin the reference sequence PrcferabK . residue positions w hich are not identical differ b> conserv ative ammo acid substitutions lkwe\er, eonsenatne substitutions arc not included as a match w lien determining sequence identit} ,
"Sequence homology ", as used hcrctn. refers to a method of determining the relαledncss of wo sequences. "Jo determine sequence homologj , wo or more sequences arc optima Uj aligned, and gaps arc introduced if neccssan HOWCΛ CΓ. in contrast to "sequence ideotit\ '\ consen atnc amino acid substitutions are counted as a match when detcmiimng sequence homologj in other words, to obtain a polypeptide or poh nucleotide inn ing 95% sequence homologj n ith a reference sequence, K5')o, preferably 90%, e\eπ more preJcrabl) 95% of (lie amino acid residues or nucleotides in the reference sequence must match or comprise a conscn am c substitution %\ ith another amino acid oi nucleotide, or a number of ammo acids ot nucleotides up to i *% preferabl yp to J0% c\en more preferøbh up Io ">% oi the total ammo aud residues 01 nucleotides not meludmg c-onsen am e substitutions in the reference. sequence ma\ be inserted into the refucnce sequence Preferαbh the homolog sequence wompi ises. at leas>t a stiekh of M) ev en mnu ptefuabK .U Last 100 e\en more ^ prcferabK at least 2*0 and e\uj mote prefcrabK at least ^)O nucleotides
\ consols αine sαbslilution rofct^ to the substitut io an ammo acid KSiduc ot [Riwϊcottdc w ith another amino <κ ιd icbiduc or nucleotide lia\ mg s.innlai ckikK ietibttw^ or μioperlies niclαdiug si/e h\droplιobicit\ etc such that the mciall futκuonabt\ doe*, not change sigmftcauth ϊ&oiaicd means aheied tπ ihc hand of man IJ oni its naiurai state t e if it occuis in tiαtutc it ha* been changed ot icmoxcd fiom sts ongniϋl cm ironment or both For
«,\ampic a poh nucieotidc O) poh peptide iiauoalh present in a irung or^anj^m is not isolated but the ssme poh nucleotide or poh peptide separated from the coexisting male! ials of sts nstuta) state i* isolated a* the term i* cinplo\ ed herein Thus the immunogenic composition as used herein also icicrs to a composition thai comprises PC V2 ORI 2 protein wheiem said PCV 2 OR! 2 piotcm is am one of those desL-πbed abo\e Prefeiabh said ?C\ 2 DlU 2 protetn is
0 a poh peptide compmmg the sequence of SbQ ID NO 4^ SbQ ID NO 6
SLQ ID NO 9 SLQ lD NO 10 OJ SLQ i D NO 1 1 iiϊ aπ\ poK peptide that is at least 80° <• liomologoits to the pohpepttde of 0 i«) am immunogenic poition of tiie poh peptides oi 0 and oi ii) i\ i the immunogenic portion of HI) comprising at lu&t 10 contiguous ammo acsds included in the sequences of SLQ ΪD NO * SLQ ID NO 6 SLQ ID \O Q SFQ lD KO 10 or SFQ ID XO I l v) a poh peptide thai is encoded b\ a DNA comprising the sequence of SBO
ID MO 3 Oi SEQ ID NO 4 v i) am poK peptide thai is encoded In a poK nucleotide that is at least 8<*°o homologous to the polynucleotide of \ ) v i i) am immunogenic portion of the polj peptides encoded bj the poh nucleotide of \ ) and or \ i) v iii) the immunogenic poiUoii of MO therein polynucleotide coding for said immunogenic ponion comprises at least "<() contiguous nucleotides, inc luded in {he sequences of SFQ JD \0 3, ot SFQ ID KO 4 Prcfctabh am of those immunogenic portions hα\c the immunogenic ciwaαeπsties. of PC\ 2 OR $-2 pi otc ui Jhst « encoded b\ the sequence of SFQ ID NO 3 or SFQ 10 KO 4
According to a futmei aspect, PCV2 ORF2 piotem is pimided in the immunological composition at an antigen inclusion ICΛ CI effective for inducing the dciucd uninunc response, nameh tedυcmg the incidence of lessening the se\erit\ of ot prev enting one ot more cluneal stgns icsυltmg from PCV2 infection Prefesabh {he PC V2 OR^-2 prolejn mcluston ic\cl ii at least <> 2 μg antigen mi of the final immunogenic composition (ug mJi more prefeiabh from abotit O 2 to abotit 400 μg ml, still moie preferabh from about O "* to about 200 μg ml, e\en inoic prefer abh from about O 35 to about 100 μg ml, still mose prefesabh from about 0 4 to about 50 ug/ml. still snore preferabij from about 0 45 to about 30 μg. mi sull more prcfetabh from about 0 6 to about 15 μg %l e\ en more prcfcrabU from about 0 7*» to about H μg nii, c\cn more preferabh from about 1 0 to about Ci μg<'ml. still more preferabh fiom about 1 3 to about 1 0 μg ml, cscn iπoie preferabh fiom about 1 4 to about 2 5 μg nsl c\en more prefesabh fsosn about I 5 to about 2 0 μg ml and most piefcrabh about S 6 μg/nil According to a further aspect, the OR.F2 antigen inclusion level is at least 0.2 μg/ PCV2 0RF2 protein as described above per dose of the final antigenic composition (μg/dosc). more preferably from about 0.2 to about 400 μg/dose, still more preferably from about 0.3 to about 200 μg/dose. even more preferably from about 0.35 to about 1Ou μg/dose, sliii more preferably from about 0.4 to about 50 μg/dose. still more preferably from about 0.45 to about 30 μg/dose. still more preferably from about 0.6 to about 15 μg/dose, even more preferably from about 0.75 to about S μg/dose. even more preferably from about 1.0 to about 6 μg/dose, still more preferably from about 1.3 to about 3.0 μg/dose, even more preferably from about 1.4 to about 2.5 μg/dose. even more preferably from about 1.5 to about 2.0 μg/dose. and most preferably about 1.6 μg/dose.
The PCV2 O.RF2 polypeptide used in the immunogenic composition in accordance with the present invention can be derived in. any fashion including isolation, and purification, of PCV2 O.RF2. standard protein synthesis, and recombinant methodology. Preferred methods for obtaining PCV2 OR.F2 polypeptide are provided in U S. Patent Application Serial No. 1 1/034,797, the teachings and content of which are hereby incorporated by reference. Briefly, susceptible cells are infected with a recombinant viral vector containing PCV2 O.RF2 DNA coding sequences. PCV2 OR.F2 polypeptide is expressed by the recombinant virus, and the expressed PCV2 OR.F2 polypeptide is recovered from the supernate by filtration and inactivated by any conventional method, preferably using binary ethyjcnimine, which is then neutralized to stop the inactivatbn process.
The immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 protein described above, preferably in concentrations described above, and ii) at least a portion of the viral vector expressing said PCV2 ORF2 protein, preferably of a recombinant baculovirus. Moreover, the immunogenic composition can comprise i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ii} at least a portion of the viral vector expressing said PCV2 ORF2 protein, preferably of a recombinant bacuiov trαs. and iii) a portion of the ceii culture supemate.
The immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ii) at least a portion of She viral vector expressing said PCV2 OR.F2 protein, preferably of a recombinant baculovϊrus. and Hi) a portion of the cell culture; wherein about
90% of the components have a si/.e smaller than 1 μm.
The immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ti) at least a portion of the viral vector expressing said PCV2 ORF2 protein, iii) a portion of the cell culture, i\) and inactivating agent to inactivate the recombinant viral vector preferably BEl, wherein about 90% of the components i) to iii) have a size smaller than 1 μro. Preferably. BEl is present in concentrations effective to inactivate the baculovinis. Effective concentrations are described above.
The immunogenic composition as used herein also refers to a composition that comprises t) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, it) at least a portion of the vira! vector expressing said PCV2 ORF2 protein, iii) a portion of the ceil culture, iv) an inactivating agent to inactivate the recombinant viral vector preierabiy BET. and v) an neutralization agent to stop the inactivalion mediated by the inactivating agent, wherein about 90% of the coinpo.ne.nis i) to iii) have a size smaller than J. μm Preferably, if the inactivating agent is BEL said composition comprises sodium thiosulfate in equivalent amounts to BEl. The polypeptide ss incorporated into a composition thai can be administered to an animal susceptible to PCV 2 infection in preferred forms., the composition inaj also include additional components known io those of skill in the art (sec also Remington's Pharmaceutical Sciences ( 1990), 18th cd. Mack Publ , Easton ) Additionally , the composition ma\ include one or more Λ eterinarj -acceptable earners ^s used herein, "a \etcπnar> -acceptable earner" includes an\ and all solvents, dispersion media, coatings, adjin anls, stabilizing agents, dduenfe. piemen ath es. anubactei ial and antifungal agents, isotonic ageins, adsotption delaung agents, and the like In a preferred embodiment, the immunogenic composition conjpns.es PC V2 ORF2 protein as piovjded herewith, preferabh m concentrations described above, which is mixed with an adjm ant. picferabh CarbopoL and pin siological salme
Those of" skill in the art Λ\ ill understand that {he composition used herein. ma\ incorporate known injectable, physiologically acceptable sterile sohitton*; For preparing a rcad> -Jo-usc solution for parenteral injection or mfusion. aqueous isotonic solutions, such as e g saline or corresponding plasma protein solutions, are readih available In addition, the immunogenic and utccuie compositions of the present (m ention can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include nater. saline, dextrose. efhanøl. glycerol, and the like Isotonic agents can include sodtuni chloride, dextrose, manmtoi. sorbitol, and lactose, among others. Stabsls/ers include albumin and alkali salts of elirj leiϊdiamintetracetic acid, among others
"ΛdjuΛanis" as used herein, can include aluminum hulrovule and aluminum phosphate, saponins e- g , Quil A. QS-21 (Cambridge Biotech Inc , Cambridge MΛ>. GPl- OjOO (Galerøca Pharmaceuticals. Inc.. Birmingham. ΛLh water-m-oil emulsion. oιl-tn-\uιter emulsion, vaiet-m-oil-in-watci emulsion The emulsion can be based in particular on light liquid paraffin oil (Euiopcan Pliatmacopca ij pe), isoprenoid oil such as &quaiaue oi squalene oil resulting from theoligoraerization of alkeαes, in particular of isobuiene or decene; esters of acids or of alcohols containing a linear slkyi group, more particularly plant oils, ethyl oleate. propylene glycol dKcaprylate/caprate), glyceryl trj-(capryiate/caprate) or propylene glycol dioleate; esters of branched laity acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifierss to form the emulsion. The emuSsifiers are preferably noniotiic sinfaciatits, in particular esters of sorbitan, of niatinide (e.g. anlrydroπiatnsitαl oleate), of glycol, of poiyglycerol, of propylene glycol and of oleic, isostearic, ricinoieic or hydroxystearic acid, Λvhich are optionally ethoxylsted. and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L 121. See Hunter et al. The Theory and Practical Application of Adjuvants (Ed.Slεwsrl-T.uH, D.E.S.). JohiiWiley and Sons, NY. pp51-94 (1995) and Todd et a!., Vaccine 15:564-570 (3997).
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.
A further instance of an adjuvant is a compound chosen from the polymers of acrylic or Hiethacrylic acid and the copolymers of inaleic anhydride and aikenvl derivative. Advantageous adjuvant compounds are the polymers of acrylic or røethacrylic acid which are cross-linked, especially with polyaikeny! ethers of sugars or polyalcohols. These compouads are known by the term carbomer (Phameisropa 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 hydroxy! groups. preferably not more than 8. the hydrogen atoms of at least three hydroxy Is being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing fromn 2 to 4 carbon atoms c g \ m\ ls. aSh ls and othei etln lerucaJh unsaturated gjoups I he misahnated radicals ma\ tiiemseh es contain other subsUHients. such as rncthv i The piodυcls sold undcs the name Caibopol, (BF Goodπcli Ohio, USA) ate μarticularh appiopπale The\ are erobb-linked w ith an alh 1 buciose oi \wth alh l penlaen thπtoi Among them tlicic mav be mentioned Carbøpol 974P, 914P and 97} P Most μicfeπed is the UAC of Carboμol, in μarttαilai the UΛC of Caibopol O7IP prefcrabK in amounts of about ^00 ug to about 5 mg pt-t dose, c\cn more ptefcϋed m an amount of about 750 μg to about 2 5 ing per dose and most picicncd in an amount of about 1 mg pet dose
Further sunαblc αdjii\anis include but aic not intuted to {he RΪB1 ad]u\ anl s\ stera (Ribi iiic ), Biock co-poh tnci (,(MR\ Atlanta GA), SAF-M (Chπon, Fmowilie C A) moπophcsphoiλ ! lspid \ ΛM idJπc lipid-amtoe ad|y\ ant beat-labile cntεtoto\Jπ (torn F coli (lecomb)tunt or othcnv iso* cholera toxin, I MS 1314, or muionn i drpepUde ansong monv othei s
Prcfεtabh the adjuv ant is added m an amount of about 1Ou μg to about H) mg pei dose Hcsi more prefeiabh {he adpj^ant is added in an amount of about 100 ug to about 10 mg pei dose Ih en more prcferabh the adjmant is added in an amount of about 500 μg to about * mg per dose ϊ\en more preferabh . the adim ani is added m an amount of about 7^0 μg to about 2 ^ mg pei dose Most prefeiabh , the adμπanf is added in an amount of about J mg per dose -VddUsosialh , the composition can meludc one or nsoie pharmaceutical-acceptable carriers Λs u<>e4 herem "a pharntaccυUcal-dcecptablc caπicτ" includes ao> and all iohcnts dis>pers)ioπ media, coatings stabili/mg age-nts diluenb, prcser\ ati\es>, antibacterial and antifungal agents, isotonic agents adsorption dela\ mg agents, and the hkc Most ptefciabh the composition prov ided herew ith, contains PC\'2 ORΨ2 μiotein teccneicd from the supcπiate of ;»? vttro cultured ccih. whercm said cells v\cie infected viih a iecombtnant \ual \ color oontaming PC V2 ORl: 2 DN \ and expicsstng PC V2 ORi 2 protein and therein said ceH cuitinc \Mκ> neaied vuth about 2 to about X niM BLl prcieuibh vαth about *> ra\i B£I to inactivate the \iraϊ vector and an equnalcm concentration of a neiitiaii/auoit agent picferabh sodium thiosulf<&, bokitmn in a final coiicenu<rtιon of aboul 2 to about 8 mM prcfcrabK of about ^ m\t
The present .m ention also tϋatc^ to an immunogenic composition thai compttscs i) am oi the PC\ 2 CSRf 2 proicms described abo\ e preferabl y ooncentrauons descnbed abo\c ϊi) al leas-l a portion of tiic \ ua! \eUor cvprcfewng Mid ΨCV2 QRF2 protein tϊt) a poiuon of the cell culture n ) a« snacm ating agent to inactivate the iecoinbinant ^ nal %ccto prefeiabh Bt I and \) an ncutrali/ϋtion agent fo stop the tnactnation mediated In the tnactπatiβff agent picfcrsbh sodram thipsulfatc m cqui\alcnJ amounts to BM snd \i) a suitable aditπsnt profit abh C srbo}X3l 97ϊ m araounts dc&cπbcd abo\c whuem about (Mf <> of the component1? rt to m) hav e s >?s/c smaUεi Jhsn 1 μm According to a ftiiihcr aspect ihss imtiuinogctnc composition further unnpπses s phanwseeui ical acceptabSe sail prcierabh a phosphate salt m phv sioiogicalh acceptable concentrattons Picicrabh the pH of said immunogenic composition is adjusted to a pin Mϋiogical pli mediimg between about 6 5 and
The immunogenic composition as used hetein also refei s to a composition thai comprises pes one mi S) at least 1 O ug of PC V2 ORi 2 piotcm descπlx'd abtπe ii) at least a [X)HiOt) of bacuioMrus c\ptess)t)g said PC V 2 0RF2 protein iii) a pouion of the cell uiltute iv ) tilxnft 2 So 8 m\i BFT -\) sodmni thiosulfate in cqtiι\ aknl amounts, to BFl and ^ i) about ! ing Caibopoi O7S and \iϊ) phosphate sail sn a pliv biological i\ acceptable concentration λsheiem about 'JO0^ of the components ι) to uO h<n e a si/e smailei than 1 uin and the pH ot said immunogenic composition is adjusted to about 6 ^ to 7 ^ The immunogenic compositions can further include one or more other immunoniothilatory agents such as. e.g.. interieiikiiis. interferons, or other cytokines. The immunogenic coin positions can also include Gεntaniiein and Merthioiate. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan, the present invention contemplates compositions comprising from about 50 μg to about 20(H) μg of adjuvant and preferably about 250 μg/ ml dose of the vaccine composition. Tints, the immunogenic composition as iised herein also refers to a composition that comprises from about l ug/ml to aboui 60 μg/nil of antibiotics, and more preferably less than about 30 μg/nil of antibiotics. The immunogenic composition as used herein also refers to a composition that comprises i) any of the PCV2 ORF2 proteins described above, preferably in concentrations described above, ti) at least a portion of the viral vector expressing said PCV2 ORF2 protein, iii) a portion of the cell culture, iv) an inactivating agent to inactiv ate the recombinant viral vector preferably BEl. and v) an neutralization agent to stop the mactivation mediated by the inactivating agent, preferably sodium thiosulfate in equivalent amounts to BEi; vi) a suitable adjuvant, preferably Carbopol 971 in. amounts described above: vϋ) a pharmaceutical acceptable concentration of a saline buffer, preferably of a phosphate salt, and viii) an anti- microbioϊogical active agent: wherein about 90% of the components ϊ) to iii) have a size smaller than S μm, It has been surprisingly found, that the immunogenic composition comprising the
PCV2 ORF2 protein was highly stable over a period of 24 months, ϊt has also been found the immunogenic compositions are very effective in reducing She clinical symptoms associated with PCV2 infections. Tt was also discovered, that the immunogenic compositions comprising the recombinant baculovirus expressed PCV2 ORF2 protein as described above, are surprisingly more effective ύian an immunogenic composition comprising the whole PCV2 virus in an inactivated font), or isolated viral PCV 2 ORF2 antigen. In particular, it has been surprisingly found, tiiat the recombinant baciiløvirus expressed PCV2 ORf;2 protein is effective in very low concentrations., which means in concentrations up to 0.25 μg/dose. This unexpected high immunogenic potential of the PCV2 ORF2 protein is increased by CarbopoL Examples 1 to 3 disclose in detail the production of PCV2 GR F2 comprising immunogenic compositions.
The immunogenic composition as used herein also refers to Ingelvac i. CireoFLEX"* 1. {Bochringer Tngeϊheira Vcimcdica. Inc., St Joseph, MO, USA), CircoVaeOt- (Medal SAS, Lyon, France! CircoVenl (intervet inc.. Miiisboro. DE. USA), or Sitvaxyn PCV-2 One Dose,f (Fort Dodge Animal Health, Kansas City . RA, USA.).
Administration of the immunogenic composition
The composition according to the invention may be applied intradenrially, intratrachealh", or iniravagmalh'. The composition preferably may be applied iπϊramnscularly or intranasally. most preferably intramuscular! Iy, ϊπ an aniπia! body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous or by direct injection into target tissues. For systemic application, the intravenous, intravascαiar, intramuscular, intranasal, intraarterial intraperitoneal, oral or intrathecal roαtci are preferred. A more local application can be effected subcutaneouily, intradermals, intracutaneous!}?, intracardtally, ijrtralobaHy. iniraπϊeduilarϊy. ϊnlrapumionarily or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, epithelial tissue). Depending on {he desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also iittentuttcnth fos instance on a dash basis, for several d<π s necks or months and in different dosages.
Prdcrabh at least one dose of the immunogenic compositions as described abov e is intramuscula administered to the subjec t need ϊheieof \t cording to a Au the: <i>>pcc l ^ the PC\ -2 antigen ot the immunogenic composition contpt isiπg an\ such PCV-2 antigen as desc ribed abmc is formulated and administered in one ( I I ml pet dose Thus accoidtng to J fiiilhcr aspect the pjcbtnt im cnlion aϊbo alales. in a 1 ml unmunngentς. t ompniition coinpn^iiig PC VJ antigen as desc ribed hereii for icducing or lessening K raplisdcnopatln h mphojd depiction <md or muHmαcicotcd giant imiuxΛ tcs in pigs infected with S5C V2 ^ccoidmg to a furthet aspect aα-oiding to Λ iαrthei a&pect the pies^nt iincntion also relates to a 1 mϊ tinmuoogcnic conipcsuion coinpπstog PC\ -2 aotsgcn a* dcsu ibed herein foi reducing ot lessening K raphadenopstln m t-onibmatJon with one ot a muitipic oi the following "n itiptpitis in pigs O ) intcr^itial pncυniomα w ύh tmcrlobular edema (2) vutancøys palloi oi sciei us (3) mottled atropine lnεts (4) gastπc ulcεts (^) ncphmis and (G) reproductt\e disordej«i e g abortion stiUbtrUM mummies
\ctordmg to a further aspect at least one iurthei adtnimsudtion of at least one dose of the jmmuKogeim, composition as desscnbed abo\ e is gn en to a subject m need thcreoi whcfctii the second oi am further administration is gnc« at least 14 da\ ς be\ ond the mtltal or am former αdmimsuations Prefesabh the immunogenic composition is administered w ith an mnauπe siumilant Prefeiabh said msraune siumilant is gι\en at least ftuee Piefcrabh at least 3 da\ s more prcicrabh at least *> da^ s c\ en more psefcrabh at least 7 da\ s die in between the fii&t and the second or am fia the-r adπunnitwtiOK oi the immune stimulant Prcfesabh the imnπine stmπilant is gnen at least ] o din s pretesabK I* ikn ^ c> cn more prcierøbh 20 even mote picferabl\ at least 22 da\ s be\ond the initial adimtusUauoii of the immunogenic composition provided hciein \ prcienred munαne sumu ldnt ss Jot example k-c\ hoJc limpet hensocv asun (KLH) ptefesabh emulsiUed with incomplete ficitnd s aditn aiU (KJLIi ICl Λ> However it is herew ith iindcisiood that am otlici immune stnnulant know n to a person skilled HΪ the art oan also be used The terra iranmiK stumiiani as used huein means aπ\ agent or composition that can tnggu the nninunc response picferabK without ntitiδung or increasing a bpecifk nnntutK response for example the mimuite response agam&l a speciilc pathogen It is furthet instmctcd to tsdmnmter the immune stimulan tn a buitabk do^t
\torcos et it lias also been surpmmgh round that the iintnuiiogcuie potential of the immunogenic compositions, usαl herein ptvfeiabK those that t-ompnse recombinant bduilounjs expicss^d PC\ 2 ORF2 protein e\en mor*. pjcferablv in combmauon with C'atbopol can be lnrihcr confiimcd In the sdrainistrαuoii of the lngc!\ ϊk, PRRS \\L\ •\<i(,C)tκ {i.ec Fxampic >) PC V 2 cluneal signs and disease manifesiations atv greajK magnified when PRRS wfcctton is ptescnt Ho\\e\ei the immunogenic compositions *; and \a«.cjnaiio» sitategies ss prm tded herew ith Serened th« cfleet great h and more than expected In other voids an unexpected sujcrgistic eftect was observed when animals pjdenibh piglets were treated w nh am of the PC\ 2 ORF2 immunogenic, compositions as proΛ idcd hcrew Uh and thc lngeh dt PRRS Ml \ \acune ^Bochi mgei ingeiheim)
BRIEF DESC R IPT ION OF T H E DR AW INGS
I-igtiic 1 is> a schematic flow diagram of a piefcncd construction of PC\r2 ORP2 recombinant KIUSICA iru& and
Ligs 2a and 2b are each schematic How diagrams of how to produce one of the compositions os«.d in accordance v\ ith the present invention DE TAILED DESCRIP T ION OF THE PREF ERRED EMBODIMENT S The following examples set forth psefcned materials and pioocduies in accoi dance vwih the μicscnt im cmion Although am methods and materials similar or equivalent to those described herein can be uv,d in the piactice or testing of (ho present iincnjinn the prcfct tcd inelhixk dev ices and materials aic now desc ribed It is Iu be understood liowev ci that these examples arc provided b\ %\a\ of illusitaiion onK and noilπriji ilierem should be deemed a limitation upon (he otcwll bcopc of the intention
EX AMPL E 1 This example compares the rciatnc \ (elds of 0RF2 using methods of the present imcmioti w ith tneJhods that me known m the pπoi ait Foui lOOOmϊ sptoner ilasks «eic each seeded \uth approxtmatelv 1 O\ 1OC Sf* ceUs/ml m 3t.Mk«l of mseα set uni fiec medui F\ccl] 420 ( I RH Btoseicnces ϊtic ϊ cncxs KS) The itisitcr cell cnltare is identified as SF* {SpOitapicn ff iivipCiM) MasJei Cell Stock, passage 19 ϊ otwl\ S 12-0^W The celk used to geneiate the St* Mastei Cell Stock -ivere obtained frøiw Fiotcm Sciences Coipoiation. Itic Vicπden, Cϊ J lie SF- cell line !or tins example was confined between passages 19 and 59 Other passages w ill work foi puηxises of ihc present linetition. bnt m order to scale the pi occss tip for Luge scale production at least IV passages wtH probabh be nccessan and passages bev ond 5(> ma\ hax e an cfiect on expresstoti although this was not tm esttgated In more detail the initial Sl - cell cultures from liquid nitsogcn storage wete grown m LvccH 420 media m suspension m stcnle spunicr ilasks w ith constant agitation The cuituies were grown m 100 jnL to 250m L spumer flasks w ith 25 to i ^O ml of Excell 42* * s.erum-free media When the cells had multiplied to a cell deπsm of 1 0 - 8 O x 10υ ceils mL tbc\ were spht to new v essels w ith a planting deiisiK of 0 ^ - 1 5 x Hf cells niL Subsequent expansion outHircs were grown in spinner flasks up to 3f> Itlej s MI si/e or m stainless steel bsos eaoloi >s of up to 300 liters foi a pes sod of 2-7 An s at 2^ - 29K
AXler seeding the flasks wcic incubated at IT3C for four hunts Subscquenth each flask Md^ seeded -vuili s ι«,ombιrωnt baculot inib containing the PCV 2 0RF2 gene ( SEQ ID \O 4) The recombinant bac ulowiits containing the PCV2 ORF2 gene was genetated as follow s the PC X'2 ORF2 gene ftoin a North Ajncπcan strain of PCV2 was PCR antplificd to tniuain a V ko/ak's sequence (^EQ ID NO 1 > and <t 31 EcoRl site (SEQ ID ^O 2), and cloned imo tlic μ&FM-T-Fas\ \ cctot {Promcga Nϊaclison W l ) Then it \\αs> s>ubscquci!tK excised and subcloncd into l!κ tiansϊcj v ector pλ'ϊ H 92 ( BD Biosci^nces Phai min^cn Sat) Diego C -Y) The subcloncd pomon is rcpicseutcd heiem as SFQ ΪD XO ? The p\ 1 1392 pln^tntd ootitsitijug the PCV 2 ORF2 gene was designated N4?~064\ and then co-iroiisfcoicd with BaculoGoldJC (BD Bioscicnces Phomiiugcn) baciiim iius D\ Λ into Sf- inscci ecus. (Protein Saen.cc*; \!cnden CT) to gctiesate the recombinant baciilo^ iras containing the PCV 2 ORF2 gene l hc new coiKtiuct is piomϊed hereto as SbQ ID \O 8 The recombinant baculox πus containing the PCV'2 OR 1-2 gene was pϊaqae-pm ilied and Master Seed Virus (XiSV) w as piopagatcd on the Sϊ ^ cell line ahquotted, and sioicd at -7O0C The MSV was posttneh identified as PCV2 0RF2 baculo\mis b\ PC R-Rt LP using baculo\ iπis specific pi nners Insect cells infected wtth PCX 2 ORF2 baαύoMms to generate XiSV oi W orking Seed Virus e\prcs<? PC\r2 ORf 2 antigen as. dcteoied b\ poh clonal serum or monoclonal antsbodses m an mdu ca Ouorcsceiit anUbtxh assa\ Addiuonalh the tdenittj of liic PC \r2 ORF2 baαtkn inis \\a<> confmncd b\ λ-temimal amino nctά sequencing The PCX 2 0RF2 bauiioλ uui MSX was also tested for puπft in accoidance -v\«h 9 C f R 1 P 27 (e) 1 13 28 and 3 13 51^ Lach iccombuωtn baeυkn jrus seeded into lite spinner flasks, had van ing multipϊ femes of infection (MOIs) Flask 1 was seeded w ith 7 ^2mϊ of 08H Mf)I seed, ilαs.k 2 was seeded with "< OhiiL of O ><>MO1 seed flask 3 vαs seeded with I ^mL ol 0.18MOI seed; and flask 4 was seeded with 0.75ml of 0.09MGi seed. A schematic flow diagram illustrating Jiie basic steps used to construct a PCV2 0RF2 recombinant baeuiøvirus is provided herein as Figure S .
After being seeded with (he baculo virus, the flasks were then incubated at 2? & 2 X for 7 days and were also agitated at lθl)rpm (hiring that time. The flasks used ventilated caps to allow fot air flow. Samples from each flask were taken every 24 hours for the next 7 days.
After extraction, each sample was eentrifuged. and both the pellet: and the supernatant were separated and then mieroftiiered through a 0.45-1 ,0 μm pore size membrane.
The resulting samples then had the amount of OR.F2 present within them quantified via an ELTSA assay. The ELlSA assay was conducted with capture antibody Swine anti- PCV2 Pab IgG Prøt G purified (diiuted 5 :250 in PBS) diluted to 1 :6000 m 0 05 M Carbonate buffer (p K 9.6). 100 μL of the antibody was then placed in {lie wells of {he inictrotiter plate, sealed, and incubated overnight at 37ϋC. The plate was then washed three times with a wash solution which comprised 0.5ml.. of Tween 20 (Sigma, Si Louis. MO). 10OmL of H)X D- FBS (Gibco Iπvitrogen. Carlsbad, CA) and 899.5mL of distilled water. Subsequently. 250 μL of a blocking solution (5g Carnation Non-fat dry milk (Nestle. Glendale, CAj in 1 OmL of D-PBS QS to 10OmL with distilled water) was added to each of the wells. The next step was to wash the test plate and then add pre-dihited antigen. The pre-dilυted antigen was produced by adding 200 μL of diluent solution C0.5mL Tween 20 m 999.5mL D-PBSj to each of the wells on a dilution plate. The sample was then diluted at a 1:240 ratio and a 1 :480 ratio, and 100 μL of each of these diluted samples was then added to one of the top welis on the dilution plate (i.e. one top well received 100 μL of the 1:240 dilution and the other received 100 μL of the 1 :480 dilution). Serial dilutions were then done for the remainder of the plate by removing K)O μL form each successive well and transferring it to the nest well on the plate. Each well was mixed prior to doing the next transfer. The test plate washing included w ashmg the plaic three tmses with the wash buffer. The plate was then sealed and incubated for an hour al 37X before being washed three more times w ith the wash buffer. The detection antibod> used was monoclonal amibod\ to PCV ORF2 St w as diluted to ! 300 in diluent solution, and 100 μL of the diluted detection aiitiboch w as then added to the wells The plate was then scaled and incubated dor an hour at 3 T5C before being washed three times w ith the wash buffer. Conjugate diluent was then prepared b> adding normal rabbit scrαm (Jackson Immuuorc search. West Gtwe, PA) to the diluent solution to \% concentration. Conjugate antibod> Goat anti-mouse (H + ] )-HRP (Jackson imnninorescareh) was diluted in the conjugate diluent to L K)J)Oo. l oo μL of the diluted conjugate antibod> was then added to each of the veils The plate was. then sealed and incubated for 45 mmutes at 3?^C before being washed three times with the wash buffer. 100 μL. of substrate (TMB Peroxidase Substrate, Kirkgaard and Pern. Laboratories (KPLs. Gaithcrsbcrg. M D), raised w ith an equal volυme of Peroxidase Substrate B (KPD was added to each of the wcHs The plate was incubated at room temperature for 15 minutes 100 μl. of IN HCL solution was then added to all of the wells to stop the reaction. The plate was then run through an ELlSA reader The results of this assaj are provided m Table 1 below :
Figure imgf000032_0001
Figure imgf000033_0001
These results indicate that when the incubation lime is extended, expression of ORF2 into the supernatant of the centrifoged cells and media is greater than expression in the pellet of the centrifoged ceils and media. Accordingly, aiiøwmg the ORF2 expression to proceed for at least 5 da\ s and reeo\ering it us the supemate rather than atknvusg expression to proceed for less ihan 5 da\ s and reeo\ eruig QRF2 from ihc cells, pan icles a great increase in ORF2 \ tdds. and a significant improvement cncr prior methods.
EXAMPL E 2
This example pres ides data as to the effieacs of the im cniion claimed herein A. 10u{)mL spuHicr flask Mas seeded with approximate!) 1.0x10' Sf- ceHs/ml m 30UmL of Exeell 420 media The flask \sas then incubated at 271C and agitated at lOOtpm Subsequent Iv . the flask s\as seeded %\ ith K) ml of PCV2 ORF2 Bac p * <> (the recombinant baculos iriis contamin^ the PCV2 ORF2 gene passaged 6 additional times in the Sf) insect ecus) \ irus seed \\ ith a 0 1 MOf after 24 hours of racubation..
The iiask was then incubated at 27"C for a total of 6 das s After incubation, the fiask was then eentrifiiged and three samples of the resulting supernatant were harvested and inactivated. The supernatant was inactnated b> bringing its temperature to 37 -*- 20C. 'I o the first sample, a 0 4M solution of 2-broπ.iocth% ieiieainm.c h\ drobromidc winch had been cv cUzed to 0 2V1 bmary ethh lennnine (BEi) in 0.3N NaOH was added to the supernatant to {»ι\e a final concentration of BEl of 5mM. To the second sample. 1 OmM BIH was added to the supernatant. To the third sample, no BEl was added to the supernatant The samples were then strrred continuous!} for 48 hrs. A i .0 M sodium tluosuifatc solution to grve a final minimum concentration of 5 m.M was added to neutralise an} residua! BEI. The quantity of ORi'2 in each sample was then quantified using the same ELl SA assav procedure as described in Evainple 1. The results of this tπa> be seen in Table 2 below :
Figure imgf000034_0001
Figure imgf000035_0001
This example demonstrates that neutralization with BF.1 does not remove or degrade significant amounts of the recombinant PCV2 ORF2 protein product. This is evidenced by the fact that there is no large loss of ORF2 in the supernatant from the BEI or elev ated temperatures. Those of ski!! in the an will recognize that, the recovered ORF2 is a stable protein product.
EXAMPLE 3 This example demonstrates that the present invention is scalable from small scale production of recombinant PCV2 OR.F2 to large scale production of recombinant PCV2 ORF2. 5,0 x Mf cells/ml of SF> cells/m! in 700OmL of ExCeIl 420 media was planted in a 2000OmL Λpplikon Bioreactor. The media and cells were then incubated at 27°C and agitated at !00RPM for the nesX 68 hours. At the 68!h hour, 4i,3mL of PCV2 ORF2 Baculoviπis MSV+3 was added to 7000ml.. of ExCeIl 420 medium. The resultant mixture was then added to the bioreactor. For the next seven days, the mixture was incubated at 27°C and agitated at 100RFM. Samples from the bioreactor were extracted even1 24 hours beginning at day 4, post-infection, and each sample was centrifuged. The supernatant of the samples were preserved and the amount of ORF2 was then quantified using SDS-PAGE densitometry. The results of this can be seen in "fable 3 below: Table d L-ase 3ι t-Oi »78-1- F
! Formatted; Swecte b (Sweden)
Figure imgf000036_0001
EXAMPLE 4
This example tests the efficacy of seven PCY2 candidate uiccmes and further defines parameters following exposure to a virulent strain of PCV2. One hundred and eight esarean derived colostrum deprived (CDCD) piglets, 'M4 da> s of age. ucre randomly di\icied into 9 groups of equal si/e Table 4 sets forth the General Slud\ Design for this e
Figure imgf000036_0002
Figure imgf000037_0001
vORF2 :: isolated vira lRF2: rORF2 ::: iecombiniint hϋctilovπus ex])resse_! ORP2: ktllec! whole cet! virus ~ PCV2 virus grown in suitable cell culture
Seven of {he groups {Groups 1 - 7) received doses of PCV 2 ORF2 poly peptide, one of the groups acted as a challenge control and received no PCV2 ORF2. and another group acted as the strict negative control group and also received no PCV2 GRF2. On Day 0, Groups i through 7 were treated with assigned vaccines. Piglets in. Group 7 were given a booster treatment on Day 14. Piglets were observed for adverse events and injection site reactions following vaccination and on Day 19, piglets were moved to the second study site. At the second study site. Groups S -S were group housed in one buiiding vhiie Group 9 was housed in a separate building. All pigs -received keyhole limpet hemoevaniti (KLH (/incomplete Freund~s adjuvant (ICFA) on Days 21 and 27 and on Day 24. Groups 1-8 were challenged wish a virulent PCV2.
Pre- and post-challenge, blood samples were collected for PCV2 serology. Post- challenge, body weight data for determination of average daily weight gain (ADWG). and clinical symptoms, as well as nasal swab samples to determine nasal shedding of PCV2, were collected. Oft Day 49, all surviving pigs were necropsied, kings were scored for lesions, and selected tissues were preserved in formalin for imtmmoMstotliemtstsy (IHC) testing at a lster date.
Materials and Methods
This was a partially blinded vaccination-challenge feasibility study conducted in
CDCD pigs, 9 to 14 days of age on Day 0. To be included in the study. PCV2 ΪFA titers of sows were < 1 : 1000. Additionally, the serologic status of sows were from a known PRRS- negative herd. Twenty-eight (28) sows were tested tor PCV2 serological status. Fourteen
(14) sows had a PCV2 titer of < 1000 and were transferred to the first study site. One hundred ten ( 1 10) piglets were delivered by cesarean section sisrgeries and were available for this study on Day -4. On Day -3, J 08 CDCD pigs at the first study site were weighed, identified with ear tags, blocked by weight and. randomly assigned to 1 of 9 groups, as set forth above in table 4. If any iesi animal meeting the inclusion criteria was enrolled in the study and was later excluded for am season Use Jmestigatos and Monitor consulted m ordci Io determine ilic use of data collected fiom tiic annual in the final anal) sis I he date of w hich enrolled piglets wen: excluded and the reason for exclusion was documented Imtωlh , no sows, WCJC excluded Λ total of 1**8 of an as dibble 1 10 pigs. s\eic sandornh assigned to one of V groups on Daj -1 The two smallcM pigs iNos 17 and 1 ^) were not aligned to a gioup and wcic available as exitas, if needed Dunnji the course of the studs scv cral aiumals weie removed Pig 82 (Giotip l>) on Da\ -1 Pig Ko 56 (Gioup 6) on Da\ 3 Pig Xo 51 (Oioup V) on Da\ 4 Pig Xo 28 {Gioup X) on Da\ 8 Pig No 6$ (Gioup S) on Da\ 7 and S'lg No 93 {Gioup 4) on D<n 9, were each found dead prior to cballcngc fhcsc si\ p)gs were not included in the final stiwh icsuHs Pig no 1 7 (one of ihc extra pigs) was. asstgtied to Gioup c> The remamtog e\tia ptg No 19 was excluded from the stah
The formulations gι\en to each of the gioups were as. foliows Group 1 was designed to administer ImI of Mtal ORF2 <Λ1)RF2) containing 16 μg ORF2/mϊ This was done b\ miving 10 24 ml of urai ORF2 (256 ug/25 μg/ml - 10 24 ml \ ORF2> w sth 3 2 mϊ of 0 5"i> Catbopol and 2 ^6 ml of phosphate buflered saline at a pH of 7 4 This produced l t> ml of formulation for group 1 Group 2 was designed to ddmimstcr Im! of \ORF2 containing S μg \ORF2/ml f his, was done h\ mi\mg 5 12 ml of \ORS 2 ( 128 μg 2^ μg/ml = ^ 12 ml \ORV21) w ith 3 2 ml of o 5% Catbopol and 7 t>8 ml of phosphate buffered sahue at a pH of 7 4 This produced f (> m! of formulation for gioup 2 Gioup 3 was designed to administer ImI of \ ORl 2 containing 4 μg \ORl 2 ml Hiss was done in tnsxing 2 ^6 ml of \ ORl 2 (64 μg''25 μg ml ~ 2 % ml Λ OR1 2) w ith 3 2 ml of O 5% Caibopoi and 10 24 ml of phosphate. buffered saline at a pϊl of 7 4 This produced lf< mi of foi mulation foi group ^ Group 4 \\as> designed to administer I mi of recombinant ORF2 (rtORF2) containing 16 ug rORF2 ml Fliss w as done b\ miMng 2 23 mi of rORF2 {^ U μg 2Η) μg ml ~ 2 23 ml r()RF2) w ith 6 4 ml of 0 Λ Caibopoi and 23 37 ml of phosphate buffeted saline fit a pH of 7 4 This produced 32 ml of formulation for group 4. Group 5 was designed to administer ImS of rORF2 containing Sμg rORF2/ml, This was clone by mixing 1.11 ml of rORF2 (256 μg/230 μg/ml ~ i. i l ml rORF2) with 6.4 ml of 0.5% Carbopol and 24.49 ml of phosphate-buffered saline at & pH of 7.4. Tlits produced 32 mi of formulation for group 5. Group 6 was designed to administer Im! of rORF2 containing S μg rORF2/ml. This \va& done by mixing 0.56 ml of iORF2 ( ! 28 μg/230 μg/ml - 0.56 mi rORF2) with 6.4 ml of 0.5% Carbopol and 25.04 mi of phosphate buffered saline at s pH of 7.4. This produced 32 ml of formulation for group 6. Group 7 was designed to administer 2inl of PCV2 whoie killed ceil vaccine (PCV2 KV) containing the MAX PCV2 RV. This was done by mixing 56 ml of PCV2 KV with 14 ml of 0.5% Carbopol. This produced 70 mi of forrmtiafton for group 7. Finally group 8 was designed to administer KLH at 0 5 μg/inl or 1.0 μg/m! per 2 mi dose. This was done by mixing 40 71. ml KLH (7.0 μg protein/mi at 0.5 μg/mi ^ 570 mi (7 0 μg/tnl)(x) = (0 5){570 mi)), 244 29 ml phosphate buffered saline at a pH of 7.4, and 285 mi Freunds adjuvant Table 5 describes the time frames for the key activities of this Example.
Table 5. Study Activities
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Following completion of the in-life phase of the study, formalin fixed tissues were examined by lmmuiiohistochemistry (IHC) for detection of PCV2 antigen by a pathologist. blood samples were evaluated for PCV2 serology, nasal swab samples were evaluated for PCV2 shedding, and average daily weight gain ( ADWG) was determined from Day 24 to
Dav 49.
Animals were housed at the first study site m individual cages in five rooms from birth to approximately 5 1 days of age (approximately Day D of the study). Each room was identical in layout and consisted of stacked individual stainless steel cages with heated and filtered air supplied separately to each isolation unit. Each room had separate heat and ventilation, thereby preventing cross-contamination of air between rooms. Animals were housed in two different buildings at {lie second study site. Group 9 (The Strict negative control group) was housed separately in a converted finisher building and Groups 1 -8 were housed in converted nursery building. Each group was housed s.u a separate pen (1 .1-12 pigs per pen) and each pen provided approximately 3.0 square feet per pig. Each pen was on an elevated deck with plastic slatted doors. A pit below the pens served as a holding tank for excrement and waste. Each building had its own separate heating and ventilation systems, with little likelihood of cross-contamination of air between buildings.
At the first study site, piglets were fed a specially formulated milk ration iron) birth to appτosimatcS> ? weeks of age AU psglets were consuming solid, special mixed ration b> Da\ I'J (approximate!) 4 Ij weeks of age). At the second stud} site, all piglets were fed a custom non-medicated commercial mi \ ration appropriate for their age and weight, ad hbnum Water at both study sues w as also a\ ailabfc («/ libitum. AU test pigs vcre treated w ith Vitamin F on Da> -2, with iron injections on Da\ -1 and w ith NAXCFJ . R ( S 0 niL, JM. in alternating hams) on Da\ s 16. 17. I H and 10 In addition. Pig No 52 (.Group 9) w as treated vilh an iron injection on Da\ 3. Pig 45 (Group 6) was treated w ith an iron injection on Daj I K Pig No. 6V (Group S) was Ueatcd with NAXChLJt on Day 6, Pig No 74 (Group λ) was treated w ith desamctha/onc and penicillin on Da> S 4. and Pig No. 51 (Group 1 ) vas treated with dc\amctha/one and penicillin on Da\ 13 and wtth NAXCFL (C on Day J4 for \arious bcallh reasons
While at both studs sites, pigs were under xcteπnan care Animal beaith exannπafions were conducted on Day π and were recorded on the Health Examination Record Form. AU animals were sn good health and tnitπtionat stains before viccsnation as determined b\ obsen a{io« on Day 0 All test animals were obsen. ed to be in good health and nutritional status prior to challenge Carcasses and tissues were disposed of b> rendering Final disposition of stodx animals was records on the Animal Disposition Record
On Da\ 0. pigs assigned to Groups 1 -6 recctx ed KO inL of FCV2 Vaccines 1-6. respectheh . I VI in the left neck region using a sterile 3 π mL Luer-lock sj ringe and a sterile 2Og \ '/j" needle. Pigs assigned to Group 7 reccn cd 2 o nil of PCV 2 Vaccine No. 7 IM in the left neck region using a stcnie 3.0 niL Lυcr-lock sv πngc and a sterile 2Og x f.i" needle. On Da> 14. pigs assigned to Group 7 received 2.0 mL of PCY2 Vaccine No. 7 IM in the right neck region using a sterile 3.0 us L Luer-Joek suinge and a sterile 2Og x 'Λ" needle.
On Daj 21 all test pigs received 2 0 in L of KKH/ICFA IM in the right hain region using a sterile ? 0 mL Luer-lock s\ tinge and a sterile 2Ug xl" needle On Da> 2"? all test pigs received 2.0 ml of JvLH ICFA in the left ham region υsnsg a sterile 3 0 ml Luer-lock s> rmge and a slenle 2Og \ 1 " needle.
On Daj 24, pigs assigned to Groups 1 -8 receiv ed S 0 mϊ. or PCV2 ISUVDi, challenge materia! (? 1 1 log^ TCϊDv/mL) IM in the left nock region using a sterile 3 0 niL l.ucr-lock ij ringc and a iterile 2Og \ J " needle. An additional ! 0 mϊ. of the same material
\\a& administered IX to eacli pig {*> 5 ml, |>cr nostril) using a sterile 3.0 ml . Liter-lock sw inge and nasal canula
Test pigs v ere observ ed daih for overall health and adv erse events on Day -4 and from Day (Ho Day ϊ*> Observations were recorded on the Clinical Observation Record. All test pigs were observed from Day 0 to Day 7. and Group 7 « as further obscn ed from Da> 14 to 21. for injection sue reactions Average daily wctght gain was determined by weighting each pig on a calibrated scaie on Dsj s -3, 24 and 4s-). or on {lie day that a pig was found dead after challenge. Body weights were recorded on the Body Weight Fonn Day -3 body weights were utilised to Mock pigs prior to randomization Da> 24 and Day 49 weight data nas utiϊi/cd to determine the average daih weight gain (ADWCi) for each pig during these time potnts For pigs that died after challenge and before Day 4'λ the ADWG was adjusted to represent the ADWG from Da> 24 to the day of death,
In order to determine PC V 2 serology , \cnotis whole blood was collected from each piglet from the orbital venous sinus on Daj s -3 and 14 For each piglet, blood was collected from the orbnal venous sinus by inserting a sterile capiHaπ tube- into the medial canthus of one of the c\ cs and draining approximately 3Jt in L of whole blood into a 4.0 mL Serum Separator Tube (SST) Gn Day s 24. 31. and 49, Λenous whole blood from each pig w as collected from the anterior Λcna cava using a sterile IHg \ i "/ Vacuiainer needle (Seeton Dickinson and Company , Franklin Lakes, NCΛK Jersey ), a Vacαtamcr needle holder and a I ? mL SST Blood collections at each time point v\eie recorded on the Sample Collection Record Blood in each SST was allowed to dot each SST was then spun down and the serum harvested. Harvested serum was transferred to a sterile snap tube and stored at -70÷ KP C until tested at a later date. Serum samples vcre tested for the presence of PCV2 antibodies by BΪV1-R&D personnel. Pigs were observed once daily from Day 20 io Day 49 for clinical symptoms and clinical observations were recorded on tlie Clin tea! Observation Record.
To test for PCV2 nasal shedding, on Days 24. 25. and (hen every other odd numbered stud)' day up to and including Day 49, a sterile dacron swab was insetted intra nasally into either the left or right nostril of each pig (one swab per pig) as aseptically as possible. swished around for a few seconds and then removed. Each swab was then placed info a single sterile snap-cap tube containing 1.0 mi, of EMEM media with 2% IFBS. 500 imils/mL of PenicJliin. 500 μg/mL of Streptomycin and 2.5 μg/mL of Fungizone. The swab was broken off in the tube, and the snap tube was sealed and appropriately labeled with animal number, study number, date of collection, study day and "nasaS swab." Sealed snap tubes were stored at -40 ± I Cf C until transported overnight ou ice to BiVJ-St. Joseph. Nasal swab collections were recorded on the Nasal Swab Sample Collection Form. BlVl-R&D conducted quantitative virus isolation (Vl) testing for PCV 2 on nasal swab samples. The results were expressed in logics values. A value of 1.3 logs or less was considered negative and any value greater than i .3 logs was considered positive. Pigs thai died ("Nos. 28, 52, 56. 69. 82, and 93) at the first study site were neeropsicd to the level necessary to determine a diagnosis. Gross lesions were recorded and no tissues were retained from these pigs. At the second study site, pigs that died prior to Day 49 (Nos. 45. 23. 5K, 35), pigs found dead on Day 49 prior to euthanasia (Nos. 2, 43), and pigs eulhani/ed on Day 49 were necropsicd. Any gross lesions were noted and the percentages of lung lobes with lesions were recorded on the Necropsy Report Form. } ram each of Use 103 pigs necropsicd at the second stud} sste, a ussue sample of tomii. hiiig. heart, lnci, niescntenc luiiph nock. kidncv and inguinal hnipfa node was placed into a single cotitattiei with buffeted 10% formalin while another tmue sample from the same aforementioned Qi gam. was placed into a Whirl -pak ( Vl-Tccli Diagnostics Lid , ^ Thchuiil L K) and each Whπl-pak was placed or) tec Fach container Λ\as propci K labeled Sample collections ΛK CIC recotdcd on the Necrops\ Rcpoil Form Allci wards foi rnalni-fivcd lib&oe sampks and a Diagnostic Rcqut'&l Fotm were stibmitk'd fot IHC toiitig ITϊC lebling \\a<- conducted in accoidance v\ itϊi standard !SU labotston pioceduro^ for tcccn ing samples sampie and shdc ptcpsiattπu and staining techniques Ftesh ussiies in VVhirl-paks ^eie shipped with ice packs lo the StudK Monitor for stotage (-70^ - i<r C) and possible future use Formahn-fj\εd tissue*; were evainmed b\ a pathologist for detection of PC V2 rn JHC and scoi cd u&ing the following scoring s-s slein 0 ~ None, 1 - Scant posime stanitng few sites 2 = \Jodeiate positive statnmg multiple sites and 3 - Abundant positπc staining diffyse lhioughoul She tissue f>ue to the fact that the pathologist could not positπeK differentiate tngmusl 1 K from mesenteric L\ icsυlts for these tissues were smiph labeled as Ljmph Node and the score gneii the highest scote fot each of the ft\o tissues per animal
Results
Results, fot this example are give nbel ow is noted that one pig from Group V died bcfoie Da\ 0, and 5 snore pigs died post-\accmatiotι (1 pig from Group 4, J ptg fioin Gioup ft. 2 pigs fiom Gioup 8, and 1 pig from Group (» Post-nioitcm cxammatioπ indicated all six died due to underly ing infections that weie not associated w ith vaccination or PMWS
Additional!} no ad\ ctse c\ cots oi uyecuon sstc i eacttotis « es e noted v* tth an\ groups
\\eιage daiK ΛK eight gain (ADW(Jl icsiilts arc picsented below in Table 6 Gioup V the sti K i ncgaU\ e control group had the highest ADWG ( I 06 -i. o 1 ? ibs/dax >. followed b\ Group 5 (0.94*- 0.22 lbs/day), which received one dose of 8 μg of rORF2. Group 3, which received one dose of 4 μg of vORF2. had the lowest ADWG (0.49 * 0.21 lbs/day), followed by Group 7 (0.5Oi 0. S 5 lbs/day), v hich received 2 doses of killed vaccine.
Table 6. Summary of Group Average Daily Weight Gain (ADWG)
Figure imgf000047_0001
vORF2 - isolated virai ORF2, rOR.l l - iccoirtbitinrtt h»culovirus. exjircsstsi ORF2: killed vvhoJe cef! virαs ::: PC'VZ virus grout) in suitable cell culture
PCV2 serology results ate presented below in Table 7. All nine groups were seronegative for PCV2 on Day -3. On Day 14. Groups receiving vORF2 vaccines had the highest liters, which tanged from 1.87.5 to 529.2. Pigs receiving kiHed viraS vaccine had the next highest titers, followed by the groups receiving rORF2 vaccines. Groups 8 and 9 remained seronegative at this time. On Day 24 and Day 31 , pigs receiving vORF2 vaccines continued to demonstrate a strong serological response, followed closely by the group that received two doses of a killed viral vaccine. Pigs receiving rORF2 vaccines were slower So respond serologically and Groups 8 and 9 continued to remain seronegative. On Day 49, pigs receiving vORF2 vaccine. 2 doses of Ae killed virai vaccine and tlie lowest dose of rORF2 demonstrated She strongest serological responses. Pigs receiving 16 μg and 8 μg of rORF2 vaccines had slightly higher IFA titers than challenge controls. Group 9 on Day 49 demonstrated a strong serological response.
Table 7. Summary of G roup PCV2 IFA Titers
AVERAGE IFA TITER
Figure imgf000048_0001
vORF2 - isolate,! vm' ύ O RF 2: rOKf 2 ~ recombinant baculfwjj-us expressed ORP2; killed whole cell virus - F'C'V? virus grown in suitable cdl culture
*For calculation purposes, a <100 ΪFA titer was designated as a liter of "50"; a >6400 TFA uter was designated as a titer of " 12,800". **Day of Challenge ***Day of Necropsy
The results from the post-challenge clinical observations are presented below in Table 8. This summary of results includes observations for Abnormal Behavior, Abnormal Respiration. Cough and Diarrhea. Table 9 includes the results from the Summary of Group Overall incidence of Clinical Symptoms and Table 10 includes results from the Summary of Group Mortality Rates Post -challenge. The most common clinical symptom noted in this study was abnormal behavior, which was scored as mild to severe lethargy. Pigs receiving the 2 lower doses of vORF2, pigs receiving 16 μg of rORF2 and pigs receiving 2 doses of KV vaccine had incidence rates of > 27.3%. Pigs receiving 8 μg of rORF2 and (he strict negative control group had no abnormal behavior. None of the pigs in {his study demonstrated any abnormal respiration. Coughing was noted frequently ήi all groups (0 to 25%}, as w as diarrhea (0-20%). None of the clinical symptoms noted were paϊhognomic for PMWS,
The overail incidence of clinical symptoms varied between groups. Groups receiving any of the \ORF2 vaccines, the group receiving J.6 μg of rOR.F2, ihe group receiving 2 doses of KV vaccine, and the challenge control group had the highest incidence of overall clinical sy mptoms (>36.4%). The strict negative controi group, the group receiving 8 μg of rORF2 and the group receiving 4 μg of rORF2 had overall incidence rates of clinical symptoms of 0%, 8,3% and 9.1%, respectively.
Overall mortality rates between groups varied as well. The group receiving 2 doses of KV vaccine had the highest mortality rate (16.7%); while groups that received 4 μg of vQRF2. 36 μg of rOR.F2, or 8 μg of τORF2 and the strict negative control group all had 0% mortality rates.
Table 8. Summary of Group Observations for Abnormal Behavior, Abnormal Respiration, Cough, and Diarrhea
Figure imgf000050_0001
Figure imgf000051_0001
vOR F2 ::: isolated viral OK F2; i()RF2 ::: recombinant baeiitovirns expressed ORF2. killed, whole eel] s'jrus - PCV2 virus armvn in suitable cell culture
'Total number of pigs in each group thai demonstrated any abnormal behavior tor at least one day
'Total number of pigs in each group that demonstrated any abnormal respiration for at least one day
■Toial number of pigs in each group that demonstrated a cough for at ieasi one day
*I'otal number of pigs in each group that demonstrated diarrhea for at least one day
Table 9. Summary of Group Overall Incidence of Clinical Symptoms
Figure imgf000051_0002
Figure imgf000052_0001
vORF2 = isfϊUfteϋ viral OHYl: rORF2 ■= recombinant baeulwims expressed ORF2; killed whole cell virus. - PC V 2 virus grown in suitable ceil culture
'Total number of pigs in each group Jhst demonstrated any clinical symptom for st least one day
Table 10. Summary of Group Mortality Rates Post-Challenge
Figure imgf000052_0002
Figure imgf000053_0001
PCV2 nasal shedding results are presented below in Table S.1. Following challenge on Day 24. 1 pig in Group 7 began shedding PCV2 on Da\ 2?. None of {he other groups experienced shedding nntii Day 33. The bulk of nasal shedding was noted from Day 35 to Day 45. Groups receiving any of the three vORF2 vaccines and groups receiving either 4 or 8 μg of rORF2 had the lowest incidence of nasal shedding of PCV2 {< 9, 1%). The challenge control group (Group 8) had the highest shedding rate (80%). followed by the strict negative control group (Group 9), which had a» incidence rate of 63.6%.
Figure imgf000053_0002
Figure imgf000054_0001
v()RF2 - isolated vital ORF2 rORP2 ~ recombinant baculovirus expressed ORP2: killed whole eel! virus ::: PCV2 virus grown in suitable cell culture
The Summary of Group incidence of Icterus. Group Incidence of Gastric Ulcers, Group Mean Lung Lesion Scores, and Group Incidence of Lung Lesions are shown below in
Table! 2. Six pigs died at the first test site doting the post-vaccination phase of the study
(Group 4, Η:::i ; Group 6. N::: i ; Group 8, !Ψ::2; Oioup 9, N:::2). Four out of six pigs bad fibrinous lesions in one or more body cavities, one pig (Group 6) had lesions consistent villi clostridial disease, and one pig (Group 9) had no gross lesions. None of the pigs that died during the post- vaccination phased of the study had lesions consistent with PMWS,
Pigs that died posi-chailenge and pigs euthanized on Day 4V were neeropsied. At necropsy, icterus and gastric ulcers were not present in any group. With regard to mean % lisig lesions. Group 9 had Sowest mean % lung lesions (0%), followed by Group 1 with 0.40 * 0.50% and Group 5 with 0.68 * 1.15% Groups 2. 3, 7 and 8 had the highest mean % lung lesions (≥ 7 27%) Each of these fotir groups contained one pig with % lung lesions >71.5%. which skewed the results higher for these four groups. WiJh She exception of Group 9 with 0% lung lesions noted, {he remaining 8 groups had < 36% lung lesions. Almost all Sung lesions noted were described as red/pmple and consolidated.
Table 12, Summary of Group Incidence of Icterus, Group Incidence of Gastric Ulcers, Group Mean % Lung Lesion Scores, and Group InckSence of Lung Lesions Noted
Figure imgf000055_0001
vORF2 = isolated viral ORF2; vORF2= : recombinant baculovirus expressed ORF2; KV or killed whole ceil virus ~ PCV2 virus grown in suitable ceil culture
The Summary of Group UlC Positive incidence Results is shown in Table 13. Group 1 (vORF2 - 16 μg) and Group 5 (rORF2 - 8 μg) had the lowest rate of HiC positive rcstiils 06.7%). Group 8 (Challenge Controls) and Group 9 (Strict Negative Controls) had the highest rate of IHC positive results. 90% and 90,9%, respectively,
Table 13. Summary of Group IHC Positive Incidence Rate
Figure imgf000056_0001
vORF2 -= isolated viral f)RP2; )<>RP2 ::: reeoπibinatn hacitlovirus exptessed ORF2: KV or killed whole cell virus ==• PCV2 virus t'rown in sEntiibfe cell culture Post-challenge. Group 5, which received one dose of H μg of rORF2 antigen, outperformed the other 6 vaccine groups. Group 5 had tiic highest ADWG (0.94 * 0.22 lbs/clay X the lowest incidence of abnormal behavior (0%). the second lowest incidence of cough (8.3%), the lowest incidence of overall clinical symptoms (8,3%). the lowest mortality raic (0%), the lowest rate of nasal shedding of PCV2 (8.3%), the second lowest rate for mean % iung lesions (0.68 ± 1.15%) and the lowest incidence rate for positive tissues 06.7%). Groups receiving various levels of rORF2 antigen overall outperformed groups receiving various levels of vORF2 and the gtoup receiving 2 doses of killed whole ceil PCV2 vaccine performed the worst. Tables 14 and 15 contain summaries of group post-challenge data.
Table 14. Summary of Group Post-Challenge Data - Part 1
Figure imgf000057_0001
Figure imgf000058_0001
vORF2 ~ isolated viral ORV2: rORi' 2 = recombinant bacυiovirυs expressed ORF2; KV or killed whole ce!) virus ::- PCV2 virus s>rosvn in sEiititble ce!) culture
T able 15. Summary of Group Post-Challenge Data - Part 2
Figure imgf000058_0002
Figure imgf000059_0001
Results of this study indicate that all further vaccine efforts should Focus on a rORF2 vaccine. Overall, nasal shedding of PCV2 was delected post-challenge and vaccination with a PCV2 vaccine resulted in a reduction of shedding, ϊmmunohislαchenϊistrv" of selected, lymphoid tissues also served as a good parameter for vaccine efficacy, whereas large differences in ADWG, cϊiiiical symptoms, and gross lesions were not detected between groups. This study vas complicated by She fact that extraneous PCV2 was introduced at some point during the study , as evidenced by nasal shedding of PCV2. PCV2 seroconversion and positive IHC tissues in Group 9. the strict negative control group.
Discussion
Seven PCV 2 vaccines were evaluated in this study, which included three different dose levels of vORF2 antigen administered once on Day 0, three different dose levels of rOR.F2 antigen administered once on Day 0 and one dose level of killed whole cell PCV2 vaccine administered on Day 0 and Day 14, Overall Group 5, which received 1 dose of vaccine containing 8 μg of τORF2 antigen, had the best results. Group 5 had the highest ADWG. the lowest incidence of abnormal behavior, the lowest incidence of abnormal respiration, {he second lowest incidence of cough, {he lowest incidence of overall clinical symptoms, the lowest mortality rate, the lowest rate of nasal shedding of PCV2, the second lowest rate for mean % iisig lesions and the lowest incidence rate for positive IHC tissues.
Interestingly, Group 4, which received a higher dose of rORF2 antigen than Group 5, did not perform as well or better than. Group 5 Group 4 had a slightly lower ADWG, a higher incidence of abnormal behavior, a higher incidence of overall clinical symptoms, a higher rate of nasai shedding of FCV2, a higher mean % lung lesions, and a higher rate for positive Ϊ1TC tissues than Group 5. Statistical analysis, which may have indicated that the differences between these two groups were not statistically significant, was not conducted on these data, but there was an observed trend that Group 4 did not perform as well as Group 5. Post-vaccination. 6 pigs died at the first study site. Four of the six pigs were from
Group 8 or Group 9. which received no vaccine. None of the six pigs demonstrated lesions consistent with PMWS, no adverse events were reported and overall, all seven vaccines appeared to be sale when adm inistered to pigs approximately 1 1 days of age. During the post-vaccination phase of the study, pigs receiving either of three dose levels of vOR.F2 vaccine oi killed whole cell vaccine had the highest IFAT lev els, while Group 5 had the low est Tl -V f Ie\ els μist puot to challenge of the Λ accuse groups
Although not fonnalh prov en, the predominant route of transmission of PCV2 to \ outJg swinec shorlK aftct \scatiuig is believed to be In oronasal direct contact and an efficac ious, sacune that reduces nasal shedding of PCV2 jtn a pioduuion setting would help ^ cotitiol the spread of infection Gtoups receiving one of three \ORF2 anngcn lc\cK and the gioup icc^n nψ H μg of rORF2 had the lowest incidence talc of nasal shedding of PCV2 (8 3"o) E\peet«ih the challenge cøirtiol gtoup had the highest incidence tate of nasal shedding (8O0O)
Gross lesions in pigs \uth PMW S secondαr\ to PC\ 2 infccuon ts pjcoih consist of gcncrah/cd Is mphadcnopαth\ in combination w ith one O) a muhtpiϋ of the following ( 1) iπierstniaS pticυraoiiis w tih mtcrJobuJsr edema (2) culsacou*; pallor oi icterus C-^ mottled αfcophjc h\cπ (4) gastπc iiiccrs pi neplmtis and ψ) icpiochxcuxe disotdcπ e g αbojuon stillbirths muromic*; etc A.f nccrops\ jcteru*; hepatitis ncphrtrts and gastric ulceis «eic not noted in am gioups and h mphadeoopatin was not spccificsJh examined foi l hc mean % Jung lesion scwcs -\aned between groups lhe gioup recen ing 1<> μg of ^ORJt* 2 antigen had the lowest mean % king lesion score (O 40 1 0 50%) followed b\ the gjoup that receded H μg of rORF2 (0 <<8 ^ 1 15°«) As expected the challenge1 eontro! gtoup had the highest mean 1O lung lesion scoic {9 88 -r 29 2%) Jn all foui gioυps the mean ιl υ lung lesion scores were clc\aιed due to one pig m each of these gjoups that had x en high lung legion scores Most of the lung lessons warn described as red purple and consolidated ϊj ptcalh . lung lesions associated \MUI PMWS mo dc&cnbed as> tan and non -collapsible vuth interlobular edema The lung lesions noted in this, stud} were either not associated w ith PG \ 2 infection ot a second pulnsoπan infectious agent maj ha\ e been ptesenl W tthsn the conteM of this studs the "o iung lesion scores ptobabh do not reflect a tiue nieasuic of the amount of lung mfecuoa due to PC V 2 Other researchers have demonstrated a direct correlation between the presence of
PCV2 antigen by IHC and histopathologic Histopsthology on select tissues was not conducted with this study. Group 1 ( 16 μg of vORF2) and Group 5 (8 μg of rORF2) had the lowest incidence rate of pigs positive for PCV2 antigen (8.3%), while Group 9 (the strict negative control group - 90.9%) and Group 8 (the challenge control group - 90.0%) had the highest incidence rates for pigs positive for PCV2 antigen. Due to She non-subjective nature of this test IHC results are probably one of the best parameters to judge v accine efficacy on.
Thus, in one aspect of the present invention, the Minimum Portcctive Dosage (MPD) of a I ml/1 dose recombinant product with extracted PCV2 ORF2 (rORF2) antigen in the CDCD pig mode! in the face of a PCV2 challenge was determined. Of {lie three groups that received varying levels of rORF2 antigen. Group 5 (8 μg of rOR.F2 antigen) clearly had the highest level of protection. Group 5 either had the best results or was tied for the most favorable results with regard to all of the parameters examined. When Group 5 was compared with die oiher six vaccine groups post-challenge. Group 5 .had she highest ADWG (0.94 .* 0.22 lbs/day), the lowest incidence of abnormal behavior (0%). the second lowest incidence of cough (8.3%). the lowest incidence of overall cluneal symptoms (8.3%), the lowest mortality rate (0%), the lowest rate of nasal shedding of PCV2 (8.3%), the second lowest rate for mean % lung lesions (0.68 * 1.15%) and the lowest incidence rate for positive IHC ussues (16.7%). In another aspect of lite present invention, the MPD of a 1 snl/l dose conventional product that is partially purified PC V2 ORF2 <vORF2) antigen in the CDCD pig model in the face of a PCV 2 challenge was determined. Of the three groups that received varying levels of vORF2 antigen. Group 1 (16 μg of vORF2) had the highest level of protection. Group I outperformed Groups 2 and 3 with respect to ADWG, mean % lung lesions, and THC. Groups 1 and 2 (8 μg of vORF2 antigen) performed equally with respect to overall incidence of clsntcal s\ mptoms. Group 3 (4 ug of \ORF2 antigen; huά the lowest mortal itj rate and al I three gioups perfoinied equalh w ith respect to nasal shedding Oscrsll. \ORF saccules did not pet form as well as tORF > accincs
In s si another aspect of the pit-sent invention (he efficacy of a inaiiimini dose of a 2m i 2 dose Conventional Killed PCVl vaccine in the CDCD ptg model in the face of a PCΛ 2 challenge was dctci mined Of the seven -vaccines ev aluated in tins stud\ the killed ΛK hole cell PCV2 v accine perfbimed (he woist Piglets teet-i\mg two doseb of killed uboie cell PCV 2 \ ace me had the lowest ADWG tlic second highest talc of abnormal bcha\ iot OS 30Ii), the second highest oxeiall incidence of clinicai sv inptoins (58 3%), the htghesi mortabtv i<ite ( I f) T1O) the second highest incidence of nasal shedding (41 7%), highest mean fO lung lesions (9 88 -*■ 29 2°u) a high incsdence of !ung lesions noted (7^'O) stid a inodeiate JHC incidence iate m tissues (41 ?%) HOWCΛCΓ, it was still effective a; inv oking an immune response
In still another aspect of the ptescnt nncntton nasal shedding of PCV2 was assessed as an efTicac\ paranictei and the prev ious PC V2 etϊicae> parameteis from prex ious studies WQK reconfinned Resuits from this studs indicate thai nasal shedding of PC\ 2 oocuis fblkming mtia nas>al challenge and that PC\ 2 vaccines reduce nasal shedding of PC\ 2 post- challenge Hu thcrmore, results fioni thts studs and seports in the literatinc indicate that THC should continue to be ev aluated m future PC V2 vaccine tπais as well Some additional conclusions ausing front this stucK arc that Is mphadeπopath\ is one of the hailmaiks of PVlWS Λ.nothei one of the hallnutiks of PMWS is Ij mphoid depletion and nuiltinttcleated/gMnt histioc> tcs Λdditioiulh no ad\ ers>e e\ents or injection site ieacUoiss weie noted for an\ of the 7 PC V2 saccmes and all 7 PC\ 2 \accmcs appeared to be safe vv hen administered to \ oung ptgs EXAMPLE 5
This example tests the efficacy of eight PCV2 candidate vaccines and reconfirms PCV2 challenge parameters TtOHi earlier challenge studies following exposure to a virulent strain of PCV2. One hundred and fifty {150} cesarean derived colostrum deprived (CDCD) piglets, 6- 16 days of age. were blocked by weight and randomly divided ink) 10 groups of equal size. Table 16 sets forth the General Study Design for this Example.
Figure imgf000064_0001
Figure imgf000065_0001
The vaccine formulations given to eacli group were as follows. PCV2 Vaccine No. 1, administered at 1 x 2 ml dose to Group 1 > w as a high dose ( 16 ug/2 ml dose) of inactivated recombinant ORF2 anligen adjuvanted with IMS 13 ! 4 ( 16 iig rORF2 - IMS 13 ! 4). PCV2 Vaccine No. 2, administered at 1 x 2 ml dose to Group 2, was a high dose ( 16 κg/2 ml dose) of a partialis purified ViDO R- I generated PCV 2 ORF2 antigen adjnvanted with Carbopoi { i(> ug vOR.F2 - Carbopoi). VC.V2 Vaccine No 3, administered at 1 \ 2 ml dose to Group 3, was a high dose ( 16 ug/2 ml dose) of inactivated recombinant O.RF2 antigen adJKvanted with Carbopoi ( 16 ug rORF2 - CarbopolV PCV2 Vaccine No. 4. administered at 1 x S. ml dose Io Group 4. was a high dose {16 ug / 1 nil dose) of a partially purified VlDO R- 1 generated PC V 2 ORF2 antigen adjuvanted with Carbopoi (16 ug \ORF2 - Carbopoi). Vaccine No. 5. administered at 1 x 2 nil dose to Group 5. was a 4 ug/2 ml dose of an inactivated recombinant 0RF2 antigen adjuvanted with Carbopoi {4 ug rORF2 - Carbopoi). PCV2 Vaccine ^No. 6. administered at 1 x 2 ml dose to Group 6, was a 1 itg/2 mi dose of an inactivated recombinant ORF 2 antigen adjuvanted with CarbopoS ( 1 ug rORF2 - Carbopoi). PCV2 Vaccine No. 7. administered at I x 2 ml dose to Group 7, was a low dose ("0.25 ug/2 ml dose) of inactivated recombinant ORF2 antigen adjm anted with Carbopol (0.25 ug rORF2 ••• Carbopol). PCV2 Vaccine No. 8, administered at 1 x 2 nil dose to Group 8, was a high dose {pre-inaciivaikm titer > S.0 log/2 ml dose) inactivated Conventional Killed VlDO R-I generated PCV2 Strove antigen adjuvaπted with Carbopol (>S.u log KV - Carbopol). On Day 0, Groups 1-8 were treated with their assigned vaccines. Groups 1-3 and 5-8 received boosters of their respective vaccines again on Day 14. The effectiveness of a single dose of 16 μg of vOKF2 - Carbopol %\as tested on Group 4 which did not receive a booster on Day 14. Piglets v ere observed for adverse events and injection site reactions following both vaccinations. On Day 21 the piglets were moved to a second study site where Groups 1 -9 were group housed in one building and Group 10 was housed in a separate building. Ail pigs received keyhole limpet hemocyanin emu.lss.iled with incomplete Frεund's adjuvant (KLH/ΪCfA) on Days 22 and 28. On Day 25. Groups i -c> were challenged with approximately 4 logs of virulent PCV 2 virus. By Day 46, very few deaths had occurred in the challenge control group, in an altempJ to immunosfinmlate the pigs and increase the virulence of the PCV 2 challenge materia!, all Groups were treated with JNGKLVACt? PRRSV MLV (Porcine Reproductive and Respirator}' Vaccine, Modified Live Virus) on Day 46.
Pre- and post-challenge biood samples were collected for PCV2 serology. Post- challenge, body weight data for determination, of average daily weight gain (ADWG) and observations of clinical signs were collected. On Day 50, all surviving pigs were necropsied, gross lesions were recorded, lungs were scored for pathology, and selected tissues were preserved in formalin for examination by lmmiinoliistochemistry (IHC) for detection of PCV2 antigen at a later date. Materials and Methods
This was a panially-blind vaccination-challenge feasibility study conducted in CDCD pigs. 6 to 16 days of age on Day 0. To be included in the study, PCV2 IFA titers of sows were < 1 : 1 UOO. Additionally, the serologic status of sows were from a known PRRS-πegative herd. Sixteen ( 16) sows were iesied for PCV2 serological status and all sixteen ( 16) had a PCV2 titer of < HK)O and vcre iransferred to the firsi study site. One hundred fifty { I SO} piglets were delivered by cesarean section surgeries and were available for this study on Day -3. On Day -3, 150 CDCD pigs at the first study site were weighed, identified wish ear tags, blocked by weight and randomly assigned to 1 of 10 groups, as set forth above in table Ϊ.6. Blood samples were collected from all pigs Jf any test animal meeting the inchfsion criteria was enroHed in the study and was later excluded for any .reason, the Investigator and Monitor consulted in order to determine the use of data collected from the animal in {lie final analysis. The date of which enrolled piglets were excluded and the reason for exclusion was documented No sows meeting die inclusion criteria, selected for die study and transported to the fire! study sue were excluded. No pigiets were excluded from the study, and no test animals were removed from the study prior to termination. Table 17 describes the time frames for the key activities of this Example.
Table 17, Studv Activities
Figure imgf000067_0001
Figure imgf000068_0001
Following completion of the in-life phase of the study, formalin fixed tissues were examined by ϊmmuiiohislochemistry (JHC) for detection of PCV2 antigen by a pathologist, blood samples were evaluated for PCV2 serology, and average daily weighs gain (ADWG) was determined from Day 25 to Day 50.
Animals were housed at the first study site in individual cages in seven rooms from birth to approximately 11 days of age (approximately Day 0 of the study ) Each room was identical in layout and consisted of stacked individual stainless steel cages with heated and filtered air supplied separately to each isolation unit. Each room had separate heat and ventilation, thereby preventing cross-contamination of air between rooms. Animals were housed in two different buildings at the second study site. Group 10 (The Strict negative control group) was housed separately m a converted nursery building and Groups 1 -9 were housed in a converted farrowing building. Each group was housed in a separate pen (14- 15 pigs per pen) and each pen provided approximately 2.3 square feet per pig. Groups 2. 4 and 8 u ere penned tπ thtee adiaeent pens on one ssde oi the alle\ wa\ and Gioups 1 ^ * 6 7 and 9 v. ere penned ni $i\ adiaoent pens, on tiic other side of the alie\wa\ Ilie Group separation was due to conceit) b\ the Sιιιd\ Monitor that vaccines adnnrtisietuS to Gtoups 2 4 and 8 had not been fκH\ iiiaUiλ ated Eat h |x. n wab on an <J<Λ ated de«Λ w ilh pla&Ut, slatted floors. A pit below the pent, served as a holding lank foi cxeruneiit and waste Fach bαiϊdiug had Hs own <,cρaialc heating and \entilattoti <.\ &ιcms vv itϊi littk likelihood of cro&&- iniuanimatioii of air bcmcen buildings
At the first study site piglets were fed a spcualK fot molaicd milk ration fiom birth to appro\ιniαtch ^5 weeks of age \\\ piglets were consuming solid special n»\cd ration bΛ Da\ 21 (<ippto\««ajeh 4 ' , week ^ of age) 4{ jhc second studv Mte all piglefe wete fed Λ cmioin non-mcdjcatcd commcrcisl mi\ iatioo αppioprmic ioi then age and wctght ttd ltbtmtn W atvt <it both studv πύa- was aUo ax^iKibie nti libtwm
Mi re^f pigs weic tteatcd w ith 1 0 ml of N AXOFLn J\f jti altciπafmg hsms on Dm s 19 20 and 2 ) in addition Pig No H (tftoup 1) was itcaied with u S mϊ of WXOt i & iM on Da\ I u Ptg No 13 (Qiotip 10) was ticated w ith 1 ml ol PemctHiu and 1 ml oi PRLDLF K 2λ on D<i\ iO Fig \o 4 (Group V) was treated with 1 n raL of WKCi I TK IM on Da\ i 1 am! Pigs 1 (Group I) 4 and i l were eadi treated w ith I 0 mi of N AX( H K on Da\ 14 for \ as ions health reasons
W hile <Jt both soκh sites pigs weie under \etcrman αtie Animal health examinations n ese oondueted on l)a\ -3 and were recorded on the Health Lvtmuωuon Reeoid l oi m \11 annuals were in good healtii and iπttπtional status> before ^ aceinaUon as detcnmned b\ ob^enation on Da\ 0 AU tcfet aπnnals were observed to be in good health and nutritional status pπor to challenge Carcasses and tissues wete disposed ol b\ rendering Final disposition of snιd\ animals w as recotdcd on the \nιnul Disposition Rccotd On Das s o and 14 pigs assigned lo Groups ! -"< and ^ -8 icceπed 2 0 mL of assigned PCV2 Vaccines M. respectively, IM in the right and left neck region, respectively, using a sterile 3.0 niL Luer-lock syringe and a sterile 2Og x Vj" needle. Pigs assigned to Group 4 received 1.0 mL of PCV2 Vaccine No. 2, IM in the right neck region using a sterile 3.0 mL Liter-lock syringe and a sterile 20g K VI" needle on Day O only. On Day -22 ail test pigs received 2.0 mL of KI.H/ICFA. IM in the left neck region using a sterile 3.0 ml. Luer-loek s\ ringe and a sterile 2Og x 1" needle. On Day 2H ail test pigs received 2.0 mL of KLH/ICFA in the right ham region using a sterile 3.0 mL Luer-lock syringe and a sterile 2Og x 1 " needle.
On Day 25. pigs assigned to Groups 1 -9 received 1.0 nil. of PCV2 ISUVDL challenge material (3.98 iogi-> TCIDw'ml.) IM in {lie right neck region using a sterile 3.0 ml. Luer-lαck syringe and a sterile 2ug x I " needle. An additional 1.0 nil. of the same mateπal was administered IN to each pig (0.5 mL per nostril) using a sterile 3.0 ml. Luer-lock s> ringe and nasal csnula.
On Day 46. all test pigs reccix ed 2.» mL INGELVAC*. PRRS MLV. JM, irt the right neck region using a sterile 3.0 mL LuerOlock s> ringe m\ά a stenlc 2Og x V needle. The PRRSV MLV was administered m an attempt to increase virulence of the PCV2 challenge material.
Test pigs were observed daily for overall health and adverse events on Da\ -3 and from Day 0 to Day 21. Each of the pigs were scored for norma! or abnormal behavior, respiration, or cough. Observations were recorded on the Clinical Observation Record. All tesf pigs were observed from Day o £o Day 7. and Group 7 was further observed from Day 14 lo 21. for injection site reactions. A\ erage daiK weight gain was determined by weighing each pig on a calibrated scale on Days -3, 25 and .50. or on the day that a pig w as found dead after challenge Body weights were recorded oti the Body Weight Form. Day -3 body weights v. ere utilized to block pigs prior to randomization. Day 25 mid Da\ 50 weight data was utilized to determine the average daily weight gain (ADWG) for each pig during these time points. For pigs that died alter challenge and before Day 50. the ADWG was adjusted to reptcseru the A.DWG from Day 25 to the day of death.
In order to determine PCV2 serology, venous whole blotxl was collected from each piglet from the orbital venous sinus on Days -3 and S 4. For each piglet blood was collected from the orbital venous sinus by inserting a sterile capillary tube into the medial eanthus of one of the eyes and draining approximately 3.0 niL of whole blood into a 4,0 mL Serum Separator Tube (SST). On Days 25, 32, and 50. venous whole blood from each pig was collected from the anterior vena cava using a sterile 20g x 1 W Vacutainer& needle (Beclon Dickinson and Company. Franklin Lakes, New Jersey), a VaeculainerJC needle holder and a \3 inL SST Blood collections at each time point were recorded on. the Sample Collection Record. Blood in each SST was allowed to clot, each SST was then spun down and the serum harvested. Harvested semni was transferred to a sterile snap rube and stored at -70* ] (f C until iesied at a later date Serum samples were tested for die presence of PCV2 antibodies by Bϊ Vl-R&D personnel .
Pigs were observed once daily from Day 22 to Day 50 for clinical symptoms and scored for normal or abnormal behavior, respiration or cough. Clinical observations were recorded on the Clinical Observation Record.
Pigs Nos. 46 (Group 1 ) and 98 (Groups 9) died at the first study site. Both of these deaths were categorized as bleeding deaths and necropsies were not conducted on these two pigs. At the second study site, pigs that died after challenge and prior to Day 50, and pigs euthanized on Day 5i\ were necropsied. Any gross lesions were noted and the percentages of lung lobes with lesions were recorded on the Necropsy- Report Font).
From each of the pigs necropsied si the second study site, a tissue sample of tonsil, lung, heart, and mesenteric lymph node was placed into a single container with buffeied 10% fourtaSm. while anothes tissue sample from lhc same aforementioned organs was placed into a Whu∑-pak ft. {M-fech Diagnostics Ltd . rhelwail t;K.) and each Whiri-psk# was placed on iec Each container was. proper!) labeled Sample collections, were accorded on (lie Necropsj Report Form Afterwards, foi malm-fixed tissue samples and a Diagnostic Request Foim were submitted lor IHC testing ΪHC icsimg was conducted m accordance w ith slandatd laboratory procedures for receiv ing samples, maniple and slide preparation, and warning techniques. Fresh (issues in Whiri-paks » were shipped %\ήh ice packs to the Stuch Monitor fot storage (-7O0 ± 10° C) and possible future ιis>c
Formahu-fj\ed tissues %\ ere examined In a pathologist foi detection of PCV2 b> JHC and scoicd u&ing the follow «ig scoπng s^ stera S) ~ None, 1 - Scant jxjsitπe stanung. few sites 2 = λJodeiate positive siammg. mυitiplc *uJes and 3 - Abundant positive siaiiutig, diffuse throughout the tissue Fot auah ϋcal purposes., a scoie of <> was considered
" negative " and a score of greater than 0 w as considered " posttπ e "
Results
Results for this example are give ncicm It ss noted that Pigs No 46 and VH died on da\ s 14 and 2^ respeetn eh These deaths were categorized as bleeding deaths Ptg No 1 1 tϋroup π Λ\as panting w\ύ\ raptd respiration on Day 15 Otherw ise all ptgs i\cre nonυal for l>ehaMor. respiration and cough during this, observ ation period and no sy stemic adverse ev ents were noted w ith am groups No injection site jeactjons were noted following \accmation on Da\ 0 Follow ing \ aecination on Dn\ 14, seven (7) out of fourteen ( 14) Group 1 pigs (50 0%) had swelling w ith a score of "2" on Da> 15 Four (4) out of fourteen (14) Group 1 (28 6" 0) sttll had a swelling of "2" on Da> 16 "None of the other groups cxpeuenecd injection sue reactions follow ing either vaccination Av erage dαil) weight gain (ADWG) results ate presented below tu Table lκ Pig Nos. 46 and 9K that died from bleeding were excluded from group results. Group 4, which received one dose of 16 ug vORF2 •• Carbopol had the highest ADWG- (1.16 * 0.26 lbs/clay). followed by Groups 1, 2, 3, 5, 6, and 10 which had ADWGs that ranged from 1.07 ± 0,23 lbs/day to U l ± 0.26 lbs/day. Group 9 had the lowest ADWG (0.88 ± 0.29 lbs/day >, followed by Groups S and 7, which had ADWGs of 0.93 ± 0.33 lbs/day and 0.V9 ± 0,44 lbs/day . respectively ,
Table 18. Summary of Group Average Daily Weight Gains (ADWG)
Figure imgf000073_0001
vOR P2 :: isolated viral ORF2_ iORI;2 :: recomimiant baculoviπis expressed OJΪ.P2, K V ot killed whole cdf virus - SX.'V? virus grown in suitable cell ciilhire
PVC2 serology results are presented below in Table 19. All ten ( 10) groups were seronegative for PCV2 on Day -3. On Day 14. PCV2 titers remained low for all ten ( U)) groups (range of 50-113), On Day 25, Group 8. which received Jiie whole cell killed virus vaccine, had the highest PCV2 titer (4617), followed by Group 2. which receiv ed 16 ug vORF2 - CarbopoL Group 4, which received as single dose of 16 itg vORF2 - CarbopoL and Group 3, which receiv ed 16 ug rORF2 — Carbopol, which had titers of 2507, 1920 and 1503 respectively. On Day 32 (otic week post challenge), titers for Groups 1 -6 and Group 8 ranged from 2360 to 7619, while Groups 7 (0.25 ug rORF2 - Carbopol), 9 (Challenge Control ), and 1.0 (Strict negative control) had titers of 382, 129 and 78 respectively . On Day 50 (day of necropsy), ail ten O 01 groups demonstrated high PCV2 titers (> 3.257).
On Days 25, 32, and 50. Group 3, which received two doses of 16 ug rORF2 - CarbopoL had higher antibody tilers than Group I. which received two doses of .16 ug rORF2 ~ TMS 1314. On Days 25, 32 and 50. Group 2. which received two doses of 16 ug vORF2. had higher titers than Group 4, which received only one does of the same vaccine. Groups 3, 5, 6, 7. which received decreasing levels of rORF2 - Carbopol, of 5.6, 4, 1. and 0 25 ug respectively, demonstrated correspondingly decreasing antibody titers on Days 25 and 32.
Table VX Summary of Group PCV2 IFA Titers
Figure imgf000074_0001
Figure imgf000075_0001
vORF2 -= isolated viral OE.P2; rOE£P2 ::: recombinant haculovirus exptessed ORF2: KV or killed whole ceil virus ~ PCV2 virus arown in suitable ceil culture
*For calculation purposes, a ≤I OO !FA titer was designated as a titer of "50"; a >640ft IFA tiler was designated as a ύter of " i 2.8(H)". **Day of Challenge ***Day of "Necropsy
The results from the post-challenge clinical observations are presented below. Tabic 20 includes observations for Abnormal Behavior. Abnormal Respiration. Cough and Diarrhea. Table 21 includes (he results from the Summary of Group Overall Incidence of Clinical Symptoms and Table 22 includes results from She Summary of Group Mortølily Rates Post-challenge, Hie incidence of abnormal behavior, respiration and cough post- challenge were low in. pigs receiving 16 ug rϋRF2™iMS 1314 (Group S.), S.6 ug rORF2~ Carbopol (Group 3). S. ug rORF2-Carbopo} (Group 6). 0 25 ug rORF2~Carbopol (Group 7). and it) pigs in the Challenge Control Group (Group 9). The incidence of" abnormal behavior, respiration, and cough post-challenge was zero in pigs receiving 16 ug vOR.F2~Carhopol (Group 2), a single dose of lόug vORF2~Carbopoi (Group 4). 4 ug r€)RF2-Carbopol (Group 5). >fc log KV-Carbopoi (Group 8), and in pigs in the strict negative control group (Group U)). The overall incidence of clinical symptoms varied between groups. Pigs receiving 16 Ug \ORF2--Carbopol (Group 2). a single dose of 16 ug vORF2 -Carbopoi (Group 4), and pigs in the Strici negative control group (Group 10) bad incidence rates of 0%: pigs receiving 16 Ug rORF2-Carbopol (Group 3), and 1 tig rORF2-Carbopoϊ (Group 6) bad incidence rates of 6.7%; pigs receiving S 6 iig rORF2-ΪMS 1314 (Grøiip S ) had an overall incidence rate of 7. 1%. pigs receiving 4 ug rORF2-Carbopo3 (Group 5), 0.25 og tORF2— Carbopol {Group 7), and >8 log KV vaccine had incidence rates of 13.3%; and pigs in the Challenge Control Group (Group 9} bad an incidence rate of 14.3%.
Overall mortality rates between groups varied as well. Group 8. which received 2 doses of KV vaccine had the highest mortality rate of" 20.0%; followed by Group 9. the challenge control group, and Group 7, which received 0.25 ug tORF2-Carbopol and had mortality rates of 14.3% and 13.3% respectively Group 4. which received one dose of 16 υg vORF2-Carbopol had a 6 7% mortality rate. All of the other Groups, 1. 2. 3. 3, 6, and 1.0, had a 0% mortality .rate.
Figure imgf000076_0001
Figure imgf000077_0001
'Total number o pigs in each group l at demonstrated any abnormal be avior or at least one day
"Total number of pigs in each group that demonstrated any abnormal respiration for at least one day
Total number of pigs m each group that demonstrated a cough for at least one day
Figure imgf000077_0002
Figure imgf000078_0002
vORF2 - isolated vtra! ORF2, rORI-'2 = recombinant baculovirus expressed OR.F2; KV c>t killed whole cell virus ::: PCV 2 virus grown in suitable cell culture
'Total number of pigs in each group {hat demonstrated any clinical symptom for at least one day
Table 22. Summary of Group Mortality Rates Post-Challenge
Figure imgf000078_0003
v(>RF2 ~ isolated viral ORF2; K)RF 2 = rweombinanl fcaculovirib ysptosεd ()R1V2: KV or kUJwd whole ce!) virus. - PC?V2
Figure imgf000078_0001
aτown in suitable ce!) cultute The Summary of Group Mean Percentage Lung Lesions and Tentative Diagnosis is given below in Table 23. Group 9. the challenge control group, bad the highest percentage lung lesions with a mean of 10.81 ± 23.27%, followed by Group 7, which received 0.25 ug rORF2-Carbopol and had a mean of 6.57 ± 24.74%. Group 5. which received 4 ug τGRF2- Carbopol and had a mean of 2.88 ± S.88%, and Group 8, which received She JCV vaccine and had a mean of 2.Ul ± 4.98%, The remaining six (6) groups had Sower mean percentage htug lesions that ranged from 0.1. 1 ± 0.3K% to 0.V0 ± 0.15%.
Tentative diagnosis of pneumonia varied among the groups. Group 3. which received two doses of 16 ug rORF2~Carbopoi, had the lowest tentative diagnosis of" pneumonia, with 13.3%. Group 9, the challenge control group, had 50% of die group tentatively diagnosed with pneumonia, followed by Group 10. the strict negative control group and Group 2. which received two doses of lf> ug vORF2-Carbopol, with 46 TYa and 40% respectively, tentatively diagnosed with pneumonia. Groups 1. 2, 3. 5, 9. and 10 had 0% of the group tentatively diagnosed as PCV2 infected; while Group 8, which received two doses if KV vaccine, had the highest group rate of tentative diagnosis of PCV2 infection, with 20%. Group 7. which received two doses of 0.25 ug rt~)RF2-Carbopol. and Group 4. which received one dose of 16 ug vORF2-Carbopol had tentative group diagnoses of PCV2 infection in 13.3% and 6.7% of each group. respectively.
Gastric ulcers were only diagnosed in one pig in Group 7 (6,7%); while the other 9 groups remained tree of gastric ulcers. Table 23. Summary of Group Mean % Lung Lesion and Tentative Diagnosis
Figure imgf000080_0001
\t>RF2 - iaolsued \iral ORF2. rt>RF2 - njcombmant baculovirtK expressed ORF2L KV or killed whole ceil virus. - PCV2 virus grown in suitable ceil eulUire
Tlie Summary of Groap IHC Positive Incidence Results is shown below in Table 24. Group ! ( 16 tig iORF2 - IMS 1314} had (he lowest group rate of IHC positive results with 0% of the pigs positive for PCV2, followed by Group 2 (16 tig vOR.F2 - Carbopol) and Group 4 (single dose 16 ug vORF2 - Carbopol)- which had gtoup IHC rates of 6.7% and 13.3% respectively. Group 9, the challenge control group, had the highest IHC positive incidence rate wish 100% of the pigs positiv e for PCV2. followed by Group 10. the strict negative control group, and Group 8 (KV vaccine), with 93 3% and 80% of the pigs positive for PCV2. respectively.
Table 24. Summary of Group IHC Positive Incidence Rate
Figure imgf000081_0001
vORF2 ::: isolated viriil fJH.P2; )<>RP2 ::: rectMiibitiam bitciilo'vin.t-5 expressed ORF2: KV or kilted whole ceil virus ~ PCV2 vints arown in suitable ceil culture
Discussion
Seven PCV2 vaccines were evaluated in this example, which included a high dose (16 μg) of rORF2 antigen adjuvanted with IMS 1314 administered twice, a high dose {16 μg) of vORF2 antigen adjuvanted with Carbopol adminislered once to one group of pigs and twice to a second group of pigs, a high dose ( 16 μ,g) of rORF2 antigen adjuvanted with Carbopol administered twice, a 4 μ.g dose of τORF2 antigen adjuvanted with Carbopol adra mistered twice, a 1 μg dose of rORF2 antigen adjuvanted witli Carbopol administered twice, a low dose (0.25 μg) of rORF2 antigen adjuvanted with Carbopol administered iwice. and a high dose {> 8 log) of killed whole cell PCV2 vaccine adjuvanted with Carbopol. Overall Group 1. which received two doses of 16 μg rORF2 - IMS 1314. performed slightly better lhan
Groups 2 through ?, which received vaccines containing various levels of cither vORF2 or rORF2 antigen adjuvanted wish Carbopol and much belter than Group 8, which received two doses of killed whole ceil PCV2 vaccine. Group 1 had the third highest ADWG { 1.KO ± 0.3« lbs/day ), the lowest incidence of abnormal behavior (0%). the lowest incidence of abnormal respiration (0%). a low incidence of cough (7 1%). a low incidence of overall clinical symptoms (7.1%). was tied with three other groups for the lowest mortality rate (0%), the second lowest rate for mean % hsig lesions (0.1.5 & 0.34%). the second lowest rate for pneumonia (21.4%) and the lowest incidence rate for positive MC tissues (0%) Group 1. was. however, the only group s.n which injection site reactions were noted, which included 50% of the vaccinates 1 day after the second vaccination. The other vaccines administered to Groups 2 through 7 performed better than the killed vaccine and nearly as well as the vaccine administered to Group 1.
Group 8. which received two doses of killed PCV2 vaccine adjuvanted with Carbopol. had the worst set of results for any vaccine group. Group 8 had the lowest ADWG (0.93 ± 0.33 lbs/day), the second highest rate of abnormal behavior (6.7%), the highest rate of abnormal respiration {6.7%), was tied with three other groups for the highest overall incidence fate of clinical symptoms (13.3%), had the highest mortality rate of all groups (20%). and had the highest positive UiO rate (80%) of any vaccine group. There was concern that the killed whole ceϊ! PCV2 vaccine may not have been fully inactivated prior to administration to Group 8, which may explain this group's poor results. Unfortunately, (fcfmm\e data w as not av ailable to confirm this concern Ov erall in the context of this example, a Conventional Killed PCV2 Λ accuse did not aid m Use reduction of PCV2 a ssoc tatcd d t se a se
•\s picMOUsh mentioned, no adveise ctcn& were associated wilh the test \ aecnies> v\ ith exception of the \ acciiio aφin anted w itb JMS i ? 14 J njection site reactions w etc noted in ^O O0O of the pigs> ! d«τv after the second vaccination w ith the v ace me fot mutated w ith IMS 1314 and m 28 6% of the pigs 2 da> s aflei the second \acc ination No reactions, were noted itj am pigs rccciΛ ing Csibopol adjι» amcd vaccines \n\ fiuthcr studies that include pigs vaccinated w ith J MS S314 adμnonfed vaccines should continue to clαsci> monitor pigs for injection sue jcaciious.
AU psgs were scrc-ijegsJrve for PC\'2 on Daj -3 arid o»h (.iroup 2 had s tttei abo\ c K)O on Da\ 14 Ou Da> 2> (da\ of challenge). Group 8 had the highest PCY2 antibod\ utei (4619) followed by Ciroup 2 (2507> With the exception of Groups 7, 9 and H) alt groups demonstrated a strong stii tbod\ ic^ponw b\ Da> 32 B\ Da\ ?0, aH gioups ujcUiding Groups 7. ^ and 10 demolish atcd a strong antibod\ response
One of the hallmarks of laic stage PC\ 2 infection and subsequent FMWS dev elopment is growth retardation in ncaned pigs and m se\ere cases, weight loss is noted Average daih weight gain of groups is a quantttatn e method of demonstrating grow th retardation or weight loss Jn this example, there was not a las go difference m \0WCi bcwccn groups Gioup 8 had the lowest -\D\\ G of 0 8H + 0 2" Ibs'daj . whtle Group 4 had the highest ADW O of I 16 ± 0 26 Ib daj Within the context of this stmh there was not a sufficient difference between groups to base future λ aecme efficacx on ADW G
Jn addition to weight loss d\ spnea, kghaτg> . palloi of the skin and sometimes icterus arc clinical symptoms associated w ith PMWS ϊn this example, abnotmal belun ior mid abnormal respiration and cough were noted infrequeailj for each group As e\ idcnced m this study, this challenge model and challenge strain do not result in overwheho ing clinical symptoms and tϊiis is not a strong parameter on which to base vaccine efficacy.
Overall, mortality rates were not high in this example and the laek of a high mortality rate in the challenge control group limits this parameter on which to base vaccine efficacy. Prior So Day 46. Groups 4 and 7 each had one out of fifteen pigs die. Group 9 had iwo out of fourteen pigs die and Group 8 had three out of fifteen pigs die. Due to the faei {hat Group 9, (he challenge control group was not demonstrating PCV2 clinical symptoms and only two deaths had occurred in this group by Day 46, Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) MLV vaccine was administered to all pigs on Day 46. Earlier studies had utilized INGELVACJC- PRRS MLV as an imrmmostinjuiant to exasperate PCV2- assøeiated PMWS disease and mortality rates were higher in these earlier studies. Two deaths occurred shortly after administering the PRRS vaccine on Day 46 - Group 4 had one death on Day 46 and Group ? had one death on Day 4? - which were probably not associated with the administration of the PRRS vaccine. By Day 50. Group 8, which received two doses of killed vaccine, had the highest mortality rate (20%). followed by Group 9 (challenge control) and Group 7 (0.25 ug rORF2 - Carbopol). wuh mortality rates of 14.3% and 13.3% respectively. Overall administration of the PRRS vaccine to the challenge model late in the post-challenge observation phase of this example did not significantly increase mortality rates. Gross lesions in pigs with PMWS secondary to PCV2 infection typically consist of generalized lymphadenopathy in combination with one or more of the following: (i) interstitial pneumonia with interlobular edema. (2) cutaneous pallor or icterus. (3) mottled atrophic livers. (4) gastric ukers, (5) nephritis and (6) reproductive disorders, e.g. abortion, stillbirths, mummies, etc. At necropsy (Day 50). ieterus. hepatitis, and nephritis were not noted in any gtoups. A gastric ulcer was noicd in one Group 7 pig, bui lymphadεnopaihy was not specifically examined for. Based on the presence of lesions thai were consistent with PCV2 infection, three groups had at least one pig tentatively diagnosed with PCV2 (FMWS). Group 8, which received rwo doses of killed vaccine, had 20% tentatively diagnosed with PCV2, while Group ? and Group 4 had 13.3% and 6.7%. respectively, tentatively diagnosed with PCV2. The tnean % lung lesion scotes varied between groups at necropsy . Groups 1. 2, 3, 4. 6 and 10 bad Sow % lung lesion scores that ranged Prom 0.1 1 ± 0.38% to 0.V0 ± 0.1.5%. As expected. Group 9. the challenge control group, had the highest mean % lung lesion score ( 10,81 i 23.27%), In four groups, the mean % lung lesion scores were elevated due to one to three pigs in each of these groups having very high lung lesion scores. The lung lesions were red/purple and consolidated. Typically, lung lesions associated with PMVVS arc described as tan. »on~eollapsible. and with interlobular edema. The lung lesions noted in this study were cither not associated with PCV'2 infection or a second pulmonary infectious agent may have been present. Within the context of this study, the % lung lesion scores probably do no reflect a true measure of the amount of lung infection due to PCV2. Likewise, tentative diagnosis of pneumonia may have been over-utilized as well. Any pigs with King lesions. some as small as 0.10% were listed with a tentative diagnosis of pneumonia. In this example, there was no sufficient difference between groups with respect to gross lesions and % lung lesions on which io base vaccine efficacy .
IHC results showed the largest differences between groups. Group I (16 μg rORF2 - IMS 1334) had the lowest positive IHC results for PCV2 antigen (0%); while Groups 9 and 10 had the highest positive IHC results with incidence rates of 100% and 93.3% respectively. Groups 3, 5, ύ and 7, which received 16, 4, 1 or 0.25 μg of rORF2 antigen, respectively, adjnvanted with Caibopol had IHO positive rates of 20%, 20%. 40% and 46.7%. respectively. Group 2, which receiv ed two doses of 16 ug vQRF2 adjuvanted with Carbopol had an IHC positive rate of 6.7%, while Gtotip 4 which received only oae dose of the same vaccine, had an JHC positive rate of 13.3%. Due to the objective nature of this test and the fact that IHC results correlated with expected results. iHC testing is probably one of the best parameters on which to base vaccine efficacy.
Thus in one aspect of the present invention, the Minimum Protective Dosage (MPD) of PCV2 rORF2 antigen adjυvarUcά with Carbopol in the CDCD pig model in the face of a PCV2 challenge is determined. Groups 3. 5. 6 and 7 each received two doses of rORF2 antigen adjuvanled with Carbopol but the level of rORF2 antigen varied for each group. Groups Λ, 5, 6 and 7 each received 16, 4, 1 or 0.25 μg of τORF2 antigen respectively . in genera!, decreasing the level of rORF2 antigen decreased PCV2 antibody titers, and increased the mortality rate, meat) % bug iesions, and the incidence of 1H.C positive tissifes Of the four groups receiving varying levels of rORF2 ~ Carbopoi, Groups 3 and 5, which received two doses of 16 or 4 μg of rOR.F2 antigen, respectively, each had an IHC positive rate of onlv 20%, and each had similar antibody titers. Overall, based on IHC positive results, the minimum protective dosage of rOR.F2 antigen administered twice is approximately 4 μg. In another aspect of the present invention, {he antigenicity of recombinant (rORF2) and VlDO R-I (vURF2) PCV2 antigens were assessed. Group 2 received two doses of 16 μg vORF2 and Group 3 received two doses of 16 μg rORP2, Both vaccines were adjuvanted with CarbopoS. Both vaccines were ftmnd io be safe and both had 0% mortality rate. Group 2 had a PCV2 antibody titer of 2507 on Day 25, while Group 3 had a PCV2 antibody titer of 1503. Group 3 had a lower mean % hmg lesion score than Group 2 (0, 1 1 * 0.38% vs. 0.90 ± (115%), but Group 2 had a lower IMC positive incidence rate that Group 3 (6.7% vs. 20%). Overall, both vaccines had similar antigenicity, but vORF2 was associated with slightly better UiO results.
In yet another aspect of the present invention, the suitability of two different adjuvants (Carbopol and IMS 1314) was determined. Groups 1 and 3 both received two doses of vaccine containing 16 ug of rOR.F2 antigen, but Group 3 received the antigen adjuvanted witli IMS 1314 while Group 3 received the antigen adjavanted with Carbopoi. Both groups had essentially the same ADWG, essentially the same incidence of clinical signs post-challenge, (he same mortality rate, and essentially the same mean % King lesions; but Group 1 had an SHC positive rate of 0% while Group 3 had an !HC positive rale of 20%. However, Group 3, which received She vaccine adjαvanted wish Carbopoi, had higher IFAT PCV2 liters on Days 23, 32, and 50 than Group 3 , which received the vaccine adjuvanted with IMS 33 14, Overall, although the PCV2 vaccine adjuvanted with IMS 1314 did provide belter WC results, it did not provide overwhelmingly tetter protection from PCV 2 infection and did induce injection site reaction. Whereas {lie PCV2 vaccine adjuvanted with Carbopoi performed nearly as well as the IMS 1314 adjuvanted vaccine, but was not associated with any adverse events.
In still another aspect of the present invention, the feasibility of PCV2 OR.F2 as a I mK 1 dose product was determined. Groups 2 and 4 boih received 36 μg of vORF2 vaccine adjuvanted with Carbopoi on Day 0. but Group 2 received a second dose on Day 14. Group 4 had a slightly higher ADVVG and a lower mean % lung lesions than Group 2. but Group 2 had higher IFAT PCV2 titers on Day 25. 32 and 50, and a slightly lower incidence rate of O1C positive tissues. All other results for these two groups were similar. Overall, one dose of \ORF2 adjuvanted with Carbopoi performed similar to two doses of the same vaccine.

Claims

We c laim
1 A method fot reducing oi lessening th«. sesei th of c hnicai SΛmptoms dbbociatcd w ith PC\ 2 infection kbboning (he muaiϊ pcnune cmot inife load of dm animal and or rαhiung the im munosuppressiv e cOXot of poiutK ctrcm tm<; mfcctjon to ptgς vompnsing admitusicπng a poicjuc cnco\ nos H JTC 2 antigen to a pig
2 T he method of Jaiπi 1 wheicm s aid antigen composes a proJeiti ciKodcci bv a D\ Λ sequence ha\ mg at least h()"o sequence idcntit\ with ORf 2 of a pore me at eo\ \roA i*, pe 2 \ n us
3 The method ol eiihei of claims i and 2 ^heicm satά porcine Cϊrco\ϊru<5 1\ pc 2 antigen J^ a ie^ombmant Ixiculov jrus cxpiessed ORl 2 antigen
4 The method of am one of cldjms 1 to 3 nlieiem <^nά porcine >,nco\ srus. l\ pc 2 amigcn is fonrasitUcd and administered m one (1) niL per do&e
^ The method of JΠ\ one of clauns 1 to 4 wherein uuά clmiuil &\ κiptoκi& <.nv selected fiom the group consisting of l\τaphadenopath\ h mptund depiction and muJlmucJcdled ot giant htsliooMcs
6 Hie method of claim 5 \%1κicin said h tnph«kiiop<itlπ lv mphoid dtpϊction aud'Or ntuHntαckatcd or giant tnsuoes tes is. m combination w ith snothct sv mptom seiectcd from the group consisting of interstitial pneumonia v ith interlobular edema, cutaneous pallor or seteras, mottled atrophic Ih ers. gastric ulcers , nephritis, pia like lesions, and reprodisctne disorders.
7 Hie method of an\ one of claims 1 to o. \s herein said administration occurs m pig-s less than 15 weeks of age
8 The method of am one of claims 1 to 7, wherein said administration occurs in pigs not older than 3 weeks of age preferabh not older ihsti 2 weeks of sgc
9 The method of aiπ one of claims i to 8. therein said administration occurs wtihra about 3 weeks of exposure to a virulent porcine ctrcox irus tj pc 2 antigen.
10 The method of am one of claims 1 to c>. wherein said composition further comprises at least one additional component selected from the group consisting of \eιerinaι> -acceptable carriers, pharmaceutical-acceptable carriers, and tmmunoiτiodulatoπ agents.
11. The method of ari\ one of claims 1 to 10, wherein said administration is selected from the group consisting of intradermal intratracheal, imπnaginat. mtτainusculaτ, intranasal, intravenous, intravascular. intraarterial. intraperitoneal, oral, intrathecal. Subcutaneous, intracutaneous, nyracardial, uitralobal. mlrsmedπUar. or sntrapulmonar
12. The method of any one of claims I to 11. wherein the administration of said porcine eircovinis type 2 antigen does not show adverse events or injection site reactions.
13. A method for improving the level of general disease resistance of pigs comprising administering a porcine circσvims type 2 antigen io a pig.
14. A process for the production of a medicament for reducing or lessening the severity of clinical sy mptoms associated wills PCV2 infection, lessening the overall porcine circovints load of an animal or reducing the immunosuppressive effect of porcine circovirus infection, said process comprising the steps of obtaining a porcine circovirus antigen, and combining said antigen with veterinary -acceptable carriers, pharmaceutical-acceptable earners, or immunomodulatory agents.
15. The process of claim 14, wherein said antigen comprises a polypeptide encoded by O.RF2 of porcine circovirus type 2.
16. The use of a porcine circovims type 2 antigen for the preparation of a medicament for reducing or lessening the severity of clinical symptoms associated with PCV2 infection, lessening the overall porcine eireovirus load of sn animal, and/or reducing the immunosuppressive effect of porcine eircovinis infection in pigs, vvhereiu said medicament is administered to a pig.
17. The use according to claim 16. wherein said antigen comprises a protein encoded by a DNA sequence having at ieasi 80% sequence identity with QRF2 of a porcine oncovirus t\pc 2 vims.
18. The use according to either of claims 16 arid S 7, wherein said porcine circoviriis U pe 2 antigen is a recombinant baculoviriis expressed ORF2 antigen.
19. The use according to any one of claims 16 to 18, wherein said porcine circøurus t\ pe 2 antigen is formulated, and administered in one ( 1) inL per dose.
20.. The use according to any one of claims 16 Io 19. w herein said clinical symptoms are selected from the group consisting of lympiiadenopatiiy. K niphoid depletion, and multinucleated or giant histiocytes.
2 ! . The use according to claim 20. wherein said lymphadenopathy, lymphoid depletion, and/or multinucleated or giant histiocytes is in combination with another symptom selected from the group consisting of interstitial pneumonia with interlobular edema, cutaneous pallor or icterus, mottled atrophic livers. gastric ulcers , nephritis, pia like lesions, am! reproductive disorders.
22. The use according to any one of claims 16 to 2 K wherein said administration occurs in pigs iess than 15 weeks of age.
23. The use according to any one of claims 16 to 22, wherein said administration occurs in pigs iioi older than 3 weeks of age, preferably not older than 2 weeks of age.
24. The use according to any one of claims 16 io 23, wherein said administration is within about 3 weeks of exposure to a virulent porcine circo virus type 2 antigen.
25. The use according to any one of" claims 16 to 24. wherein said composition further comprises ai least one additional component selected from the group consisting of veterinary -acceptable carriers, pharmaceutical-acceptable carriers, and immunomodulatory agents.
26. The use according to am one of claims 16 to 25. wherein said administration is intradermal, intratracheal, mtravagmal, intramuscular, intranasal, intravenous, intravascular, intraarterial intraperitoneal, oral intrathecal subcuumcoxis. intracutaneous, intiaeardial. iotralobai, iπtτamtxtoUar, or iiitrapuinioiiar.
27. The use according to one of claims 16 to 26. wherein the administration of said porcine cireovirus type 2 antigen does not show adverse events or injection site reactions. The use of a porcine circovirus type 2 antigen for the preparation of a medicament for improving the level of general disease resistance of pigs.
PCT/US2006/062662 2005-12-29 2006-12-28 Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs WO2007094893A2 (en)

Priority Applications (16)

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EP20184598.9A EP3766518A1 (en) 2005-12-29 2006-12-28 Pcv2 immunogenic composition for lessening clinical symptoms in pigs
CN200680053578.7A CN101426522B (en) 2005-12-29 2006-12-28 Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs
MX2016013804A MX356667B (en) 2005-12-29 2006-12-28 Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs.
PL16185663T PL3127551T3 (en) 2005-12-29 2006-12-28 Pcv2 immunogenic composition for lessening clinical symptoms in pigs
ES06850366.3T ES2625903T3 (en) 2005-12-29 2006-12-28 Immunogenic composition of PCV2 to reduce clinical symptoms in pigs
JP2008548847A JP5739602B2 (en) 2005-12-29 2006-12-28 Use of PCV2 immunogenic composition for alleviation of clinical symptoms in pigs
MX2008008311A MX2008008311A (en) 2005-12-29 2006-12-28 Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs.
DK06850366.3T DK1976558T3 (en) 2005-12-29 2006-12-28 PCV2 IMMUNOGENIC COMPOSITION TO REDUCE PIG CLINICAL SYMPTOMS.
KR1020147001544A KR20140019479A (en) 2005-12-29 2006-12-28 Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs
AU2006338182A AU2006338182B2 (en) 2005-12-29 2006-12-28 Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs
EP16185663.8A EP3127551B1 (en) 2005-12-29 2006-12-28 Pcv2 immunogenic composition for lessening clinical symptoms in pigs
EP06850366.3A EP1976558B1 (en) 2005-12-29 2006-12-28 Pcv2 immunogenic composition for lessening clinical symptoms in pigs
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