EP2488185A1 - Clones infectieux de torque teno virus - Google Patents

Clones infectieux de torque teno virus

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Publication number
EP2488185A1
EP2488185A1 EP10823754A EP10823754A EP2488185A1 EP 2488185 A1 EP2488185 A1 EP 2488185A1 EP 10823754 A EP10823754 A EP 10823754A EP 10823754 A EP10823754 A EP 10823754A EP 2488185 A1 EP2488185 A1 EP 2488185A1
Authority
EP
European Patent Office
Prior art keywords
ttv
seq
ttvgtl
polynucleotide
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10823754A
Other languages
German (de)
English (en)
Other versions
EP2488185A4 (fr
Inventor
Gregory Paul Nitzel
Robert Gerard Ankenbauer
Jay Gregory Calvert
Donna Steuerwald Dunyak
Jacqueline Gayle Marx
Nancee Lois Oien
Douglas Steven Pearce
Mira Ivanova Stoeva
James Richard Thompson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zoetis LLC
Original Assignee
Pfizer Inc
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Filing date
Publication date
Priority claimed from PCT/US2009/005662 external-priority patent/WO2010044889A2/fr
Application filed by Pfizer Inc filed Critical Pfizer Inc
Publication of EP2488185A1 publication Critical patent/EP2488185A1/fr
Publication of EP2488185A4 publication Critical patent/EP2488185A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to novel nucleotide and amino acid sequences of Torque teno virus ("TTV”), including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals.
  • TTV Torque teno virus
  • Vaccines provided according to the practice of the invention are effective against multiple swine TTV genotypes and isolates.
  • Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines.
  • vaccines that comprise, as antigen, the expressed protein of single TTV open reading frames, most particularly from ORF1 or ORF2, and also fragments of the full length ORF1 and ORF2-encoded proteins.
  • TTV Torque Teno Virus
  • transfusion-transmitted virus is generally assigned to the Circoviridae family. It is generally recognized that TTV was first isolated from human transfusion patients (see for example, Nishizawa et al., Biochem. Biophys. Res. Comm. vol. 241 , 1997, pp.92-97). Subsequently, TTV or TTV-like viruses have been identified from other mammals, including swine, and numerous strains or isolates have been published (see for example, McKeown et al. Vet. Microbiol, vol. 104, 2004, pp 1 13-1 17).
  • TTV and TTV-like viruses are very common; however the pathogenesis of TTV, and the contributions it may make to other disease states (for example, those caused by other viruses and bacteria) remains unclear.
  • TTV infections appear to be common in humans, including even in healthy individuals, and such infections are often asymptomatic, and may remain for years.
  • the general inability to propagate the virus in cell culture, and a lack of any clear mechanistic disease models, have made any overall characterization of TTV biology difficult.
  • TTV viremia is elevated in human patients afflicted with other viral diseases, (such as hepatitis or HIV/AIDS), there is also considerable medical literature suggesting that TTVs are, in fact, avirulent, and await any clear actual association with known disease states. See, for example, Biagini et al., Vet. Microbiol, vol. 98, 2004, pp. 95-101 .
  • PDNS porcine circovirus disease
  • TTV infection can potentiate numerous disease states. Accordingly, there is a need for various classes of TTV reagents, such as high affinity antibodies, and for example, peptide fragments of TTV or whole virions that are highly
  • TTV is the principle causative factor of diseases in swine
  • numerous swine diseases either require the presence of more than one virus, or that the primary effect of certain "primary" pathogens is potentiated by TTV infection.
  • numerous diseases of swine can be treated or lessened by administering anti-TTV agents to affected or potentially affected animals.
  • TTV is a small, non-enveloped virus comprised of negative polarity, single- strand circularized DNA.
  • the genome includes three major open reading frames, ORF1 , ORF2 and ORF3, which overlap, and ORF1 encodes the capsid protein.
  • ORF1 encodes the capsid protein.
  • the present invention is directed to recombinant constructs whereby TTV can be propagated in vitro, including via infectious clones. More particularly, the invention is directed to the discovery that effective vaccines can in fact be made from TTV, most particularly when the TTV antigen is the expression product of a single ORF, or a fragment thereof. In a preferred embodiment, the invention provides for ORFI protein vaccines.
  • the present invention provides a method of treating or preventing a disease or disorder in an animal caused by infection with torque teno virus (TTV), including disease states that are directly caused by TTV, and disease states contributed to or potentiated by TTV.
  • TTV torque teno virus
  • the animal treated is a swine.
  • Disease states in swine that may be potentiated by TTV, and which may also be treated or prevented according to the practice of the invention, include those caused by or associated with porcine circovirus (PCV), and porcine reproductive and respiratory syndrome virus (PRRS).
  • PCV porcine circovirus
  • PRRS porcine reproductive and respiratory syndrome virus
  • the present invention also includes the option to administer a combination vaccine, that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-TTV pathogen, with appropriate choice of adjuvant.
  • a combination vaccine that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-TTV pathogen, with appropriate choice of adjuvant.
  • the present invention also provides a diagnostic kit for differentiating between porcine animals vaccinated with the above described TTV vaccines and porcine animals infected with field strains of TTV.
  • ttvg1 -7 SEQ ID NO: 4
  • ttvGT1 -17 SEQ ID NO: 5
  • ttvGT1 -21 SEQ ID NO: 6
  • ttvgtl -27 SEQ ID NO: 3
  • ttvgt1 -178 SEQ ID NO: 7 or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
  • the invention further provides RNA polynucleotide molecules that are the complement of any such DNA polynucleotide sequence, and vectors and plasmids for the expression of any such RNA or DNA polynucleotides, and for TTV virus that is expressed from such nucleotide sequences, wherein said virus is live, or fully or partially attenuated.
  • the invention also provides a DNA vaccine that comprises a
  • the invention provides a polypeptide encoded by any of the open reading frames of the genotype 2 TTV 13 (SEQ ID NO: 1 ) or genotype 2 TTV10 (SEQ ID NO: 2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • the invention also provides a polypeptide encoded by any of the open reading frames of the (all sertotype 1 ) ttvg1 -7 (SEQ ID NO:10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), ttvgt1 -27 (SEQ ID NO: 13), and ttvgtl - 178 (SEQ ID NO:9) ORF1 polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • the present invention provides for such effective vaccines, which preferably comprise a polypeptide resultant from expression of a single TTV open reading frame, or a mixture thereof.
  • the polypeptide is expressed from ORF1
  • preferred mixtures include a combination of the polypeptides of ORF1 and ORF2, and ORF1 and ORF3.
  • polypeptide vaccines wherein the antigen is defined by (a) the first 100 N- terminal amino acids of the capsid protein of TTV13 (SEQ NO:1 ) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto; or (c) an arginine rich region thereof.
  • Figure 2 evidences successful expression of codon-optimized TTVgl ORF1 protein in E. coli, with a 6X His tag for affinity purification
  • Figure 3 provides a vector map for the Chromos construct pcTV-TTV1 -7 ORF1 (plus yeast invertase) expression plasmid from which is expressed (following integration into an artificial chromosome in CHO cells) vaccinating ORF1 protein.
  • Figure 4 provides a phylogenetic tree for various TTV strains including a compilation of percent identities.
  • Figure 5 panels A, B and C provides identification of in-common arginine rich regions of ORF1 proteins as expressed from various TTV isolates.
  • Figure 6 provides a vector map for TTVgl -178 as assembled.
  • Figure 7 demonstrates that Chromos-expressed gITTV ORF1 significantly reduced lung lesions compared to the challenge controls and reduces the magitude and duration of gITTV viremia, again compared to the challenge controls.
  • Figure 8 provides a vector map for the pCR2.1 +TTVg1 -178 construct that contains a ttvg1 -178 strain full length infectious clone.
  • SEQ ID NO:1 provides the genotype gt2 TTV 10 DNA sequence.
  • SEQ ID NO:2 provides the genotype 2 gt2 TTV 13 DNA sequence.
  • SEQ ID NO:3 provides the genotype 1 ttvgt1 -27 DNA sequence.
  • SEQ ID NO:4 provides the genotype 1 ttvgtl -7 DNA sequence.
  • SEQ ID NO:5 provides the genrotype 1 ttvgtl -17 DNA sequence.
  • SEQ ID NO:6 provides the genotype 1 ttvgtl -21 DNA sequence.
  • SEQ ID NO:7 provides the genotype 1 ttvg1 -178 DNA sequence
  • SEQ ID NO:8 provides the amino acid sequence of TTV strain AY823991 ORF1 .
  • SEQ ID NO:9 provides the amino acid sequence of TTV strain ttvgtl -178 ORF1 (TTV genotype 1 ).
  • SEQ ID NO: 10 provides the amino acid sequence of TTV strain ttvgtl -7 ORF1 .
  • SEQ ID NO: 1 1 provides the amino acid sequence of TTV strain ttvgtl -17 ORF1 .
  • SEQ ID NO: 12 provides the amino acid sequence of TTV strain ttvgtl -21 ORF1 .
  • SEQ ID NO: 13 provides the amino acid sequence of TTV strain ttvgtl -27 ORF1 .
  • SEQ ID NO: 14 provides the amino acid sequence of TTV strain gt2 TTV10 ORF1 (genotype 2).
  • SEQ ID NO: 15 provides the amino acid sequence of TTV strain gt2 TTV13 ORF1
  • SEQ ID NO: 16 provides the DNA sequence of known strain AY823991
  • SEQ ID NO: 17 provides the DNA sequence of known strain AY823990
  • SEQ ID NO:18 provides the 76057-3 TTV capsid encoding sequence, codon optimized for E. coli. as cloned into the pUC57 GenScript® vector.
  • SEQ ID NO:19 provides the 76057-4 TTV capsid encoding sequence, codon optimized for E. coli. as cloned into the Invitrogen pET101/D-TOPO® expression plasmid.
  • SEQ ID NO:20 provides the 76057-5 TTV capsid encoding sequence, codon optimized for Saccharomyces cerevisiae as cloned into the pUC57 GenScript® vector.
  • SEQ ID NO:21 provides the DNA sequence for a construct that encodes ttvgtl -7 ORF1 with a yeast invertase expression tag (Yl).
  • SEQ ID NO:22 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
  • SEQ ID NO:23 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
  • SEQ ID NO:24 provides a ttvgtl peptide sequence (numbering based on the corresponding AY823990 sequence) from the ORF1 capsid protein
  • SEQ ID NO:25 provides the amino acid sequence of TTV strain AY823990 ORF1 .
  • SEQ ID NOS:26-29 define primer sequences.
  • those familiar with the art will recognize that numerous slightly different abbreviations are commonly used interchangeably for specific serotypes, for example, gITTV, TTVgl , genotype 1 TTV, serotype 1 TTV, gtl TTV, and the like. A similar situation exists for genotype 2.
  • porcine and “swine” are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig.
  • “Mammals” include any warm-blooded vertebrates of the Mammalia class, including humans.
  • infectious DNA molecule is a DNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell.
  • isolated polynucleotide molecule refers to a composition of matter comprising a polynucleotide molecule of the present invention purified to any detectable degree from its naturally occurring state, if any.
  • the nucleotide sequence of a second polynucleotide molecule is "homologous" to the nucleotide sequence of a first polynucleotide molecule , or has "identity" to said first polynucleotide molecule, where the nucleotide sequence of the second polynucleotide molecule encodes the same polyaminoacid as the nucleotide sequence of the first polynucleotide molecule as based on the degeneracy of the genetic code, or when it encodes a polyaminoacid that is sufficiently similar to the polyaminoacid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in practicing the present invention.
  • Homologous polynucleotide sequences also refers to sense and anti-sense strands, and in all cases to the complement of any such strands.
  • a polynucleotide molecule is useful in practicing the present invention, and is therefore homologous or has identity, where it can be used as a diagnostic probe to detect the presence of TTV virus or viral polynucleotide in a fluid or tissue sample of an infected pig, e.g. by standard hybridization or amplification techniques.
  • nucleotide sequence of a second polynucleotide molecule is homologous to the nucleotide sequence of a first polynucleotide molecule if it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the
  • BLASTN algorithm National Center for Biotechnology Information, otherwise known as NCBI, (Bethesda, Maryland, USA) of the United States National Institute of Health).
  • NCBI National Center for Biotechnology Information
  • BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences- a new tool for comparing protein and nucleotide sequences.” (1999) FEMS Microbiol Lett. 174:247-250.]. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1 , and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 89:10915-10919. 1992). The percent identity is then calculated as: Total number of identical matches x 100/ divided by the length of the longer sequence+number of gaps introduced into the longer sequence to align the two sequences.
  • a homologous nucleotide sequence has at least about 75% nucleotide sequence identity, even more preferably at least about 80%, 85%, 90% and 95% nucleotide sequence identity. Since the genetic code is
  • a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.
  • a homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
  • non-silent mutations i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
  • certain conservative amino acid substitutions may be made which are generally recognized not to inactivate overall protein function: such as in regard of positively charged amino acids (and vice versa), lysine, arginine and histidine; in regard of negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and in regard of certain groups of neutrally charged amino acids (and in all cases, also vice versa), (1 ) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and
  • homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above.
  • homologous nucleotide sequences can be determined by hybridization under selected conditions.
  • the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO: 1 (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO: 1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2xSSC/0.1 % SDS at 42°C (see Ausubel et al editors, Protocols in Molecular Biology, Wiley and Sons, 1994, pp.
  • hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe.
  • the hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51 .
  • a second nucleotide sequence is homologous to SEQ ID NO: 1 (or any other sequence of the invention) if it hybridizes to the complement of SEQ ID NO: 1 under highly stringent conditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 xSSC/0.1 % SDS at 68°C, as is known in the art.
  • isolated polynucleotide molecules and the isolated RNA molecules of the present invention include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.
  • ttvg1 -7 SEQ ID NO: 4
  • ttvGT1 -17 SEQ ID NO: 5
  • ttvGT1 -21 SEQ ID NO: 6
  • ttvgtl -27 SEQ ID NO: 3
  • ttvgt1 -178 SEQ ID NO: 7 or a fragment thereof than encodes the TTV capsid protein or a fragment of said protein;
  • the invention also provides a polypeptide encoded by any of the open reading frames of the genotype 2 TTV13 (SEQ ID NO: 1 ) or genotype 2 TTV10 (SEQ ID NO:2) polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • the invention also provides a polypeptide encoded by any of the open reading frames of the (all sertotype 1 ) ttvg 1 -7 (SEQ ID NO: 10), ttvGT1 -17 (SEQ ID NO: 1 1 ), ttvGT1 -21 (SEQ ID NO: 12), ttvgt1 -27 (SEQ ID NO: 13), and ttvgtl - 178 (SEQ ID NO:9) ORF1 polynucleotides, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
  • polypeptide is expressed from ORF1 , and preferred mixtures include a combination of the polypeptides of ORF1 and ORF2, and ORF1 and ORF3.
  • TTV polypeptide-based vaccines wherein the antigen is defined by: (a) the first 300 N-terminal amino acids of the ORF1 capsid protein of TTV13 (SEQ NO:1) or TTV10 (SEQ ID NO:2); or (b) an amino acid sequence that is at least 90 percent identical thereto;
  • the DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of the viral genes and their encoded gene products.
  • Knowledge of a polynucleotide encoding a viral gene product of the invention also makes available anti-sense polynucleotides which recognize and hybridize to
  • fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to a specific RNA (as determined by sequence comparison of DNA encoding a viral polypeptide of the invention as well as (ii) those which recognize and hybridize to RNA encoding variants of the encoded proteins.
  • Antisense polynucleotides that hybridize to RNA DNA encoding other TTV peptides are also identifiable through sequence comparison to identify characteristic, or signature sequences for the family of molecules. Such techniques (see Example 8) are further of use in the study of antigenic domains in TTV polypeptides, and may also be used to distinguish between infection of a host animal with remotely related non-TTV members of the Circoviridae.
  • Example 4 provides guidance as to effective codon optimization for enhanced expression in yeast and E. coli for the constructs of the invention.
  • Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release.
  • Diluents include water, saline, dextrose, ethanol, glycerol, and the like.
  • Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
  • Stabilizers include albumin, among others.
  • Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
  • Vaccines of the present invention may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others.
  • adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, MT), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta GA), QS-21 (Cambridge Biotech Inc., Cambridge MA), SAF-M (Chiron, Emeryville CA), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant.
  • Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1 % (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 ⁇ g/ml Quil A, 100 ⁇ g/ml cholesterol, and 0.5% (v/v) lecithin.
  • Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1 % (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 ⁇ g/ml Quil A, and 50 ⁇ g/ml cholesterol.
  • Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
  • Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, with may be additionally lipidated, such as those described in
  • ORFI TTV protein is formulated with 5% AMPHIGEN®.
  • Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention.
  • sustained release formulations include virus, infectious DNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like.
  • biocompatible polymers such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like.
  • the virus, plasmid, or viral vector can be microencapsulated to improve
  • Liposomes can also be used to provide for the sustained release of virus, plasmid, viral protein, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Patent
  • An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, viral protein plasmid or viral vector, and then increasing the dosage while monitoring the effects.
  • An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine.
  • Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like.
  • the actual dosage is preferably chosen after consideration of the results from other animal studies (see, for example, Examples 2 and 3 below).
  • One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination.
  • the timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above.
  • the effective dose amount of virus, protein, infectious DNA molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated.
  • the dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 10 1 to about 10 9 pfu (plaque forming units), more preferably from about 10 2 to about 10 8 pfu, and most preferably from about 10 3 to about 10 7 pfu.
  • the dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about O. ⁇ g to about 100mg, more preferably from about ⁇ g to about 10mg, even more preferably from about 1 ( ⁇ g to about 1 mg.
  • the dose amount of an infectious DNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 ⁇ g to about 100mg, more preferably from about ⁇ g to about 10mg, even more preferably from about 10 ⁇ g to about 1 mg.
  • the dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 10 1 pfu to about 10 9 pfu, more preferably from about 10 2 pfu to about 10 8 pfu, and even more preferably from about 10 3 to about 10 7 pfu.
  • a suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
  • Suitable doses for viral protein or peptide vaccines range generally from 1 to 50 micrograms per dose, or higher amounts as may be determined by standard methods, with the amount of adjuvant to be determined by recognized methods in regard of each such substance.
  • an optimum age target for the animals is between about 1 and 21 days, which at pre-weening, may also correspond with other scheduled vaccinations such as against Mycoplasma hyopneumoniae.
  • a preferred schedule of vaccination for breeding sows would include similar doses, with an annual revaccination schedule.
  • anti-TTV antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, porcine, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a TTV polypeptide of the invention.
  • the term "specific for” indicates that the variable regions of the antibodies of the invention recognize and bind a TTV polypeptide exclusively (i.e., are able to distinguish a single TTV polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) to interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA
  • antibody refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen.
  • Immunoglobulins are serum proteins composed of "light” and “heavy” polypeptide chains having "constant” and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
  • Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab')2, as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences. Diagnostic Kits
  • the present invention also provides diagnostic kits.
  • the kit can be valuable for differentiating between porcine animals naturally infected with a field strain of a TTV virus and porcine animals vaccinated with any of the TTV vaccines described herein.
  • the kits can also be of value because animals potentially infected with field strains of TTV virus can be detected prior to the existence of clinical symptoms and removed from the herd, or kept in isolation away from naive or vaccinated animals.
  • the kits include reagents for analyzing a sample from a porcine animal for the presence of antibodies to a particular component of a specified TTV virus.
  • Diagnostic kits of the present invention can include as a component a peptide or peptides from ORF1 , 2, or 3 which is present in a field strain but not in a vaccine of interest, or vice versa, and selection of such suitable peptide domains is made possible by the extensive amino acid sequencing as provided for in Examples 1 and 2 of the Specification.
  • kits of the present invention can alternatively include as a component a peptide which is provided via a fusion protein.
  • the term "fusion peptide" or "fusion protein” for purposes of the present invention means a single polypeptide chain consisting of at least a portion of a TTV virus protein, preferably of ORF1 , and a heterologous peptide or protein.
  • DNA was purified from porcine serum using a DNA blood mini kit (Qiagen) per manufacturer's protocol. DNA was eluted from the columns in 50 ⁇ _ Tris- EDTA buffer. DNA was then amplified via random primed rolling circle
  • Plasmid DNA was isolated from transformed colonies and digested with EcoRI to confirm presence of an approximately 2.7 kB insert. Four clones (4, 7, 10 and 13) were selected and submitted to ACGT, Inc. for sequencing.
  • TTV13 SEQ ID NO:2
  • TTV10 SEQ ID NO: 1
  • TTV genotype 2 AY823991 DNA sequence
  • TTV13 (137) TCTGCAAAAAAGAGGAAATAAATTTCATTGGCTGGTCCATAAGTCCTCAT
  • TTV10 (148) TCTGCAAAAAAGAGGAAGTAAATGCTATTGGCTAAATCTGAAGTCTTCAT AY823991 (187) TAGAATAATAAAAGAACCAATCAGAAGAACTTCCTCTTTTAGAGTATATA
  • TTV10 (348) CGGAGTCAAGGGGCCTATCGGGCGGGCGGTAATCCAGCGGAACCGGGCCC
  • TTV10 (398) CCC-TCCATGGAGGAGAGATGGCTGACGGTAGCGTACGCCGCCCACGGAT
  • TTV10 (447) TATTCTGCGCCTGCAGTAAGCCCAAAGACCACCTTGAAAAATGCCTTTCC
  • TTV13 (537) AGGTGGAGACGCTACTTTCGATATCGGTATCGACGCGCTCCTCGCCGCCG
  • TTV10 (547) AGGTTCCAGCGCTACTTTCGATATCGGTATAGACGCGCTCCTCGCCGCCG
  • TTV13 (587) CCG CACAAAGGTAAGGAGACGGAGG AGGAAAGCTCCGGTCATAC
  • TTV10 (597) CCGACGCTACAAGGTAAGGAGACGGAGGGTTAAAAAGGCTCCGGTCATTC
  • TTV13 (631) AATGGAACCCTCCTAGCCGGAGGACCTGCCTCATAGAGGGGTTCTGGCCG
  • TTV10 (647) AATGGTTCCCCCCAACAGTCAGAAACTGTTTTATCAAGGGAATCTGGCCG
  • TTV13 (881 TAGACATCCTTGGAGAAACTATATAGTGACTTGGGATCAGGACATTCCTT
  • TTV10 (897 TAGACACACAACCAGATCCTACGTAGTAACATGGGACCAAGACATACCAT
  • TTV13 (931 GTAAACCTTTACCATATCAGAACTTACACCCATTATTAATGCTATTAAAA
  • TTV10 (947 GTAAACCTTTACCATACACAAATTTACATCCATTTGTAATGCTTCTAAAA
  • TTV13 (981 AAACAACACAAATTAGTACTCTCACAACAAAACTGTAACCCTAACAGAAA
  • TTV10 (997 AAACATCATAAAGTAGTTCTAAGCAAACAAGACTGTAATCCTAGAAAAAT
  • TTV13 (1031 ACAAAAACCTGTAACTTTAAAATTCAGACCGCCACCAAAACTAACTTCAC
  • TTV10 (1047 GGACAAACCAGTCACCTTAAAAATAAAGCCACCACCAAAACTCACATCAC
  • TTV13 (1081 AATGGAGACTAAGTAGAGAATTAGCAAAAATGCCACTCATTAGACTAGGA
  • TTV10 (1097 AGTGGAGACTAAGCAGAGAATTATCAAAAATACCGCTCTTAAGACTAGGA
  • TTV13 (1131 GTTAGTTTTATAGACTTAACAGAACCGTGGCTAGAAGGTTGGGGAAATGC
  • TTV10 (1147 GTTTCTTTAATAGACTTCAGAGAACCATGGGTTGAAGGTTTTGGAAATGC
  • TTV10 (1197 ATTCTTTAGTACTTTAGGATATGAAGCAGATAAAAGCAATTTAAAAACAA
  • TTV13 (1231 CAAATTGGTCACAAATTAAATATTACTGGATATATGATACAGGAGTAGGA
  • TTV10 (1247 GCGCTTGGTGCCAATGTAAATACTTCTGGATATATGATACCGGAGTAAAT
  • TTV10 1423 TTTGGAAGATCTGAAAGAGACTTAAAAGCACTAGCAACTTCAAACACAAA
  • TTV10 (1523 CTGTAATAGGATGGGCTAGCAGTAACAACACAGCACAAGATAGTACACAA AY823991 (1577) AACAGTGGTGGATCAACATCAGCTATACAAGGTGGATATGTAG CA
  • TTV13 (1623) TGGGC-AGGAGGACAAGGAAAACTAAATCTAGGAGCAGGATCAATAGGAA
  • TTV10 (1811) TTGCCCTCTTCGGACCCTTGGTAGAAAAAGCAAA-CTGGGAAGGCCTAGA
  • TTV10 (2057) TCCTCAATGCGTGGGATTATGACTATGATGGAATTGTTAGAAAAGACACT
  • TTV10 (2154) GTACCCGCTCGCTGGACCCAAAACAGAGAAATTGCCCTCCTCAGACGAAG
  • TTV13 (2221) ACGAAGAGAGCGTTATCTCAAGCACGAGCAGTGGATCCGATCAAGAA
  • TTV10 (2304 CCTCCAGCATGTCCAGCGACTGGTGAAGAGATTCAGGACCCT ATAGA
  • TTV13 (2365 TAAATACAGAAACCTAGCAGACCCCTCACTCAATGTCACAGGACACATGG
  • TTTV10 (2351 CAAATACAGAAACTTAGCAGACCCCTCATTAAATGTCACAGGACATTTTG
  • TTV10 (2401 AACACTTCTGCCGCTTACACTATAAAAACATAGCAGAAATCAGAGCTAGA
  • TTV10 2451 AATGCCAAAAAAAACCTCAATAAACTATACTTTTCAGACTAAAAGAAG--
  • TTV13 2515 TCCTGTGTCCAATCTATTTTTTTAAACACCCTTCAAAATGGCGGGAGGGA
  • TTV13 2565 CACAAAATGGCGGAGGGACTAAGGG TG N
  • TTV10 2533 TAGCGGGGGGGACC CCCCTGCACCCCCCCATGCGG
  • TTV13 (2638 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
  • TTV10 (2570 GGGCTCCGCCCCCTGCACCCCCGGGAGGGGGGGAAACCCCCCCTCAACCC
  • TTV10 (2620 CCCGCGGGGGGGCAAGCCCCCCTGCACCCCCCC
  • TTV 13 shows 92% identity when compared with previously published AY823991 sequence. However, TTV10 only show 76% similarity between either AY823991 or TTV13 and may be considered a separate genotype.
  • TTVlOOrfl (1 MPFHRYRRRRRRPTRRWRRRRFQRYFRYRYRRAPRRRRRYKVRRRRVKKA
  • TTV130RF1 (1 MPYRRYRRRRRRPTRRWRHRRWRRYFRYRYRRAPRRRR-TKVRRR-RRKA
  • TTVlOOrfl 151 DIPCKPLPYTNLHPFVMLLKKHHKVVLSKQDCNPRKMDKPVTLKIKPPPK
  • TTV130RF1 149 DIPCKPLPYQNLHPLLMLLKKQHKLVLSQQNCNPNRKQKPVTLKFRPPPK
  • TTV130RF1 (199 LTSQWRLSRELAKMPLIRLGVSFIDLTEPWLEGWGNAFYSVLGYEAIKEQ
  • TTVlOOrfl (251 LKTSAWCQCKYFWIYDTGV NHVYWMLNKDAGDNAGDLITNQNS
  • TTV130RF1 (249 GHWS WSQIKYYWIYDTGVGNAVYWMLKQDVDDNPGKMASTFKTTQGQH
  • TTVlOOrfl (296 IAHIEQIGEGYPYWLYFFGRSERDLKALATSNTNIRNEFNTNPNSKK
  • TTV130RF1 (299 PNAIDHIELINEGWPYWLYFFGKSEQDIKKEAHS-AEIAREYATNPKSKK
  • TTVlOOrfl (343 LKIAVIGWASS NTAQDSTQGANTPIEGTYLISHVLQTSGH TAGAAQ
  • TTV130RF1 (348 LKIGIVGWASSNFTTPGSSQNSGG-NIAAIQGGYVAWAGGQGKLNLGAGS
  • TTV130RF1 (397 IGNLYQQGWPSNQ WPNTNRDETNFDWGLRSLCILRD MQLGNQELDDEC
  • TTVlOOrfl (440 TMFALFGPLVEKAN-WEGLEKIPELKPELKDYNILMRYNFRFQWGGHGTE
  • TTV130RF1 (447 TMLSLFGPFVEKANPIFATTDPKYFKPELKDYNLIMKYAFKFQWGGHGTE
  • TTVlOOrfl (489 TFKTSIGDPSQIPCPYGPGEAPQHLVRNPSKVHEGVLNAWDYDYDGIVRK
  • TTV130RF1 (497 RFKTTIGDPSTIPCPFEPGDRFHSGIQDPSKVQNTVLNPWDYDCDGIVRK
  • TTV130RF1 (547 DTLKRLLELPTETEEEEKAYPLLGQKTEKEPLSDSDEESVISSTSSGSDQ
  • TTV130RF1 (597 EEETQR--RKHHKPSKRRLLKHLQRWKRMKTL-- Amino Acid Alignment of TTV10 TTV13 ORF with Published Sequence
  • TTV10 ORF demonstrates only 65% homology to the published sequence and may represent a unique phenotype of TTV
  • primers were designed to clone the ORF from TTV10 and TTV13 for expression in baculovirus using the
  • TTV13Rev121 1 5' cgt act cga gtc aca gtg ttt tea tec (SEQ ID NO:26); TTV13For121 1 : 5' eta ggt acc atg cct tac aga cgc tat (SEQ ID NO:27)
  • the TTV insert in pGem was isolated by EcoRI digestion, gel- purified and re-circularized using standard ligation conditions. Following an overnight ligation at 4°C, ligase was inactivated at 65°C, and the reaction was purified using QuiQuick purification kit (Qiagen) following the manufacturer's protocol.
  • TTVORF13 was the PCR amplified using re-circularized TTV13 genomic DNA with Expand Hi-Fidelity® enzyme (Roche) using the above described TTV13 forward and reverse primers (0.15 ⁇ each), 0.2 mM dNTP's in 1 X Hi Fidelity enzyme buffer. PCR conditions were: 1 cycle at 4 min, 95°C; 35 cycles with 94°C, 15 sec denaturation, 55°C, 30 sec anneal, and 68°C 1 .5 min extension; and 1 cycle of 72°C, 7 min extension.
  • TTVORF10 was PCR amplified using re-circularized TTV10 genomic DNA with Expand Hi-Fidelity® enzyme (Roche) using the above described TTV10 forward and reverse primers (0.15 ⁇ each) 0.2 mM dNTP's in 1 X Hi Fidelity enzyme buffer. PCR conditions were: 1 cycle at 4 min, 95®°C; 35 cycles with 94°C, 15 sec denaturation, 56°C, 30 sec anneal, and 68°C 1 .5 min extension; and 1 cycle of 72°C, 7 min extension.
  • PCR products were purified using QiaQuick PCR purification kit (Qiagen) following the manufacturer's protocol. Both PCR TTV10Orf1 anfd TTV130rf1 products and the Gateway entry plasmid, pENTR3C, were digested with Kpnl. Digested DNA was purified using QIAquick PCR amplification kit and
  • TTV10 ORF1 or the TTV130RF1 DNAs were ligated into pENTR3C using standard ligation procedures. Following a 2 hour ligation at room temperature, ligated DNA was used to transform chemically competent E. coli DH5a. Transformed colonies were selected using Kanamycin. Plasmid was purified from transformed E.coli and ORF1 DNA insertion was verified by restriction fragment analysis.
  • pENTR3C plasmids containing TTV10 ORF1 or TTV13 ORF1 were then inserted into Invitrogen destination vectors pDESTI O or pDEST 20 encoding a His6X or a GST protein N-terminal to the TTV Orf1 reading frame.
  • Recombinant pDEST vectors containing the open reading frame of TTV Orf1 were used to transform DHI OBac E. coli.
  • Recombinant bacmid DNA was isolated and used for transfection of SF9 cells following standard protocol.
  • Recombinant baculovirus containing the native Orf1 were isolated by plaque purification.
  • Standard PCR was used to incorporate a BamH1 restriction site upstream from the initiation codon in TTV10 Orf1 or an Xbal restriction site upstream from the initiation codon in TTV Orf13. These constructs were cloned into pFastBac transfer vector and used to transform E. coli DHI OBac. The resultant
  • Recombinant baculovirus containing the native Orf1 were isolated by plaque purification. Confirmation of recombinant baculovirus was performed using PCR.
  • TTVOrfl 0 Full-length TTVOrfl 0 was also cloned into a PGex-6p-1 vector for expression of a GST-fusion protein in a bacterial system.
  • the TTV ORFs contain an arginine rich amino terminus.
  • the arginine rich segment was removed from TTVOrfl 3 at a convenient restriction site (EcoR1 ) located at nucleotide 368 of the Orf1 open reading frame and was in frame with the GST coding region of pGex-6p-1 . This clone resulted in the removal of 100 amino- terminal amino acids containing a highly enriched arginine segment.
  • Total cellular DNA from porcine bone marrow was amplified by rolling circle amplification following procedures described above, except that single- stranded binding protein was added to improve the efficiency of the amplification reaction.
  • Amplification products were digested with EcoR1 , purified using a
  • AY823990 (844 GAAGACCAGTACGGATACCTCGTACAATACGGGGGAGGTTGGGGAAGTGG ttvgl-7 ... (850 GAGGACAACTATGGATACTTAGTACAGTATGGAGGTGGTTGGGGTAGCGG ttvGTl-17... (850 GAAGACAACTACGGCTACTTAGTACAGTACGGAGGAGGTTGGGGGAGCGG ttvGTl-21... (850 GAGGACAACTATGGATACTTAGTACAGTATGGAGGTGGTTGGGGTAGCGG ttvgtl-27... (850 GAGGATCAATACGGATACCTGGTGCAATACGGTGGAGGTTGGGGAAGTGG
  • AY823990 (2491 TTACTAATGGGGGGGGGTCCGGGGGGGGCTTGCCCCCCCGCAAGCTGGGT ttvgl-7 ... (2499 TATTATTTTGGGGGG--TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGGGT ttvGTl-17... (2500 TTATTTGTAGGGGGGG-TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGGGT ttvGTl-21... (2499 TATTACTTTGGGGGGG-TCCGGGGGGGGCTTGCCCCCCCGTAAGCTGTGT ttvgtl-27... (2497 TTATTTTTTGGGGGG--TCCGGGGGGGGCTTGCCCCCCCCCGAAAGCTGGGT
  • TTVgt1 -27 demonstrates the greatest homology with published sequence, AY823990, demonstrating 91 % identity.
  • TTVgt1 -7,17, and 21 demonstrate 85- 87% identity.
  • TTVgt1-7 and TTVgt1 -21 share 99% nucleotide identity Orf1 Amino Acid Alignment
  • Ttgl-170rfl MAPARRWRRGFGRRRRRYRKRRWGWRRRYWRYRPRYRRRRWWRRRRRSV
  • Ttgl-270rfl (1) MAPTRRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV ttgl-210rfl (1) MAFARRWRRRFGRRRRRYRKRRYGWRRRYYRYRPRYYRRRWLVRRRRRSV
  • Ttvgl-70rfl (51) YRRGGRRARPYRISAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD
  • Ttgl-170rfl (51) YRRGGRRARPYRISAFNPKIMRRWIRGWWPILQCLRGQESLRYRPLQWD
  • Ttgl-270rfl (51) YRRGGRRARPYRVSAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD ttgl-210rfl (51) YRRGGRRARPYRISAFNPKVMRRWIRGWWPILQCLKGQESLRYRPLQWD
  • Ttgl-170rfl (101) VEKSWRIKTDLEDNYGYLVQYGGGWGSGEVTLEGLYQEHLLWRNSWSKGN
  • Ttgl-170rfl (151) DGMDLVRYFGCIVYLYPLKDQDYWFWWDTDFKELYAESIKEYSQPSVMMM
  • Ttvgl-70rfl (201) AKRTKIVIARSRAPHRRKVRRIFIPPPSRDTTQWQFQTDFCNRPLFTWAA
  • Ttgl-170rfl (201) AKKTKIVIARSRAPHRRKVRKIFIPPPSRDTTQWQFQTEFCNKPLFTWAA
  • Ttgl-270rfl (201) AKRTRIVIARDRAPHRRKVRKIFIPPPSRDTTQWQFQTDFCNRKLFTWAA ttgl-210rfl (201) AKRTKIVIARSRAPHRRKVRRIFIPPPSRDTTQWQFQTDFCNRPLFTWAA
  • Ttgl-170rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPAQ tgl-270rfl (251) GLIDMQKPFDANGAFRNAWWLEQRTEQGEMKYIELWGRVPPQGDSELPKK ttgl-210rfl (251) GLIDLQKPFDANGAFRNAWWLEQRNEAGEMKYIELWGRVPPQGDTELPLQ
  • Ttvgl-70rfl (301) TEFQKPSGYNPKYYVNPGEEKPIYPVIIYVDMKDQKPRKKYCVCYNKTLN
  • Ttgl-170rfl (301) KEFQKPDGYNPKYYVQAGEEKPIYPIIIYVDKKDQKARKKYCVCYNKTLN
  • Ttgl-270rfl (301) SEFTTAT-DNKNYNVNDGEEKPIYPIIIYVDQKDQKPRKKYCVCYNKTLN ttgl-210rfl (301) TEFQKPSGYNPKYYVNPGEEKPIYPVIIYVDMKDQKPRKKYCVCYNKTLN
  • Ttgl-170rfl (351) RWRAAQASTLKIGDLQGLVLRQLMNQEMTYIWKEGEFTNVFLQRWKGFRL
  • Ttgl-170rfl (551) AASSRALSADTPTEAAQSALLRGDSEKKGEETEETTSSSSITSAESSTEG
  • Ttgl-270rfl (550) PASTRALCADTPTEATQSALLRGDSEKKGEETEETTSSSSITSAESSTEG ttgl-210rfl (551) AASSRALSADTPTEATQSALLRGDSEKKGEETEETSSSSSITSAESSTEG
  • Hydrophobicity plots of the proteins demonstrate 5 areas of hydrophilicity, which may indicate surface-exposed regions that are potentially antigenic. Two of these regions are at the amino terminus and at the carboxy terminus, and are both arginine-rich and highly conserved. A highly conserved hydrophilic region between amino acids 190 and 232 was observed and may potentially serve as antigenic site. The remaining hydrophilic regions between amino acids 295 and 316, and between amino acids 415 and 470 are also be antigenic.
  • the putative start codons for ORF1 and coding region are as follows: ttvgtl -27 nt 517-2435; ttvg1 -7 nt 517- 2435; ttvgtl -17 nt 517—2436; ttvgtl -21 nt 517-2439; ttv10 nt 487-2346; and ttv13 nt 477-2363.
  • the putative start codons for ORF 2 and coding region are as follows: ttvgtl -27 nt 428-646; ttvg1 -7 nt 428-643; ttvgtl -17 nt 428-643; ttvgtl - 21 nt 428-646; ttv10 nt 404-610; and ttv13 nt 394-597.
  • the processes were monitored for cell density and viability, and infection was monitored through monitoring of cell size and virus titration. Protein expression was monitored through SDS-PAGE, Coomassie gel analysis and Western blotting. To ensure proper control, negative and positive controls were maintained throughout all experiments. Although all experiments were able to confirm expression of the target protein, optimal conditions were found when utilizing SF9 cells maintained in ExCell 420 media (Sigma, SAFC) with a cell density of 2x10 6 cells/ml and an MOI of 0.1 , with the process terminated following a three day infection. The majority of the recombinant expressed protein can be located within the cell pellet although some resides in the resultant supernatant.
  • Figure 1A sample lanes were as follows (from right to left)
  • Lane 5 (see the arrows in Figure 1 B) demonstrates a unique reaction to a ⁇ 69kD and 49kD protein in the native TTV ORF1 expression utilizing anti-TTV ORF1 rabbit polyclonal antibody.
  • antigen provided only TTV sequence and was not tagged.
  • Figure 6B sample lanes were as follows (from right to left)
  • a liver was collected aseptically from a caesarean-derived, colostrum deprived (CDCD) pig.
  • the liver tissue was tested for g1 and g2 TTV in unique qPCR assays and confirmed to be positive for only gITTV.
  • a 10% (wt/vol) liver homogenate was then prepared in media containing antibiotics and antimycotics. Finally, the homogenate was clarified by centrifugation, designated as gITTVpO and frozen at -70C. The resulting gITTV homogenate was tested to be free of extraneous viruses, bacteria and mycoplasma via routine testing.
  • the present study was conducted to evaluate the efficacy of three TTV vaccine candidates administered at ⁇ 7 days of age, and again at weaning (-21 days of age) followed by a challenge at ⁇ 5 weeks of age.
  • TTV is a small, non-enveloped virus with a single-stranded circular DNA genome of negative polarity.
  • the genome includes an untranslated region and at least three major overlapping open reading frames.
  • Porcine TTV is ubiquitous and PCR-detection of the virus in serum samples collected from various geographical regions shows prevalence in pigs ranging from 33 to 100%.
  • Krakowka et al., AJVR (2008) 69: 1623-1629 reported that g1 -TTV inoculated pigs had no clinical signs but developed interstitial pneumonia, transient thymic atrophy, membranous
  • formulations can be numerically or statistically differentiated when compared to challenge control groups.
  • seronegative females without a history of disease caused by PRRSV or M hyo were sourced from Lincoln Trail / Puregenic Pork, Alton, IL, and transported to the Pfizer Animal Health Research Farm in Richland, Ml at approximately 3 weeks pre-partum. If necessary, sows were induced to farrow within a 2 or 3 day period using injectable prostaglandin (Lutalyse®). Normal piglets from these sows were allotted to study according to the allotment design. Pigs were randomized to treatment by litter and each litter had at least one piglet assigned to each treatment. Housing: During the vaccination phase, pigs were housed with their mother with no cross-fostering, in BL-2 isolation facilities.
  • Pigs remained housed by litter until the time of 2 nd vaccination.
  • Post-second vaccination pigs were moved to a further facility and housed in two rooms (one room contains NTX (non-vaccinated and non-challenge controls) animals, the second room vaccinates), and each room contains 4 or 8 pigs per pen.
  • NTX non-vaccinated and non-challenge controls
  • Vaccine was masked using a numeric code prior to vaccination. The investigator, vaccine administrator and study personnel were masked to treatment and did not have access to the masking code unless treatment information was required for the welfare of an animal. Investigational Veterinary Products
  • gITTV pass 1 was derived from liver homogenate tested positive (7.6x10e8 to 1.6x10e9 DNA copies/2ml_) for gITTV and negative for g2TTV by qPCR. An appropriate number of bottles were removed from the freezer and thawed shortly before challenge. An aliquot was then removed from one of the bottles, and held for retitration at a later time.
  • Challenge stock was transported on ice to the research facility and maintained on ice during the challenge procedure.
  • a challenge dose equals 2.0ml_ of stock solution (2.0 mL intraperitoneal). The dose was delivered to each pig is therefore expected to be 7.6x10e8 to 1 .6x10e9 DNA copies/2mL. Following challenge, an aliquot of challenge stock was kept for titration to confirm challenge dose.
  • Body Weights All pigs were weighed Day 0, the day of challenge (Day 28) and at necropsy. All weights were recorded.
  • Vaccination At approximately 7 days of age (Day 0), -10 randomly allotted pigs per treatment group (Groups T01 thru T04) were vaccinated as described in Table 1 . Pigs were injected in the right neck with a single dose syringe (2.0 mL intramuscular (IM) dose) of IVP, or a 2 mL IM dose of control according to allotment. A second dose of the same IVP or control was administered in the left neck at the time of weaning (-21 days of age).
  • IM intramuscular
  • Blood Sampling Prior to Day 0, Day 14 (prior to vaccination) and Day 28 prior to challenge (as well as Day 31 , 34, 37, and 40), a blood sample was collected, using 5 mL or 9 mL serum separator tubes (dependant on body weight), from all pigs for gITTV status (qPCR-Pfizer-VMRD Laboratory Sciences). Serum samples were aliquoted by site personnel to at least three separate tubes and were stored at -80 C. Table 2: gITTV qPCR analysis to be performed on sera by time point
  • Rectal Temperatures post challenge were recorded once per day on Day 28 prior to challenge as well as Day 31 , 34, 37, and 40.
  • Necropsies On Day 40 all animals were euthanized and necropsied. Upon necropsy, lung lesions were scored using the following methods: 1 ) a numeric score (0, 1 , 2, 3) and 2) the percentage of consolidation for each lobe (left cranial, left middle, left caudal, right cranial, right middle, right caudal, and accessory) was scored and recorded as percent of lobe observed with lesions. Liver, kidney, thymus and lymph nodes were also scored. A blood sample was also taken prior to euthanasia. Tissues were collected as indicated in the following table:
  • the percentage of total lung with lesions was transformed and analyzed with a general linear mixed model with fixed effects, treatment, and random effect litter. Linear combinations of the parameter estimates were used in a priori contrasts after testing for a significant (P ⁇ 0.10) treatment effect. The 10% level of significance (P ⁇ 0.10) was used to assess statistical differences.
  • qPCR data will be transformed prior to analysis with an appropriate log transformation.
  • the transformed titers will be analyzed using a general linear repeated measures mixed model analysis. Pairwise treatment comparisons will be made at each time point if the treatment or treatment by time point interaction effect is significant (P ⁇ 0.10).
  • Treatment least squares means, 90% confidence intervals, the minimum and maximum will be calculated and back-transformed for each time point. Descriptive statistics, means, standard deviations, and ranges, will be calculated for each treatment and day of study, pre-challenge. Study Results and Discussion
  • T01 Chromos expressed gI TTV ORF1
  • T02 Baculovirus expressed g2TTV ORF1
  • T04 Challenge controls
  • the challenge virus was comprised of infectious gITTV, it may not be surprising that the genotype 2 ORF1 from Baculovirus did not provide very substantially lower lung lesions as compared to the challenge controls. It is however interesting to note that while not substantial, it did offer numerically lower lung lesion scores compared to the challenge controls, thereby indicating that some level of cross protection is possible between different TTV genotypes upon optimization of dose and adjuvant selection.
  • Example 4 Codon optimization and recombinant expression glTTV ORF1 as a full length protein with a 6His tag, and detection thereof by an antibody.
  • the TTVgl nucleotide sequence was submitted to GenScript (Piscataway, New Jersey, USA) for codon optimization and gene synthesis for both E. coli and Saccharomyces cerevisiae. In both cases, the codon optimized gene was cloned into the GenScript pUC57 vector, as product.
  • GenScript OptimumGeneTM codon optimization analysis involves analysis of numerous parameters including codon usage bias,GC content, CpG dinucleotide content, mRNA secondary structure, identification of possible cryptic splicing sites, presence of premature polyA sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motifs (ARE), inhibition sites (INS), repeat sequences of various kinds (including direct, reverse and dyad), and also restriction sites that may interfere with cloning.
  • Translational performance may be additionally improved via translational initiation Kozak sequences, Shine-Dalgarno sequences, and to increase efficiency of translational termination via stop codons.
  • SEQ ID NO: NOS 18-20 provide TTV capsid gene that were codon optimized for both Escherichia coli (NOS: 18-19) and Saccharomyces cerevisiae (NO: 20).
  • the sequences for E. coli are very similar, however, to clone the gene into the commercial pET101/D-TOPO expression vector (Invitrogen) to create 76057-4 (SEQ ID NO:19), additional CA nucleotides had to be added at the N- terminus.
  • the pET101/D-TOPO expression vector also has a C-terminal V5 tag and 6X-His for purification, although the sequences for 76057-3 (SEQ ID NO: 18) and 76057-4 are otherwise identical.
  • the expressed codon-optimized TTVgl protein is approximately 68 kD in size, relative to the 63kD protein, due to the addition of a 10 amino acid protective peptide at the amino terminus, and 32 amino acids corresponding to the V5 epitope and a 6X His tag at the carboxy terminus ( Figure 2).
  • sequence for 76057-5 (SEQ ID NO: 20) has been codon optimized for S. cerevisiae, and it thus differs slightly from the E. coli sequences. In addition, this sequence lacks a 10 amino acid protective peptide at the N-terminus (which was added to the E. coli sequence), and it also has flanking restriction
  • the protective peptide of ten amino acids was added to N-terminus of the TTVgl sequence for expression in E. coli. since this has been shown to increase protein stablility when fused to the amino terminus. Restriction sites have been engineered such that the peptide can be removed for evaluation of the full length protein. Expression of the codon optimized TTVgl was evaluated in the pET101/D-TOPO vector with and without the protective peptide N-terminal fusion. The TTVgl sequence codon optimized for S. cerevisiae was also subcloned into a pESC-Trp vector with the potential for producing suface-expressed protein in yeast that can be used to elicit an antibody response in vivo.
  • Rabbit polyclonal antibodies were raised against Baculovirus expressed g2 TTV GST-ORF1 protein prepared in Example 2. Two rabbits were
  • the rabbit antibody did not, however, respond to the E.coli expressed g2TTV ORF1 that had the 100 A.A. N-terminal arginine-rich region removed from the amino terminus as described in Example 2. This may suggest that a major antigenic epitope may be in the 100 amino acid region that was missing in the truncated g2 TTV ORF1 , and that there is homology between g1 and g2 TTV in this region.
  • Monoclonal antibodies can be generated against full-length g1 TTV ORF1 , or other g1 TTV antigens.
  • Other potential immunizing antigens include g1 TTV whole virus, g2 TTV GST-ORF1 (Baculo), g1 TTV GST-truncated ORF1 (E coli), and g2 TTV GST-truncated ORF1 (E coli).
  • a peptide library can be generated to identify linear epitopes that are antigenic. For example, 18mer peptides, with a 10AA overlap, can be utilized to cover the TTV genome.
  • the peptides can then be utilized in Western blots or ELISA's to determine their overall reactivity to the gI TTV ORF1 or g2TTV ORF1 monoclonal and/or polyclonal antibodies so that immunogenic domains can further be identified.
  • Rabbit polyclonal antibodies may also be raised against three g1 TTV ORF1 peptides cross-linked to KLH, and subsequently screened using peptide- ovalbumin conjugates.
  • the peptide-KLH conjugates can also be used to produce monoclonal antibodies.
  • multiple g1 TTV ORF1 peptides copies may be conjugated together, including from different strains.
  • peptides were generated (CPC Scientific), they were then conjugated to KLH or ovalbumin (by the Proteos Co).
  • the KLH- conjugated peptides were used for immunization of rabbits, while the Ovalbumin conjugated peptides are used for screening the serum (i.e., to detect antibodies to the peptides and not the carrier protein).
  • TTV (459-479): DFGHHSRFGPFCVKNEPLEFQ (21 aa, pi 6.9)
  • CTWKRLRRMVREQLDRRMDHKRQRLH 26 aa, pi 13
  • Each of the three peptides has a single cysteine residue present in the sequence to enable selective peptide coupling to a carrier protein.
  • a further and highly preferred peptide is constructed by using the peptide sequence corresponding to residues 601 -620 of SEQ ID NO:9 (20AA, pi 13) except that a cysteine residue is used at the N-terminus in replacement for Asn 601 .
  • This peptide is also likely surface exposed in the native protein.
  • the Chromos ACE system is a protein expression platform that consists of three main components.
  • the first component is a neutral, functional mammalian artificial chromosome called the Platform ACE, which resides in the genetic material of a modified Chinese Hamster Ovary (CHO) cell line.
  • the second component is the ACE targeting vector, which is a plasmid used for loading target genes onto the Platform ACE.
  • the third element is a site-specific, unidirectional integrase, which catalyzes the direct and specific loading of the target gene onto the Platform ACE. Additional information concerning the ACE System can be found of the website of Chromos Molecular Systems, Inc. of Canada, or by contacting the company directly at 604-415-7100 where the technology is available for license.
  • the Chromos ACE system has a number of significant advantages over traditional protein production platforms. The first of these is speed. The first of these is speed.
  • Chromos ACE system allows for the rapid, efficient and reproducible insertion of selected genes.
  • the second advantage is expression. High level constituitive protein expression is achieved over time.
  • a third advantage is stability.
  • the Chromos ACE system allows selective and controlled protein expression. Briefly, restriction sites were added to both ends of the TTV7 ORF1 gI DNA using PCR. Additionally, the sequence for yeast invertase was added to the 5' end of a separate PCR preparation. The amplified sequences were then treated with restriction enzymes and sub-cloned into the plasmid pCTV927. The DNA sequence was verified by ACGT Inc.
  • CHk2 (Chinese Hamster Ovary) cells were then transfected with the plasmids using Lipofectamine 2000 (Invitrogen), and selective pressure was added using hygromycin B. Ten single-cell clones were analyzed for TTV protein production using SDS PAGE and Western Blotting. More specifically, the ACE Targeting Vector pCTV-TTV7ORF1 +YI was generated as follows (see Figure 3).
  • the gene TTV7ORF1 was obtained as a PCR product.
  • a primer was designed to contain the yeast invertase secretion signal and the restriction site EcoRV at the 5' end of the gene.
  • a second primer was designed to contain the restriction site Kpnl at the 3' end of the gene.
  • the plasmids pCTV-TTV7ORF1 +YI and pSIO343, which coded for TTV7ORF1 /yeast invertase and the unidirectional lambda integrase, respectively, were transfected into the Chk2 cell line, which contained the Platform ACE.
  • the transfected cells were named Chk2-TTV7ORF1 +YI . These cells were seeded in 96-well plates and monitored for the formation of single-cell clones. Media containing Hygromycin was added to each 96-well plate to select for cell clones that contained the ACE targeting vector. Once single-cell clones were identified, twelve of them were expanded into 24-well plates, and then to 6-well plates.
  • Figures 4 and 5 provides a 7-way amino acid alignment of ORF1 (capsid proteins) from 5 TTV gt1 viruses of the present invention and two TTV gt2 (or gt2-like) viruses of the invention.
  • ORF1 capsid proteins
  • TTV10 and TTV13 TTV10 and TTV13
  • TTV fragments (1900bp and 2200bp), which together span the entire TTV circular genome, were separately cloned into separate pCR 2.1 TA (Invitrogen) cloning vectors.
  • PCR primers were designed using the consensus sequence that was generated from strains of the present invention (ttvgt1 -27, -7, -17 and -21 ), and also from published sequences (AY823990(g1 ) and AB076001 -(Sd-TTV31 )).
  • Primer pairs that correspond to the sequence at 680s and 2608a or 1340s and 764a were used to amplify PCR products from DNA that was extracted from liver homogenate samples of pigs infected with TTV challenge strain. These PCR fragments were cloned into Invitrogen's pCR2.1 - TOPO TA vector using directions that were supplied with the kit. Clones were subsequently used to generate DNA sequences across the entire 2880 base genome and the sequence was found to be 86% homologous to published sequences GQ120664.1 and AY823990.1 .
  • gI TTV is a single-stranded DNA (ssDNA) virus. Fragments of gI TTV are converted to double-stranded DNA (dsDNA) using polymerase chain reaction (PCR). The dsDNA fragments of gI TTV are then cloned into pUC-based plasmid cloning vectors and transformed into E. coli. The fragments of gITTV are less than 1 full-length dsDNA equivalent of the gI TTV genome. Amplification of gI TTV dsDNA concatemers.
  • Concatemers of full-length g1 TTV dsDNA genome equivalents are generated using ⁇ 29 polymerase amplification kits (e.g., illustra TempliPhi).
  • Full-length gITTV dsDNA fragments are generated by digestion of the concatemers at appropriate restriction endonucleases (RE) sites.
  • RE restriction endonucleases
  • These full-length gI TTV dsDNA fragments can be cloned into plasmid vectors.
  • the concatemers or the uncloned fragments (resulting from RE digestion) can be used without immediate cloning in subsequent molecular biology constructions (see below).
  • Tandem duplications of the gI TTV genome Plasmid constructs encoding tandem duplications of the gI TTV genome are next generated. The tandem duplications in the constructs are approximately greater than 1 .2 copies of full- length dsDNA equivalents of the the gI TTV genome.
  • the tandem duplications in plasmids are generated using (1 ) subcloning employing appropriate RE sites, (2) PCR assembly of tandem duplications, or (3) other molecular biology methods.
  • the templates for the generation of the tandem duplications are the gI TTV dsDNA fragments and/or the full-length gI TTV dsDNA clones (yielded by ⁇ 29 polymerase amplification).
  • tandem duplication plasmid constructs are not identical to the gITTV virus.
  • the tandem duplication constructs are dsDNA while the virus is ssDNA, the constructs encode >1 .2 full- length dsDNA equivalents of the gI TTV genome while the virus has only one full- length equivalent, the construct contains interrupting plasmid sequences while the virus has only viral sequences.
  • the tandem duplication plasmid constructs are introduced into pigs (by inoculation, injection, electroporation, or other methods of introduction) or introduced into tissue culture cells (by transfection, electroporation, or other methods of introduction) where the plasmid construct recombines at homologous sequences to regenerate a unit-length dsDNA equivalent of the gI TTV genome.
  • the dsDNA equivalent of the gITTV genome is a presumed replicative intermediate of the gI TTV viral life cycle. The presence of this presumed dsDNA replicative intermediate will lead to the production of the bona fide ssDNA gI TTV.
  • Plasmid constructs directing in vivo transcription of gITTV ORFs can be made, such as the fusion of transcriptional promoters (e.g., CMV) to gITTV ORFs.
  • plasmid constructs directing the in vitro generation of gITTV ORF transcripts can be made, such as the fusion of transcriptional promoters (e.g., T7) to gITTV ORFs followed by use of in vitro transcription kits.
  • Either gITTV ORF-expressing plasmids or gITTV ORF- expressing RNA transcripts can be co-injected into pigs or co-transfected into cells along with the tandem duplication plasmid constructs to yield gITTV virus.
  • gITTV virus production Detection of gITTV virus production. To date, whole gITTV virus cannot be propagated in tissue culture cells. The generation of gITTV virus is detected by immune reagents (e.g., a-g1TTV antibody) or by molecular methods (e.g., qPCR).
  • immune reagents e.g., a-g1TTV antibody
  • molecular methods e.g., qPCR
  • Total DNA was isolated (DNEasy Blood and Tissue Kit, Qiagen, Valencia, CA ) from a frozen liver homogenate sample (200 microliters) derived from a prior TTV challenge study.
  • the DNA was then PCR-amplified using forward and reverse primers selected to overlap at the unique EcoRI site of the swine TTV 1 - 178 genome.
  • the forward primer: for TTVg1 -178 was selected as positions 1399 to1428 (ACGG... CCAA) from SEQ ID NO:7
  • the reverse primer: was selected to correspond to base positions 1443 (5')-to 1416 (3') ATAT... TTGT (opposite strand) from SEQ ID NO:7.
  • PCR conditions were as follows: 1 cycle of denaturation at 94°C for 1 minute; 35 cycles of 94°C, 30 seconds; 55°C, 30 sec; 72°C, 3 minutes; followed by a final 10 minute extension at 72°C.
  • the resultant ⁇ 2.8kb fragment was cloned into pCR2.1 vector using a TOPO TA cloning kit. (Invitrogen, Carlsbad, CA). Upon sequence verification, this plasmid (named pCR2.1 +TTV_178) was found to contain the entire TTV 1 -178 genome sequence.
  • pCR2.1 +TTV_178 vector was then linearized with EcoRI in order to release the full length TTV genome, which was then transfected into human embryonic kidney (293), baby hamster kidney (BHK-21 ), swine testicular (ST) and porcine kidney (PK) cells lines using Lipofectin (Invitrogen, Carlsbad, CA). The transfection was allowed to proceed for a total of 5 days at which time the cells were fixed, and then used for IFA staining to determine if the TTV DNA provided expression of ORF 1 protein.
  • IFA staining was accomplished with rabbit polyclonal sera that was raised against a peptide corresponding to a C- terminal region of the capsid protein (residues 601 -620 in SEQ ID NO:9), except that the N terminal residue thereof (Asn 601 ) was replaced with a cysteine residue.
  • the results indicate that the TTV-transfected DNA successfully expressed the ORF-1 protein in at least 293, BHK-21 , ST and PK cells lines.

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Abstract

La présente invention concerne de nouvelles séquences de nucléotides et d'acides aminés de Torque teno virus (« TTV »), y compris leurs nouveaux génotypes, utiles pour la préparation de vaccins destinés au traitement et à la prévention de maladies chez le porc et d'autres animaux. Les vaccins obtenus selon la pratique de la présente invention sont efficaces contre plusieurs isolats et génotypes du TTV porcin. La présente invention concerne également des anticorps monoclonaux et polyclonaux diagnostiques et thérapeutiques, ainsi que des clones infectieux utiles pour la propagation du virus et la préparation de vaccins. Des aspects particulièrement importants de l'invention comprennent des vaccins qui permettent d'obtenir, en tant qu'antigène, une protéine ORF1 du TTV, ou des fragments peptidiques de celle-ci.
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WO2000066621A1 (fr) * 1999-05-04 2000-11-09 Tripep Ab Peptides de la sequence du virus tt et anticorps monospecifiques se fixant au virus tt
WO2010044889A2 (fr) * 2008-10-16 2010-04-22 Pfizer Inc. Isolats de torque teno virus (ttv) et compositions associées

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