EP1311698A2 - Schweine-adenovirus 5 vektor und impfstoff - Google Patents

Schweine-adenovirus 5 vektor und impfstoff

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Publication number
EP1311698A2
EP1311698A2 EP01929140A EP01929140A EP1311698A2 EP 1311698 A2 EP1311698 A2 EP 1311698A2 EP 01929140 A EP01929140 A EP 01929140A EP 01929140 A EP01929140 A EP 01929140A EP 1311698 A2 EP1311698 A2 EP 1311698A2
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Prior art keywords
padv
porcine
nucleic acid
recombinant
acid sequence
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English (en)
French (fr)
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Eva Nagy
Tamas Tuboly
Miklos Nagy
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University of Guelph
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University of Guelph
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    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to a novel porcine adenovirus, PadV-5, and its use as a delivery vector or vaccine.
  • Adenoviridae Viruses in the family Adenoviridae are widely distributed all over the world. They have been isolated from humans, several other mammalian species, birds, amphibians are also known to be present in fish. Adenoviruses are generally very similar to each other with some variations, mostly between avian and mammalian viruses, in genome size and genomic organization.
  • PAdV-5 According to the limited data provided by restriction endonuclease analysis of the genome of the 1995 PAdV-5 strain, it is likely that this virus is not identical to the PAdV-5 isolated in 1990.
  • the viruses included in this application and referred to as PAdV-5 were the HNF-61 and HNF-70 strains identified in 1990.
  • PAdV Porcine adenoviruses
  • Porcine adenoviruses generally do not cause disease in swine and the proposed use of the PAdVs as viral vector vaccines (Tuboly et al. (1993)), especially where mucosal immune response is required, led to the extensive study of the genomes. So far 5 PAdV serotypes have been described (Haig et al. (1964); Clarke et al. (1967); Kasza (1966); Hirahara et al. (1990)). The restriction endonuclease physical maps of the genomes of PAdV-1-5 have been established (Kleiboeker et al. (1993); Reddy et al. (1993); Tuboly et al.
  • PAdV-4 Kleiboeker (1994); PAdV-3 (Reddy et al. (1995); PAdV-1-2 (Reddy et al. (1997)), El (PAdV-4, (Kleiboeker 1995)); PAdV-3, Reddy et al. (1998b)) and E4 (PAdV-3 (Reddy et al. (1997)) have also been carried out. Most recently, the sequence of the entire genome of PAdV-3 has been published (Reddy et al. (1998a)).
  • helper-dependent viral vectors As vaccines is more practical. So far, two viral genes have been expressed by such PAdV-3 vectors.
  • the gD gene of the Aujeszky's disease virus was inserted into the E3 region (Reddy et al. (1999b)), and the E2 gene of the classical swine fever virus was inserted near the right hand terminus of the viral genome (Hammond et al. (2000)).
  • the virus which is the subject of the present invention belongs to serotype 5 of porcine adenoviruses (PAdV-5), and was originally isolated in Japan (Hirahara et al. (1990)). There is no further report on the presence of PAdV-5 elsewhere around the world.
  • the present inventors were the first to sequence the genome of PAdV-5. The inventors further determined that at least 60% of the E3 region is not essential for virus replication, increasing the theoretical vector capacity of PAdV-5 to 2.9 kb, which is much larger than the figure given for PAdV-3 (Reddy et al. (1999b)).
  • the present invention provides an isolated porcine adenovirus serotype 5 (PAdV-5) having a nucleic acid sequence shown in Figure 7 or SEQ.ID.NO.:l, or a homolog or analog thereof.
  • the nucleic acid sequence of the PAdV-5 comprises:
  • nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b); (d) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or
  • nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions.
  • the present inventors have identified the E3 region of PAdV-5 and successfully inserted the TGEV S gene into the virus and successfully generated TGEV specific antibodies in a recipient pig using the recombinant PAdV-5 virus.
  • the present invention provides a recombinant porcine adenovirus serotype 5, comprising a heterologous nucleic acid sequence that is stably integrated into the recombinant porcine adenovirus genome.
  • the site of integration of the heterologous nucleic acid sequence is in a non-essential region, such as the E3 region, more preferably between map units at about 75 and about 82 as shown in Figure 10 or in the E3 region as shown in Figure 13 (SEQ.ID.NO.:8) or 14 (SEQ.ID.NO.:9).
  • the present invention also includes modified forms of the isolated PAdV- 5 shown in Figure 7 (SEQ.ID.NO.:l) or modified forms of the analogs or homologs as described above.
  • modified forms include an isolated PAdV-5 wherein the E3 region has been deleted.
  • the recombinant porcine adenovirus serotype 5 includes a live porcine adenovirus having virion structural proteins unchanged from those in a native porcine adenovirus from which the recombinant porcine adenovirus is derived.
  • the recombinant PAdV-5 of the invention comprises a heterologous nucleic acid sequence that encodes an antigenic determinant from an infectious agent.
  • the recombinant PAdV-5 further comprises a nucleic sequence encoding an immuno-potentiating molecule where the molecule is preferably interleukin 3 (IL-3), porcine interleukin 4 (IL4), gamma interferon ( ⁇ lFN), porcine granulocyte macrophage colony stimulating factor (GM-CSF), or porcine granulocyte colony stimulating factor (G-CSF).
  • IL-3 interleukin 3
  • IL4 porcine interleukin 4
  • ⁇ lFN gamma interferon
  • GM-CSF porcine granulocyte macrophage colony stimulating factor
  • G-CSF porcine granulocyte colony stimulating factor
  • the present invention provides a method of producing a recombinant porcine adenovirus vector for use as a vaccine comprising inserting into a non-essential region of a porcine adenovirus serotype 5 genome, at least one heterologous nucleic acid sequence preferably in association with an effective promoter sequence.
  • the heterologous sequence encodes an antigenic polypeptide and /or an immuno-potentiating molecule
  • the heterologous nucleotide sequence encoding an antigenic polypeptide encodes determinants of infectious agents and preferably the nucleotide sequence encoding an immuno-potentiating molecule is interleukin 3 (IL-3), porcine interleukin 4 (IL4), gamma interferon ( ⁇ lFN), porcine granulocyte macrophage colony stimulating factor (GM-CSF), or porcine granulocyte colony stimulating factor (G-CSF).
  • IL-3 interleukin 3
  • IL4 porcine interleukin 4
  • ⁇ lFN gamma interferon
  • GM-CSF porcine granulocyte macrophage colony stimulating factor
  • G-CSF porcine granulocyte colony stimulating factor
  • the present invention provides the use of the recombinant PAdV-5 of the invention in the preparation of a vaccine for generating and/or optimising antibodies or cell mediated immunity so as to provide or enhance protection against infection by an infectious organism in animals, where the vaccine includes recombinant porcine adenovirus serotype 5 stably incorporating, at least one heterologous nucleotide sequence, and suitable carriers and/or excipients.
  • the at least one heterologous nucleotide encodes an antigenic polypeptide, more preferably antigenic determinants of infectious agents.
  • the present invention includes a vaccine for eliciting or enhancing an immune response to an antigen comprising an effective amount of a recombinant porcine adenovirus serotype 5 comprising a nucleic acid sequence encoding the antigen, preferably in admixture with a suitable diluent or carrier.
  • the present invention provides a method of eliciting or enhancing an immune response to an antigen comprising administering an effective amount of a recombinant porcine adenovirus serotype 5 comprising a nucleic acid sequence encoding the antigen to an animal in need thereof.
  • Figure 1A illustrates a PAdV-5 map of HindJH and Mlul restriction map of the genome, m.u.: map unit, 1 m.u.: ⁇ 335 bp;
  • Figure IB is an enlargement of sequenced region of the map of Figure 1A;
  • Figure 1C illustrates reading frames of the r strand of PAdV-5;
  • Figure ID illustrates the portion of the DNA removed to generate the clones;
  • Figure 2 illustrates the alignment of the predicted ORF2 amino acid sequences of PAdV-5 HNF-70 (SEQ.ID.NO.:2) and some closely related animal adenoviruses (SEQ.ID.NOs.:3-5);
  • Figure 3 illustrates the sequence alignment of the predicted ORF3 proteins of HNF-61 (SEQ.ID.NO.:6) and HNF-70 (SEQ.ID.NO.:7);
  • Figure 4 illustrates the unrooted phylogenetic tree of pVi ⁇ protein homologues of selected animal adenovirus generated by the Clustal method
  • Figure 5 illustrates the time course analysis of PAdV-5 HNF-70 nucleic acid synthesis
  • Figure 6 illustrates the restriction endonuclease analysis of the wild type PAdV-5 HNF-70 strain (A) and its deletion mutant R- ⁇ HH (B) genomic DNA in ethidium bromide stained 0.8% agarose gel.
  • Figure 7 is the complete nucleotide sequence of porcine adenovirus serotype 5 (PAV-5) strain HNF-70.
  • Figure 8 illustrates the genome organization and putative transcription map of PadV-5. Arrows above and below the central line represent the locations of putative ORF's. The location of the major late promoter (MLP) is indicated. Late regions (L1-L6) are indicated by lines.
  • MLP major late promoter
  • L1-L6 Late regions
  • Figure 9 illustrates a phylogenetic analysis of the pVIII genes of selected adenoviruses.
  • the lengths of the branches indicate the phylogenetic distance between the viruses.
  • the scale bar represents 10 mutations per 100 sequence positions.
  • Virus names not defined elsewhere are: CELOV, CELO virus (fowl adenovirus 1); EDSV, egg drop syndrome virus; FAdV, fowl adenovirus; HEV, turkey haemorrhagic enteritis virus; MAdV, muriine adenovirus; OAdV, ovine adenovirus.
  • Figure 10 illustrates the strategy for construction of the recombinant transfer vectors.
  • Figure 11 is a Northern blot analysis of S gene expression showing total RNA extracted from cells infected with recombinant virus RPAdV-2.2S and ⁇ RPAdV-2.2Sc.
  • Figure 12 is a Western blot analysis the RPAdV-2.2S and ⁇ RPAdV-2.2Sc recombinant virus infected cells were collected at 24 hours p..i.
  • Figure 13 illustrates the nucleotide sequence (SEQ.ID.NO.:8) of the E3 region of the same strain shown in Figure 7 (PAV-5, HNF-70).
  • Figure 14 illustrates the nucleotide sequence (SEQ.ID.NO.:9) of the E3 region of the HNF61 strain of porcine adenovirus serotype 5 (PAV-5). DETAILED DESCRIPTION OF THE INVENTION I. PAdV-5
  • the present inventors have determined the complete nucleotide sequence of PAdV-5 and constructed a putative genomic map. Accordingly, the present invention provides an isolated porcine adenovirus serotype 5 (PAdV-5) having a nucleic acid sequence shown in Figure 7 or SEQ.ID.NO.:l, or a homolog or analog thereof.
  • PAdV-5 porcine adenovirus serotype 5
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques or chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid sequence refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.
  • the nucleic acid sequences of the present invention may be ribonucleic (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the sequences may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8- amino adenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines
  • the PAdV-5 nucleic acid sequence comprises: (a) a nucleic acid sequence as shown in Figure 7 (SEQ.ID.NO.:l), wherein T can also be U;
  • nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or
  • sequence that has substantial sequence homology means those nucleic acid sequences which have slight or inconsequential sequence variations from the sequences in (a) or (b), i.e., the sequences function in substantially the same manner (e.g. useful as a vector or vaccine). The variations may be attributable to local mutations or structural modifications.
  • Nucleic acid sequences having substantial homology include nucleic acid sequences having at least 65%, more preferably at least 85%, and most preferably 90-95% identity with the nucleic acid sequences as shown in Figure 7 (SEQ.ID.NO.:!).
  • sequence that hybridizes means a nucleic acid sequence that can hybridize to a sequence of (a), (b), (c) or (d) under stringent hybridization conditions. Appropriate "stringent hybridization conditions" which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1- 6.3.6.
  • the following may be employed: 6.0 x sodium chloride /sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C.
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be at high stringency conditions, at about 65°C.
  • a nucleic acid sequence which is an analog means a nucleic acid sequence which has been modified as compared to the sequence of (a), (b) or (c) wherein the modification does not alter the utility of the sequence (i.e. as a vector or vaccine) as described herein.
  • the modified sequence or analog may have improved properties over the sequence shown in (a), (b) or (c).
  • One example of a modification to prepare an analog is to replace one of the naturally occurring bases (i.e.
  • adenine, guanine, cytosine or thymidine of the sequence shown in Figure 7 with a modified base such as such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8- thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8- halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8- hydroxyl guanine and other 8-substituted guanines, other aza and deaza
  • a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecule shown in Figure 7.
  • the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
  • a further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497).
  • PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand.
  • nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • the analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.
  • the present invention also includes modified forms of the isolated PAdV- 5 shown in Figure 7 (SEQ.ID.NO.:l) or modified forms of the analogs or homologs as described above.
  • modified forms include an isolated PAdV-5 wherein the E3 region has been deleted.
  • Such a modified form can be used to insert a heterologous gene for the preparation of a vaccine as described below.
  • the present invention provides a modified PAdV-5 wherein the E3 region, or a portion thereof, has been deleted.
  • the E3 region that is deleted is as shown in Figure 13 (SEQ.ID.NO.:8) or Figure 14 (SEQ.ID.NO.:9).
  • the isolated PAdV-5 of the invention is useful in preparing a recombinant PAdV-5 vector for the insertion of heterologous nucleic acid sequences of interest and the expression of the heterologous sequence in a host.
  • the inventors have identified the E3 region of PAdV-5 and successfully inserted the TGEV S gene into the virus and successfully generated TGEV specific antibodies in a recipient pig using the recombinant PAdV-5 virus.
  • the present invention provides a recombinant porcine adenovirus serotype 5, comprising a heterologous nucleic acid sequence that is stably integrated into the recombinant porcine adenovirus genome.
  • the site of integration of the heterologous nucleic acid sequence is in a non-essential region of the viral genome, most preferably in the E3 region.
  • it is preferably between map units at about 75 and about 82 as shown in Figure 10 or in the E3 region as shown in Figure 13 (SEQ.ID.NO.:8) or Figure 14 (SEQ.ID.NO.:9).
  • the terms "heterologous nucleic acid sequence” includes one or more sequences that are not normally present in the PAdV-5 sequence in nature.
  • the heterologous nucleic acid sequences encode the antigenic determinants of infectious organisms against which the generation of antibodies or cell-mediated immunity is desirable, such as antigenic determinants of intestinal infections caused by gastrointestinal viruses; for example rotavirus and parvovirus infections, or respiratory viruses, for example influenza virus and porcine reproductive and respiratory syndrome virus (PRRSV) or that of hog cholera virus (classical swine fever).
  • infectious organisms against which the generation of antibodies or cell-mediated immunity is desirable, such as antigenic determinants of intestinal infections caused by gastrointestinal viruses; for example rotavirus and parvovirus infections, or respiratory viruses, for example influenza virus and porcine reproductive and respiratory syndrome virus (PRRSV) or that of hog cholera virus (classical swine fever).
  • PRRSV porcine reproductive and respiratory syndrome virus
  • hog cholera virus classical swine fever
  • Heterologous nucleotide sequences which may be incorporated include, but are not limited to, the antigenic determinants of the agents of: porcine parvovirus; mycoplasma hyopneumonia; porcine influenza virus; transmissible gastroenteritis virus (porcine coronavirus); porcine rotavirus; hog cholera virus (classical swine fever); swine dysentery; African swine fever virus; pseudorabies virus (Aujeszky's disease virus), in particular the glycoprotein D of the pseudorabies virus; porcine respiratory and reproductive syndrome virus (PRRSV); and porcine circovirus (Postweaning multisystemic wasting syndrome).
  • porcine parvovirus mycoplasma hyopneumonia
  • porcine influenza virus transmissible gastroenteritis virus (porcine coronavirus); porcine rotavirus
  • hog cholera virus classical swine fever
  • swine dysentery African swine fever virus
  • Heterologous nucleotide sequences more preferred for incorporation in the vectors of the invention are those expressing antigenic determinants of porcine parvovirus, porcine rotavirus, TGEV (porcine coronavirus) and classical swine fever virus. Most preferred, are heterologous nucleotide sequences expressing the antigenic determinants of TGEV.
  • heterologous nucleic acid sequences incorporated may encode immuno-potentiator molecules such as cytokines or growth promoters, for example porcine interleukin 4 (IL4), gamma interferon ( ⁇ lFN), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), FLT-3 ligand and interleukin 3 (IL-3).
  • cytokines for example porcine interleukin 4 (IL4), gamma interferon ( ⁇ lFN), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), FLT-3 ligand and interleukin 3 (IL-3).
  • IL4 porcine interleukin 4
  • ⁇ lFN gamma interferon
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • FLT-3 interleukin 3
  • heterologous nucleic acid sequence can comprise both heterologous genes coding for antigenic determinants and immuno-potentiator molecules.
  • Non-essential regions of the viral genome which may be suitable for the purposes of replacement with or insertion of heterologous nucleic acid sequences may for example be non-coding regions at the right terminal end of the genome at map units between about 75 to about 82, preferably 75.7 and 81.7, of the genome-spanning parts of the Hindlll F and D fragments.
  • the heterologous gene sequences may be associated with a promoter and leader sequence in order that the nucleotide sequence may be expressed in situ as efficiently as possible.
  • the heterologous gene sequence is associated with the porcine adenoviral major late promoter and splice leader sequence.
  • the mammalian adenovirus major late promoter lies near 16-17 map units on the adenovirus genetic map and contains a classical TATA sequence motif (Johnson, D.C., Ghosh-Chondhury, G., Smiley, J.R., Fallis, L. and Graham, F.L. (1988), Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector. Virology 164, 1-14).
  • porcine adenoviral major late promoter any other suitable eukaryotic promoter may be used.
  • those of SV40 virus, cytomegalovirus (CMV) or human adenovirus may be used.
  • the splice leader sequence of the porcine adenovirus serotype under consideration is a tripartite sequence spliced to the 5' end of the mRNA of all late genes.
  • the heterologous gene sequence may also be associated with a poly-adenylation sequence.
  • the invention includes all of the uses of the isolated PAdV-5, the modified PAdV-5 and the recombinant PAdV-5 vectors of the inventions including the use thereof as a vaccine. Accordingly, in a further aspect of the invention there is provided a recombinant PAdV-5 vaccine for generating and/or optimising antibodies or cell-mediated immunity so as to provide or enhance protection against infection with an infectious organism in animals, the vaccine comprising a recombinant porcine adenovirus serotype 5 vector stably incorporating at least one heterologous nucleic acid sequence formulated with suitable carriers and excipients.
  • the heterologous nucleic acid sequence encodes an antigenic polypeptide and/or an immuno-potentiator molecule.
  • the recombinant vaccine may include a live recombinant porcine adenovirus vector in which the virion structural proteins are unchanged from that in the native porcine adenovirus from which the recombinant porcine adenovirus is produced.
  • the vaccine may be directed against any infectious organism and/or agent, for example, infectious organisms and /or agents causing respiratory and/or intestinal infections.
  • heterologous gene sequences encoding the antigenic determinants of those infectious organisms may be incorporated into non- essential regions of the genome of the porcine adenovirus serotype 5 comprising the vector.
  • suitable heterologous nucleotide sequences may also be those of immuno-potentiators such as cytokines or growth promoters.
  • a method of preparing a vaccine for generation and /or optimization of antibodies or cell- mediated immunity so as to induce or enhance protection against an infectious organism in an animal which includes constructing a recombinant porcine adenovirus serotype 5 vector stably incorporating at least one heterologous nucleotide sequence, and placing said recombinant porcine adenovirus vector in a form suitable for administration.
  • the nucleotide sequence encodes an antigenic polypeptide, although it may also be an immuno-potentiator molecule. More preferably, the nucleotide sequences may encode for and/or express, an antigenic polypeptide and an immuno-potentiator molecule.
  • the present invention includes a vaccine for eliciting or enhancing an immune response to an antigen comprising an effective amount of a recombinant porcine adenovirus serotype 5 comprising a nucleic acid sequence encoding the antigen, preferably in admixture with a suitable diluent or carrier.
  • the present invention also provides a method of eliciting or enhancing an immune response to an antigen comprising administering an effective amount of a recombinant porcine adenovirus serotype 5 comprising a nucleic acid sequence encoding the antigen to an animal in need thereof.
  • enhancing or eliciting an immune response is defined as enhancing, improving or augmenting any response of the immune system, for example, of either a humoral or cell-mediated nature.
  • the enhancement of an immune response can be assessed using assays known to those skilled in the art including, but not limited to, antibody assays (for example ELISA assays), antigen specific cytotoxicity assays and the production of cytokines (for example ELISPOT assays).
  • Administration of an "effective amount" of the vaccine of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result (e.g., to elicit or enhance an immune response).
  • the effective amount of a compound of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage procedures may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For example an effective amount is an amount sufficient to elicit an immune response, preferably at least 10 4 TCID 50 per dose.
  • the term "animal” includes all members of the animal kingdom and is preferably a pig.
  • the vaccine may be a multivalent vaccine and additionally contain heterologous nucleic acid sequences encoding immunogens related to other intracellular viral, parasitic and bacterial infectious diseases in a prophylactically or therapeutically effective manner.
  • any one recombinant adenovirus can contain the expressible nucleic acid sequences of more than one microbial antigen and/or more than one immuno-potentiator molecule.
  • the vaccines of the present invention may additionally contain suitable diluents and/or carriers.
  • the vaccines may contain one or more other adjuvants, which can further enhance the immunogenicity of the vaccine in vivo.
  • these other one or more adjuvants may be selected from many known adjuvants in the art including the lipid-A portion of the LPS from gram negative bacteria (endotoxin), trehalose dimycolate of mycobacteria, the phospholipid lysolecithin, dimethyldictadecyl ammonium bromide (DDA), certain linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers, aluminum hydroxide, and liposomes.
  • the vaccine may also contain preservatives such as sodium azide, thimersol, beta propiolactone, and binary ethyleneimine.
  • a vaccine of the invention is suitable for administration to subjects in a biologically compatible form in vivo.
  • biologically compatible form suitable for administration in vivo means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • the vaccines may be administered in a convenient manner such as by injection (intradermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranodal etc.), oral administration, inhalation, transdermal administration (such as topical cream or ointment, etc.), or suppository applications.
  • the invention provides (i) a vaccine vector such as a recombinant porcine adenovirus serotype 5, containing DNA molecules of the invention, placed under the control of elements required for expression (if necessary); (ii) a composition of matter containing a vaccine vector of the invention, together with a diluent or carrier; particularly, (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector; (iv) a method for inducing an immune response against antigenic polypeptides in an animal, which involves administering to the animal an immunogenically effective amount of a vaccine vector to elicit an immune response, e.g., a protective or therapeutic immune response to the antigenic polypeptide; particularly; (v) a method for preventing and /or treating disease, which involves administering a prophylactic or therapeutic amount of a vaccine vector containing DNA of the invention to an animal in need; and (vi) a method for preventing and /or treating disease, which involves administer administering
  • the recombinant adenovirus or antigens may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • a suitable carrier diluent, or excipient
  • the vaccines can also be lyophilized.
  • the vaccines can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • the vaccines can contain at least one adjuvant compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.
  • Adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Patent No. 2,909,462 (incorporated herein by reference) which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms.
  • the preferred radicals are those containing from 2 to 4 carbon atoms, e.g.
  • the unsaturated radicals may themselves contain other substituents, such as methyl.
  • the products sold under the name Carbopol( (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol( 974P, 934P and 971P).
  • the copolymers of maleic anhydride and alkenyl derivative the copolymers EMA( (Monsanto) which are copolymers of maleic anhydride and ethylene, linear or cross-linked, for example cross-linked with divinyl ether, are preferred.
  • Adjuvants useful in any of the vaccine compositions described herein are as follows.
  • Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.
  • the antigen can be precipitated with, or adsorbed onto, the aluminum compound according to standard protocols.
  • Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT), can be used in parenteral administration.
  • Adjuvants for mucosal administration include bacterial toxins (e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof).
  • CT cholera toxin
  • LT E. coli heat-labile toxin
  • PT pertussis toxin
  • a purified preparation of native cholera toxin subunit B (CTB) can be of use. Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
  • a mutant having reduced toxicity is used.
  • Suitable mutants have been described (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant)).
  • Additional LT mutants that can be used in the methods and compositions of the invention include, for example Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as a bacterial monophosphoryl lipid A (MPLA) of various sources (e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri). saponins, or polylactide glycolide (PLGA) microspheres), can also be used in mucosal administration.
  • MPLA bacterial monophosphoryl lipid A
  • sources e.g., E. coli,
  • Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (for example, WO 95/2415), DC-chol (3 b-(N-(N',N'- dimethyl aminomethane)-carbamoyl) cholesterol (for example, U.S. Patent No. 5,283,185 and WO 96/14831)) and QS-21 (for example, WO 88/9336).
  • the Thl cell-mediated immune response is considered to play a pivotal role in pig defense against mycobacterial infection and the development of such immune responses is believed to involve (1) antigen presentation by antigen-presenting cells (APC) including macrophages and dendritic cells to antigen-specific T cells; (2) T cell activation and cytokine release; and (3) enhanced bactericidal activities of macrophages by cytokines (immuno- potentiator molecules) released from T cells (Munk et al. (1995)). Cytokines including interleukin- 12 (IL-12), interferon (IFN) and granulocyte- macrophage colony stimulating factor (GM-CSF) orchestrate in the development of anti- mycobacterial Thl immune responses.
  • APC antigen-presenting cells
  • IFN interferon
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • IL-12 is usually released by APC upon interaction with infectious pathogens and is a crucial Thl differentiation and activation factor.
  • APC antigen presentation and IL-12 release by APC will result in the release of Thl cytokine IFN from Thl lymphocytes.
  • IFN is a potent macrophage-activation cytokine capable of enhancing bactericidal activities of macrophages.
  • GM-CSF was originally identified as a hematopoietic growth factor but recent evidence indicates that it is a critical cytokine required for effective antigen presentation by enhancing dendritic cell differentiation and APC activation via increasing cell surface expression of MHC II and B7 molecules (Peters et al. (1996)).
  • Prime/boost protocol whereby immunization with a adenovirus recombinant expressing a foreign gene product is followed by a boost using a purified subunit preparation form of that gene product, elicits an enhanced immune response relative to the response elicited with either product alone. Accordingly, it is within the scope of the present invention to use a prime/boost protocol.
  • a methodology of prime/boost protocol is described in WO 98/58956, which is incorporated herein by reference.
  • repeated vaccination with the same recombinant adenovirus could be used as the boost.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active material is combined in a mixture with a pharmaceutically acceptable vehicle.
  • suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's).
  • compositions include, albeit not exclusively, solutions of the active material in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • pharmaceutically acceptable vehicles or diluents include, albeit not exclusively, solutions of the active material in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • U.S. Patent No. 5,843,456 incorporated herein by reference, and directed to rabies compositions and combination compositions and uses thereof.
  • compositions of the invention may be confirmed in experimental model systems.
  • Animals are conveniently inoculated with vector vaccines according to the invention at any age.
  • piglets may be vaccinated at 1 day old, breeders may be vaccinated regularly up to point of giving birth and thereafter.
  • animals are vaccinated while still not fully immunocompetent. More conveniently, animals can be vaccinated for protection against re- infection after a period of 4 weeks subsequent to initial vaccination.
  • administration is by oral delivery or intra-nasally.
  • the resulting clones were sequenced using the T7 and Sp6 specific primers at the Biological Research Institute, Szeged (Hungary), with the PRISM ready reaction dye deoxy cycle protocol (Perkin Elmer) in an automated DNA sequencer (Applied Biosystems Inc.).
  • GenBank database was performed by the Blast program for the nucleotide and deduced amino acid (aa) sequences of the predicted open reading frames (ORF). Sequence analysis was done with the Seqaid II version 3.5 and the Clone Manager version 3.12 computer programs. Sequence alignments were carried out with the Align program version 1.02 and with the DNAstar MegAlign program. The distance matrix phylogenetic analysis was carried out with the MegAlign program. DNA and RNA time course
  • RNA and DNA samples were also collected from cells treated with 50 ⁇ g/ml cytosine arabinoside (AraC, Sigma) as described (Mittal et al., 1993).
  • the start of DNA replication was detected by a dot blot method.
  • the frozen samples were thawed and mixed with an equal volume of 0.8 N NaOH, incubated at 80°C for 20 minutes. Fifty ⁇ l of the cell lysates of each time point were loaded into the wells of a dot blot filtration manifold and blotted onto Nytran membranes (Schleicher and Schuell) as described (Sambrook et al., 1989).
  • the DNA was immobilized in a UV crosslinker (Fischer) and probed with digoxigenin labeled (Boehringer Mannheim) PAdV-5 HNF-70 genomic DNA according to the manufacturers' instructions.
  • the DNA was detected in a chemiluminescent reaction (Boehringer Mannheim, Non Radioactive DNA Labeling and Detection Kit) and exposed to Kodak X-Omat AR films.
  • RNA transcription was analyzed by Northern blotting. Equal amounts of the total RNA extracted from mock and virus infected cells of each time point were separated in 1.1% formaldehyde agarose gels, transferred bidirectionally to Nytran membranes and immobilized by UV crosslinking. Prehybridization, hybridization in the presence of 50% formamide and washing of the blots were carried out as described (Sambrook et al, 1989).
  • the E3 specific probe (fig. IB) was the 1.9 kb EcoRI G fragment of the HNF-70 strain labeled with [ ⁇ 32P]dCTP (ICN) by the random primer method (Random Primer Labeling Kit, Life Technologies). Construction of E3 deleted PAdV-5
  • Plasmids containing the left (D) and right (C) terminal Mlul fragments of HNF-70 were used to construct full length genomic clones of the viral DNA by homologuos recombination in E. coli. Briefly, the terminal fragments were released from the plasmid by double digestions with BamHl-Mlul (Mlul D fragment) and H dIII-MM (Mlul C fragment). These fragments were cloned together into Bam ⁇ I-Hin ⁇ T ⁇ . digested pWE15 cosmid vector (Stratagene, modified by Ojkic et al, unpublished). The resulting clone was used in co- transformation of competent E.
  • Two deletion clones were generated, namely Mlul B- ⁇ (992-2497) and MZttl B- ⁇ (1260-2497) by removing a 1505 bp Pvull-Hpal or a 1237 bp Hindi fragment from the original Mlul B fragment, respectively (fig. ID).
  • the E3 region of the cloned full length HNF-70 DNA was replaced by the deletion clones via homologous recombination in BJ 5183 E. coli resulting in full length genomic clones with deletions in the E3 region (pR- ⁇ PH and pR- ⁇ HH).
  • the cloned DNA was transfected into ST cells using lipofectin reagent (Life Technologies) according to the instructions of the manufacturer.
  • the transfected cells were overlaid with 0.7% agarose in DMEM supplemented with 10% fetal bovine serum. Plaque formation was monitored daily and 6-7 days after transfection the plaques were transferred to fresh ST cell monolayers. Viral DNA was extracted from the infected cells by the Hirt method and analyzed with the appropriate restriction enzymes (RE).
  • RE restriction enzymes
  • the putative E3 region of both strains of PAdV-5 was located between 75.7 and 81.7 map units (m.u.) of the genome (the full genome of PAV 5 is shown in Figure 7), spanning parts of the H dHI F and D fragments.
  • the sequencing of these fragments revealed several ORFs on both the right (r) and the left (1) strand.
  • the 1 strand showed numerous ORFs but only five of
  • ORF1 and ORF2 shared an 8-nucleotide overlap (ATGACTGA, SEQ.ID.NO.:10), the stop codon of ORF1 embedded in the ORF2 coding gene.
  • ORF1 of 222 aa between 73.7 and 75.7 m.u. (nt 1-666, Figure IB) of the genome showed high homology with several of the known human and animal adenovirus pVUI protein sequences.
  • the predicted ORF1 amino acid sequences of HNF-61 and HNF-70 were identical, and only one nucleotide difference was detected at nt 546.
  • ORF2 The deduced amino acid sequence of the ORF between 75.7 and 76.7 m.u. (ORF2, 129 aa, nt 662-1048) was similar to the ORF5 of bovine adenovirus type 1 (BAdV-1, 78.3%), the 13.7 kDa protein of PAdV-3 (63.7% similarity) and also to the 13.3 kDa protein of canine adenovirus type 1 and 2 (61.2 and 58.9% similarity, respectively). ORF2 also exhibited a strong similarity ranging from 55.3% (human adenovirus, HAdV, type 7) to 61.6% (HAdV-40) to the 12.5 kDa E3 ORF of human adenoviruses.
  • ORF3 A large open reading frame (ORF3) was identified between 76.1 and 81.6 m.u. It was present in both strains of PAdV-5 (HNF-61: nt 798-2654, HNF-70: nt 798-2633) with a coding capacity of 618 aa and 612 aa, respectively.
  • Figure 3 shows the alignment of the putative ORF3 aa sequences of the strains HNF- 61 and HNF-70. These ORFs had 66.67% identity on the aa level and 77.65% homology at the nucleotide level. The first 300 aa were almost completely identical (97.3%) but from this point to approximately aa 570 no significant similarity could be detected.
  • ORF3 showed a weak similarity (39.3%) only to the ORFIO of the E3 region of BAdV-1.
  • a homology search of the Genbank database for ORF3 did not reveal similarities with any other known adenovirus sequences.
  • ORF4 A short ORF of 50 aa (ORF4) was present in the r strand of both strains, starting at nt 1159. There was a 98.6% homology between the nucleotide sequences of the ORF4 of the two strains of PAdV-5 and there was only a 2 aa difference between them.
  • the length of the putative E3 region of PAdV-5 starting at the end of ORF1 (pVHI homologue) and ending before the ATG signal of the fiber coding ORF5 was 2039 bp for HNF-61 and 2020 bp for HNF-70.
  • the full nucletide sequence of the E3 region of each of the HNF-70 and HNF-61 strains are shown in figures 11 and 12 respectively.
  • TATAAAA SEQ.ID.NO.:ll
  • TATAAAA SEQ.ID.NO.:ll
  • GC rich sequences were identified before the TATA box, GGCGG (SEQ.ID.NO.:13) at nts 57, 174, 324, 328 and GCCGG (SEQ.ID.NO.:14) at nt 106 on both strains.
  • Canonical (AATAAA, SEQ.ID.NO.:15) poly-adenylation (poly-A) sequences were located on the genome.
  • Both strains had one poly-A signal at 10 (HNF-61) or 12 (HNF-70) bp downstream of the ORF3 stop codon at nts 2664 and 2648, respectively.
  • the HNF-61 E3 region contained another canonical poly-A signal at nt 1881, which was not present in HNF-70.
  • TTGTTT SEQ.ID.NO.:16 signals were identified at nt 1588 in both strains and at nt 2698 in HNF-61.
  • EXAMPLE 2 Phylogenetic analysis The pVIII sequences of adenoviruses are generally considered to be conserved to a certain extent at the amino acid level, and can be of interest in generating phylogenetic trees. The pVIII homologue of PAdV-5 (ORF1) was used in such phylogenetic comparison with all the known animal and human pVm sequences. Figure 4 shows the result of the analysis with selected representatives of animal adenoviruses. According to the alignment the closest relative of PAdV-5 is BAdV-1 with 79% identity in this region.
  • the sizes of these transcripts were 3.2, 2.2, 1.6, 0.8 and 0.45 kb, respectively.
  • the 0.8 kb transcript was the earliest mRNA detected.
  • the 1.6 kb mRNA was the most abundant and was transcribed for the longest period of time. Transcription was not sensitive to AraC treatment.
  • EXAMPLE 4 Deletion of the E3 region
  • the deletion in pR- ⁇ HH affected a shorter segment of the E3 region leaving ORF2 intact and removing the 3' 48 nts of ORF4 and most of ORF3 between nts 1260 and 2497.
  • pR- ⁇ PH did not generate CPE in the cell culture even after repeated attempts of transfections and several blind passages of the transfected cells.
  • the pR- ⁇ HH generated virus (R- ⁇ HH) was plaque purified three times and the extracted viral DNA was analyzed with RE digestion.
  • Figure 6 shows the Hpal, EcoRI and H dlfl RE patterns of wild type HNF-70 and R- ⁇ HH DNA.
  • the original Hpal site was fused to the HincT site in R- ⁇ HH (Fig. ID) and could be cleaved by Hpal so that the 5.9 kb Hpal C fragment of HNF-70 migrated at 4.7 kb in R- ⁇ HH.
  • the size of the original 1.9 kb EcoRI fragment (the same as the one used as E3 probe, Figure IB) was 0.7 kb in R- ⁇ HH as expected.
  • the size of the 4.2 kb H di ⁇ D fragment was decreased to 3.0 kb by the 1.2 kb fragment deleted from R- ⁇ HH.
  • the E3 region of adenoviruses is considered to be the most convenient site for foreign gene insertion.
  • Studies with HAdVs clearly demonstrated that this region of the genome is not essential in virus replication, although it may play an important role in viral pathogenesis by helping the virus to evade recognition by the immune system.
  • Analysis of some animal adenovirus genomes also showed that at least part of the E3 region could be deleted without adverse effects on virus replication in vitro (Dragulev et al., 1991; Mittal et al., 1995; Evans et al., 1998; Reddy et al., 1999).
  • sequenced region presented here comprised the entire pVTH gene and the 5' portion of the fiber gene.
  • the region of PAdV-5 between the pVHI and fiber sequences is the E3 region.
  • HAdVs in general have a longer E3 region than animal adenoviruses.
  • HAdV-5 E3 The size of HAdV-5 E3 is for example 3 kb (Cladaras and Wold, 1985), whereas it is only 0.8 kb in mouse adenovirus type 1 (MAdV-1, Raviprakash et al., 1989), 1.1 kb in CAdV-1 (Dargulev et al, 1991) and 1.8 kb in BAdV-1 (Evans et al., 1998).
  • the size of PAdV-5 E3 was determined and shown to be 2 kb, representing 6 % of the 33.5 kb genome (Tuboly et al., 1995). It was also larger than the corresponding E3 of other PAdVs, of approximately 1.2 kb in PAdV 1-3 (Reddy et al, 1995 and 1996) and 1.8 kb in PAdV-4 (Kleiboeker, 1994).
  • HAdVs have a complex E3 region and code for several overlapping ORFs, the number of which vary among serotypes, whereas the E3 of animal adenoviruses is not only shorter but also more simple.
  • the HAdV-5 E3 region contains 10 ORFs (Cladaras et al., 1985) and the short E3 of MAdV-1 has only 1 ORF (Raviprakash et al, 1989). All PAdV serotypes analyzed so far have 3 ORFs (Reddy et al, 1995 and 1996; Kleiboeker, 1994). There were also three ORFs (ORF2-4) identified within the PAdV-5 E3 region.
  • ORF2 ORF2 that was homologous to the 12.5 kDa HAdV E3 protein and its counterparts in PAdVs and several other mammalian adenoviruses.
  • ORF3 of PAdV-5 was present in both strains. ORFs of such size have not been reported for other animal adenovirus E3 regions and only BAdV-1 ORFIO exhibited some homology with PAdV-5 ORF3 in the amino terminal region.
  • the comparison of ORF3 of HNF-61 and HNF-70 showed almost perfect identity of the predicted amino acid sequences at the amino terminal half and striking variability towards the C-terminal, although the very ends of these proteins also appeared to be highly conserved between the two strains.
  • Porcine adenoviruses show fewer variations in the E3 region.
  • the E3 sequences of PAdV-1-3 serotypes are very similar to each other (Reddy et al., 1996) and the analysis of several PAdV-4 isolates indicated that these viruses were genetically stable (Kleiboeker et al., 1993).
  • both viruses proved to be genetically stable when subjected to several tissue culture passages (results not shown).
  • PAdV-5 is more closely related to BAdV-1 than to any of the known PAdVs.
  • ORF2 similarity searches of the Genbank also showed a closer relationship between PAdV-5 and BAdV-1 than PAdV-5 and other PAdV-s.
  • the pVHI and ORF2 aa comparisons indicated that PAdV-5 was almost as similar to CAdVs as to PAdV-1-3.
  • PAdV- 5 may not be a direct descendant of one of the well established porcine adenovirus serotypes. It is more likely that either a strain of BAdV-1 or a hypothetical common ancestor of PAdV-5, BAdV-1, PAdV-1-3 and CAdV-1-2 has recently been introduced to the swine species.
  • PAdV-5 E3 The early expression of the PAdV-5 E3 genes, as determined by Northern blot analysis, further supported the identity of this region as an E3 region. Although detailed transcription analysis was not done, the number of AraC resistant transcripts showed that PAdV-5 has a more complex transcriptional pattern than other porcine adenoviruses. T e sequence data confirmed this assumption.
  • the promoter region of PAdV-5 E3 was localized between nts 95 and 364, with no typical CCAAT sequences but with several GC rich regions preceding the TATA box.
  • the poly-A signal for the E3 region was identified downstream of the ORF3 stop codon but the presence of the alternative TTGTTT signal in the middle part of the E3 and an additional canonical poly-A signal at nt 1881 in HNF-61 indicated the potential for a more complex transcriptional map.
  • the limited vector capacity of adenoviruses is an important issue in vector development.
  • Studies with helper independent HAdVs show that approximately 105% of the original genome size can be accommodated inside the virion (Bett et al, 1993). Viruses with larger packaged DNA are more subject to genetic rearrangements and eventually loss of the inserted foreign gene.
  • the size of the PAdV-5 genome is approximately 33.5 kb (Tuboly et al., 1995) which in theory means that a maximum of 1.7 kb foreign gene can be inserted into a helper independent PAdV-5 vector without deleting any part of the genome. It is possible to increase the size of the foreign DNA with the size of a sequence that could be deleted from the PAdV-5 genome without compromising its ability to replicate.
  • Genome organization The genome of PAdV-5 was 32621 bp in length (Figure 7), the G + C content was 50.5% and the genome structure and arrangement (Figure 8) were similar to those of published mastadenoviruses. Early regions. Early genes are required for the expression of other viral genes, replication of viral DNA, transformation of cultured cells, and influencing the immune response of the infected host (Gooding & Wold, 1990). Early regions El to E4 were identified in PAdV-5. The E1A proteins were located between nt 418 and 1084. E1A 173R
  • the retinoblastoma susceptibility protein (pRb) binding motif LXCXE (Defeo- Jones et al, 1991) with a slight difference ( 97 LDYPE), and the zinc finger motif ( 107 CX 2 CX 13 CX 2 C; Culp et al, 1988) were present in the E1A 173R protein as in PAdV-3 (Reddy et al, 1998a), or human adenovirus (HAdV) E1A proteins (Culp et al, 1988).
  • the EIB region (between nt 1180 and nt 2787) encoded two proteins in two non-overlapping ORFs, the 163R (19 kDa, small T antigen) and the 340R (38 kDa, large T antigen).
  • the EIB 163R protein showed the highest homology to the BAdV-2 EIB ORF2 159R protein (47% identity).
  • the EIB 340R protein had 37% amino acid identity with the PAdV-3 EIB 474R protein.
  • the mRNAs for DNA-binding protein are transcribed from the E2A region (located on the 1 strand between nt 21166 and 19841).
  • the putative DBP of PAdV-5 was 441 amino acids (49.8 kDa), and the N-terminal domain contained two nuclear localization signals ( 0 PKPKK (SEQ.ID.NO.:17) and 47 RRRK (SEQ.ID.NO.:18)) which were similar to those predicted for BAdV-3 ( 29 PRKK (SEQ.ID.NO.:19) and 35 RKRR (SEQ.ID.NO.:20)) and PAdV-3 ( 47 RRKR (SEQ.ID.NO.:21), 77 RRK (SEQ.ID.NO.:22)) (Reddy et al, 1998a, b).
  • PAdV-5 the 86 YSRLKYT (SEQ.ID.NO.:24) motif was identified at the same location.
  • the nuclear localization signal (NLS) RLPI(R) 4 PRI of the pTP of PAdV-5 was similar to that of PAdV-3 (RLPL(R) 4 PRP) (Reddy et al, 1998a).
  • the serine residue, involved in the initiation of DNA replication, and the flanking residues (NSGD) were also well conserved (Smart & Stilknan, 1982) at 512 NSGD in the PAdV-5 pTP.
  • ORF 7710 . 4309 with a predicted splice acceptor site at nt 7690 comprised the main body of the predicted DNA polymerase (pol) gene of PAdV-5.
  • the conserved region I (YGDTDS (SEQ.ID.NO.:25)) and two possible zinc finger motifs CEYC(X) 7 HTC(X) 10 HH and CETRCDKC(X) 23 CSVC of PAdV-5 pol were also present in PAdV-3 (Reddy et al, 1998a).
  • PAdV-5 has the largest E3 region so far reported among PAdVs. Moreover, E3 ORF4 was unique to PAdV-5. The E4 region of PAdV-5 was also larger, about 50%, than in most human adenoviruses and in PAdV-3. In addition, 8 of the 11 ORFs were unique to PAdV-5. The detailed analyses of PAdV-5 E3 and E4 regions have been described (Tuboly & Nagy, 2000; Tuboly et al, 2000).
  • HAdVs two genes coding for the LX and TVa2 proteins are classified as intermediate genes (Shenk, 1996).
  • the minor capsid component (IX) is needed for packaging the viral DNA (Ghosh- Choudhury et al, 1987) and is involved in activating the major late promoter (MLP) (Lutz et al, 1997).
  • MLP major late promoter
  • the putative IX gene of PAdV-5 (126 aa, 13.7 kDa) showed 42% identity to the BAdV-2 118R ORF-4 protein.
  • the IVa2 protein of PAdV-5 (372 aa, 42.5 kDa) had 66% amino acid identity with the TVa2 protein of HAdV-2.
  • PAdV-5 TVa2 The entire potential nucleoside triphosphate binding site GPTGCGKS (SEQ.ID.NO.:26) (Gorbalenya & Koonin, 1989) was present in PAdV-5 TVa2. Late regions. The late regions of the genome were characterized by their predicted common poly(A) sites, and their products are mainly structural proteins (Shenk, 1996). Transcription starts from the MLP, and the primary transcript is processed into several late mRNAs. For PAdV-5 six late regions (L1-L6) were predicted (Fig. 1). The putative MLP of PAdV-5 (nt 5077- 5273) was deduced by promoter prediction and sequence similarity with known adenovirus MLP sequences.
  • the canonical TATA box of the predicted MLP was located at nt 5122-5128.
  • An inverted CAAT box (nt 5084-5088), an upstream promoter element (Sawadogo & Roeder, 1985) (nt 5104-5109), initiator element (Lu et al, 1997) (nt 5150-5156), and two downstream activating elements (Leong et al, 1990) DEI (nt 5225-5235) and DE2 (nt 5240- 5255) were identified within this region.
  • the common poly(A) tail addition site of the LI region was predicted at nt 12173.
  • the putative LI 52 kDa protein (354 aa, 40.4 kDa) was most similar to the 55 kDa protein of HAdV-17 (62% identity) and pHIa (573 aa, 64.5 kDa) showed the highest identities to the pHIa of HAdV-40 (61%).
  • the putative L2 region had a common poly(A) tail addition site at nt 14172.
  • the El (penton base; 471 aa, 52.7 kDa) and pVII (147 aa, 18.9 kDa) proteins were predicted in this region.
  • the RGD motif of protein HI which interacts with surface integrins ⁇ v ⁇ 3 and v ⁇ 5 (Wickham et al, 1993) was missing from the predicted penton protein of PAdV-5.
  • the entire LDV motif which interacts with integrin ⁇ 4 ⁇ x (Komoriya et al, 1991), was present at 264 LDV.
  • the fibre-interacting domain is highly conserved in the penton base proteins of adenoviruses (Caillet-Boudin, 1989).
  • PAdV-5 the 230 SRLNNLLGIRKR (SEQ.ID.NO.:27) motif was identical to the PAdV-3 fibre- interacting domain (Reddy et al, 1998a).
  • Protein IE of PAdV-5 was most similar to that of PAdV-3 (77% identity), and pVII exhibited 54% similarity to pVII of BAdV-2.
  • One putative protease cleavage site was found in pVII at 20 MYGGA (SEQ.ID.NO.:28), exactly at the same position as in PAdV-3 (Reddy et al, 1998a).
  • the L3 region had a predicted common poly(A) site at nt 15356.
  • Protein V of PAdV-5 (374 aa, 42.4 kDa) was most closely related to the corresponding protein of BAdV-2 (57% identity).
  • the common poly(A) tail addition site of the L4 region was located at nt 19790.
  • the predicted pX protein 70 aa, 7.8 kDa had 79% amino add identity to pX of BAdV-2. There was only one protease cleavage site at 38 MSGGF (Weber & Anderson, 1988).
  • the pVI protein of PAdV-5 (233 aa, 25.2 kDa) showed the highest similarity to pVI of HAdV-40 (55% identity) and contained two sequence motifs ( 30 MNGGAFNW (SEQ.ID.NO.:29) and 219 IVGLGVRS (SEQ.ID.NO..-30)) which corresponded to the consensus protease cleavage site sequences (Russell & Kemp, 1995).
  • the protease requires a peptide (GVQSLKRRRCF (SEQ.ID.NO.:31)), which derives from the C-terminus of pVI as a cofactor for its activity (Mangel et al, 1993; Webster et al, 1993).
  • this peptide sequence was well conserved at 222 LGVRSVKRRRCF (SEQ.ID.NO.:32).
  • the predicted L5 region was characterized by a ⁇ oly(A) tail addition site at nt 26455.
  • the 100 kDa protein (722 aa) and the 33 kDa protein (219 aa) showed the highest similarity to the corresponding BAdV-3 proteins (59% and 27% identity, respectively).
  • the pVEI gene (222 aa, 24.1 kDa) has been previously described (Tuboly & Nagy, 2000).
  • PAdV-5 was phylogenetically closer to certain bovine adenoviruses, specifically to BAdV-1 (based on pVEI) and BAdV-2 (based on hexon, sequences provided by D. Ojkic, Guelph, Canada; no BAdV-1 sequences were available) than to other described porcine adenoviruses. All these findings underline the recent classification of PAdVs (Benk ⁇ et al, 1999). The poly (A) site for the L6 region was located at nt 28688.
  • the N- terminal region of the PAdV-5 fibre protein (500 aa, 53 kDa) encoded here, contained a nuclear localisation signal ( 2 KRAKR (SEQ.ID.NO.:35)) motif (Hong & Engler, 1991) and a penton base interacting "FDPVYPYG (SEQ.ID.NO.:36) sequence (Caillet-Boudin, 1989).
  • a nuclear localisation signal 2 KRAKR (SEQ.ID.NO.:35) motif
  • FDPVYPYG SEQ.ID.NO.:36
  • VA RNA Virus-associated RNA
  • VA RNAs low molecular weight RNAs
  • VA RNA X and VA RNA ⁇ RNA polymerase- ⁇ i
  • PAdV-5 The analysis of the PAdV-5 genome summarized herein indicate that the size and the genome organization of this adenovirus are similar to that of mastadeno viruses. However, unique characteristics of PAdV-5 were also identified. Most importantly the RGD motif of the penton base protein and the TLWT motif of the fibre protein were not present. Only one protease cleavage site was found in pX. Phylogenetic analysis of pVEI and hexon proteins showed that PAdV-5 was well separated from the other PAdVs but was more closely related to BAdV-1 and BAdV-2. MATERIALS AND METHODS FOR EXAMPLE 6 TO 8 Cells, viruses, viral DNA and cDNA
  • the HNF-70 strain of PAdV-5 and the cell culture adapted Purduell5 strain of TGEV were propagated in continuous swine testicle (ST) cells (McClurkin et al., 1966) as described (Tuboly et al, 1995). Virus titrations, plaque purifications and virus neutralization assays were also performed in ST cells as described (Tuboly et al., 1993).
  • Adenoviral DNA was extracted from HNF-70 infected ST cells by the method of Hirt (1967) at the peak of the cytopathic effects (CPE).
  • CPE cytopathic effects
  • the transfer vectors were generated in bacteria by homologous recombinations of the modified MluIB fragments carrying the S gene and the Rpac+ genomic clone linearized with Pad.
  • the recombinant clones were analyzed and selected by standard miniprep and RE digestion methods (Sambrook et al, 1989). Large scale DNA preparation of the clones selected for ST cell transformation was done with the Concert Nucleic Acid Purification System (Life Technologies), according to the instructions of the manufacturer. DNA transf ection and selection of recombinant viruses
  • Lipofectin (Life Technologies) mediated ST cell transfections were done as described earlier (Tuboly and Nagy, 2000) following the manufacturers' instructions.
  • the transfected cells were covered with 0.7 % agarose in DMEM supplemented with 10% fetal bovine serum. Plaque formation was monitored daily and 10 individual plaques from each transfection were transferred to Eppendorf centrifuge tubes with 1 ml of DMEM on day 7 post transfection. The tubes were frozen to -70 °C and thawed on ice. The contents were used for the inoculation of duplicate wells of ST cell monolayers in 6-well tissue culture plates.
  • Wild type and recombinant adenovirus infected, together with ur nfected ST cells were harvested at the peak of CPE formation, the proteins were separated in 10% SDS-polyacrylamide gels as described (Laemmli 1970) and transferred to nitrocellulose membranes (Sambrook et al., 1989). TGEV specific pig polyclonal antibodies (Tuboly et al., 1994) were used in 1:500 dilution to detect the proteins. The reaction was developed by the Boehringer Mannheim chemiluminescent detection kit according to the instructions of the manufacturer.
  • Blood samples were collected weekly and the clinical signs were monitored daily. The pigs were euthanized after 3 weeks and subjected to post-mortem examination. Contents from the small intestine and parts of the lung were collected, processed as described (Tuboly et al., 1993) and tested for the presence of virus and slgA antibodies. For antibody detection, the serum samples and the filtered intestinal and lung contents were heat inactivated at 56°C for 1 hour. Samples were tested in a TGEV specific IgG or IgA ELISA as described earlier (Tuboly et al, 1993) and in a TGEV-specific virus neutralization (VN) microtiter assay (Tuboly et al., 2000). The serum samples were also tested for the presence of PAdV-5 specific antibodies by a VN assay (Tuboly et al., 1993).
  • VN virus neutralization
  • Rectal swabs were collected daily to monitor virus shedding.
  • the swabs were processed as described (Tuboly et al., 1995) and the viral titers were determined in 96 well plates with ST cells.
  • the viruses isolated at day 5 p.i. were pooled in each group, and the virus was propagated in ST cells for DNA extraction and RE analysis of the viral DNA.
  • EXAMPLE 6 Transfer vectors
  • PAdV Recombinant PAdVs were plaque purified 3 times and the presence and orientation of the S gene were confirmed by RE analysis of the viral genome (data not shown) after each round of plaque purification. The expression of the recombinant proteins was monitored by Western blots. Table 1 summarizes the stability data of each recombinant virus in tissue culture. Following transfection with RPAdV-2.2S (no deletion in the E3 region)
  • S gene expression was monitored by Northern blot analysis of total RNA extracted at 2 h p.i. and every 4 h thereafter from recombinant virus infected cells and blots were probed with radioactively labeled 2.2 kb S gene DNA.
  • RPAdV-2.2S and ⁇ RPAdV-2.2Sc expressed TGEV S gene specific mRNA at approximately the same level.
  • the S gene mRNA synthesis in RPAdV-2.2S infected cells was undetected during early times of virus replication and could be detected first only at 18 hours p.i. whereas S gene specific mRNA appeared somewhat earlier in ⁇ RPAdV-2.2Sc infected cells, at 14 hours p.i. ( Figure 11).
  • Pigs orally inoculated with the recombinant viruses namely RPAdV-2.2S, ⁇ RPAdV-2.2Sc and ⁇ RPAdV-2.2Sr, remained healthy throughout the experiment and no signs of diarrhea or respiratory distress were observed.
  • the titre of virus in the rectal swabs collected daily was determined and the results are summarized in Table 2.
  • Virus was not recovered from the lungs or the small intestine of the euthanised pigs at 3 weeks p.i. (not shown).
  • a sample was considered negative after 3 blind passages in tissue culture.
  • VN test of the sera collected at the end of the experiment showed relatively high TGEV neutralizing titres (up to 1: 64) in groups injected with RPAdV-2.2S and ⁇ RPAdV-2.2Sc.
  • No TGEV specific VN antibodies were detected in the samples from pigs injected with ⁇ RPAdV-2.2Sr or wild type PAdV-5, or from the mock-infected group. Similar results were obtained in the TGEV specific ELISA to detect serum IgG.
  • PAdV-5 specific VN antibodies were present in the sera of all animals immunized with recombinant or wild type PAdV-5 but there was no evidence of such antibodies in the mock- infected group.
  • TGEV specific antibodies of class A were detected in both the lungs and the intestinal contents of the pigs immunized with RPAdV-2.2S and ⁇ RPAdV-2.2Sc.
  • the intestinal slgA was present in all of the animals by ELISA. However the slgA titres measured in the lungs were lower in all animals and pig #2 of the group injected with RPAdV-2.2S was negative.
  • human adenoviruses have been shown to be efficient vector systems for the delivery of porcine coronavirus antigens like those of the TGEV or porcine respiratory coronavirus S protein (Torres et al, 1996; Calleabut et al., 1996). Their widespread use of human adenovirus in domestic animals may be limited, mainly because of safety concerns. Animal adenoviruses, however, are mostly species-specific, presenting almost negligible risk for humans or other animal species and replicate more co efficiently that human adenovirus in the native porcine host, thereby providing a safer and more efficient delivery system in animals.
  • PAdV-3 carrying the gD gene of the Aujeszky's disease virus (Reddy et al, 1999b) and the E2 gene of the classical swine fever virus (Hammond et al, 2000) has already been developed as a recombinant virus vector.
  • PAdV-3 carrying the gD gene of the Aujeszky's disease virus (Reddy et al, 1999b) and the E2 gene of the classical swine fever virus (Hammond et al, 2000) has already been developed as a recombinant virus vector.
  • PAdV-3 carrying the gD gene of the Aujeszky's disease virus (Reddy et al, 1999b) and the E2 gene of the classical swine fever virus (Hammond et al, 2000) has already been developed as a recombinant virus vector.
  • the wide prevalence of PAdV-3 may be a limiting factor in their use as recombinant vaccines because of widespread preexisting
  • PAdV-5 to our knowledge is not present in pig populations, and has been reported only once from Japan (Hirahara et al, 1990).
  • the development of PAdV-5 into a recombinant TGEV vaccine is described in herein.
  • Five helper independent recombinant porcine adenoviruses have been constructed and tested for their stability and their ability to express the entire or the 5' 2.2 kb half of the TGEV S gene.
  • Three of the recombinant viruses carrying the 2.2 kb S gene were selected and their ability to induce TGEV neutralizing antibodies was tested by oral immunization of pigs.
  • ⁇ RPAdV-2x2.2S with two sets of the 3' truncated S gene, produced 7 out of 10 plaques that carried both inserts right after the transfection and became stable during further rounds of plaque purifications.
  • the virus did not lose any of the inserts or the PAdV sequences as detected by RE analysis (not shown).
  • the size of the genome of this recombinant virus was 109.6% of the original genome size, exceeding the expected maximum of 106.8 % (Hammond et al., 2000) described for PAdV-3.
  • the ⁇ RPAdV-4.4S virus with the full-length S gene did not yield a stable lineage, despite several rounds of plaque purification of the positive viruses.
  • the expected genome size of this virus was also 109.6% of the wild type genome but unlike the ⁇ RPAdV-2x2.2S, parts or all of the insert or the PAdV genome were constantly being lost during virus replication. This phenomenon raised questions about current theories of adenovirus genome stability.
  • the main limiting factor of the stability is believed to be the packaging capacity determined by the size and icosahedral structure of the virion. According to experiments reported herein, the size of the insert is not the only important factor influencing the stability of the genome. The sequence, structure or the orientation of the insert may also play an important role.
  • the recombinant viruses were analyzed by Western blotting to determine the size of the recombinant proteins. All of the viruses with the gene in a left to right orientation expressed a protein of the predicted size. The estimated size of the S protein in RPAdV-2.2S, ⁇ RPAdV-2.2Sc and ⁇ RPAdV-2x2.2S was 110 kD. The ⁇ RPAdV-4.4S virus preparation also expressed the expected 200 kD protein but smaller S protein fragments were also detected.
  • Titers are expressed as log2 dilutions of the samples. Dilutions for VN started with 1:2, and for ELISA with 1:10. Mock infected pigs were negative in all tests.
  • Dragulev B.P. Sira S., Abouhaidar M.G. and Campbell J.B., 1991 Sequence analysis of putative E3 and fiber genomic regions of two strains of canine adenovirus type 1. Virology 183, 298-305.
  • Protein IX a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes. EMBO Journal 6, 1733-1739.
  • Haig DA Clarke MC, Pereira MS. Isolation of an adenovirus from a pig. / Comp Pathol 1964; 74, 81-85.
  • Kleiboeker SB Identification and sequence analysis of the El genomic region of a porcine adenovirus. Virus Res 199536(2-3):259-68 Kleiboeker S.B., 1994 Sequence analysis of putative E3, pVEI, and fiber genomic regions of a porcine adenovirus. Virus Res. 31, 17-25.
  • VA RNAs from avian and human adenoviruses dramatic differences in length, sequence, and gene location. Journal of Virology 58, 600-609.
  • Viral DNA, and viral peptide can act as cofactors of adenovirus virion proteinase activity. Nature 361, 274-275.
  • the mouse adenovirus type 1 contains an unusual E3 region. /. Virol. 63, 5455- 5458.
  • Reddy PS Idamakanti N,Babiuk L, Mehtali M, Tikoo SK. Porcine adenovirus as a helper-dependent expression vector. / Gen Virol 1999a; 80: 2909-2916.
  • Reddy PS Idamakanti N, Hyun BH, Tikoo SK, Babiuk LA. Development of porcine adenovirus-3 as an expression vector. / Gen Virol 1999b; 80 (3):563-70.
  • Porcine adenoviruses types 1, 2 and 3 have short and simple early E-3regions. Virus Res. 43, 99-109.
  • Adenovirus terminal protein precursor Partial amino acid sequence and the site of covalent linkage to virus DNA. Journal of Biological Chemistry 257, 13499-13506.
  • the adenovirus protease is activated by a virus-encoded disulphide-linked peptide.
  • FIG. 1 PAdV-5 maps.
  • A HmdEI and Mlul restriction map of the genome, m.u.: map unit, 1 m.u.: -335 bp.
  • B The sequenced region enlarged. Bar indicates the EcoRI DNA fragment (between nts 616 and 2569) used as the E3 probe in Northern blotting.
  • C RF: reading frames of the r strand. The open reading frames are shown by the boxes. Numbers above the boxes are the start and stop positions of each ORF. The numbers inside the boxes indicate the putative number of amino acids encoded by each ORF of HNF-70.
  • D The dotted boxes represent the part and size (indicated below the box) of the DNA removed to generate pR- ⁇ PH and pR- ⁇ HH genomic clones.
  • FIG. 1 Alignment of the predicted ORF2 amino acid sequences of PAdV-5 HNF-70 and some closely related animal adenoviruses.
  • the adenovirus serotypes are indicated on the left.
  • the sequence identities are indicated by dots.
  • FIG. 1 Sequence alignment of the predicted ORF3 proteins of HNF-61 and HNF-70. The number of amino acids is indicated on the right, identities are shown by dots.
  • Figure 4 Unrooted phylogenetic tree of pV I protein homologues of selected animal adenoviruses generated by the Clustal method. The length of branches represents the distance between sequence pairs. Units at the bottom indicate the number of substitution events.
  • Figure 5 Time course analysis of PAdV-5 HNF-70 nucleic acid synthesis. Numbers in the middle indicate hours p.i.
  • A DNA dot blot, in the absence (AraC-) and presence (AraC+) of AraC, probed with digoxigenin labeled genomic DNA.
  • B Northern blot of E3 region transcripts, probed with the EcoRI G fragment ( Figure 1). Lines and numbers on the right show the position and size (kb) of the RNA molecular weight marker (M).
  • Figure 6 Restriction endonuclease analysis of the wild type PAdV-5 HNF-70 strain (A) and its deletion mutant R- ⁇ HH (B) genomic DNA in ethidium bromide stained 0.8% agarose gel. Lanes 1: Hpal, lanes 2: EcoRI, lanes 3: H dET. Arrowheads indicate the corresponding fragments for each digest. M: 1 kb DNA ladder.

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CN110981945B (zh) * 2019-12-08 2021-10-19 中牧实业股份有限公司 一种猪圆环病毒2型重组Cap蛋白的表达制备及应用
CN112226408A (zh) * 2020-09-30 2021-01-15 西北农林科技大学 一种猪病原或外源性蛋白特异性抗原肽筛选和鉴定的方法
CN112226408B (zh) * 2020-09-30 2024-04-02 西北农林科技大学 一种猪病原或外源性蛋白特异性抗原肽筛选和鉴定的方法

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