EP1311698A2

EP1311698A2 - Porcine adenovirus type 5 vector and vaccine

Info

Publication number: EP1311698A2
Application number: EP01929140A
Authority: EP
Inventors: Eva Nagy; Tamas Tuboly; Miklos Nagy
Original assignee: University of Guelph
Current assignee: University of Guelph
Priority date: 2000-05-03
Filing date: 2001-05-03
Publication date: 2003-05-21
Also published as: WO2001083737A3; CA2413265A1; WO2001083737A2

Abstract

The entire nucleotide sequence of porcine adenovirus serotype 5 (PAdV-5) is described as are methods of inserting heterologous nucleotide sequences, such as the TGEV S gene, into the E3 region of the virus. Vaccines and methods of preparing vaccines with the recombinant adenovirus are described as well as applications of the recombinant virus and vaccines.

Description

Title; Porcine Adenovirus Vaccine FIELD OF THE INVENTION

This invention relates to a novel porcine adenovirus, PadV-5, and its use as a delivery vector or vaccine. BACKGROUND OF THE INVENTION

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.

^• The prototype strains of the first four porcine adenovirus (PAdV) serotypes were isolated in the 1960s (Haig et al. (1964); Clarke et al. (1967); Kasza (1966)). A new serotype was identified in Japan in 1990 and designated as PAdV-5 (Hirahara et al. (1990)). The two reference strains, HNF-61 and HNF-70 of PAdV-5 were isolated from the same swineherd. In 1995, again in Japan, a different research group reported the isolation of another PAdV and after serological comparisons with PAdV-1-4 but not with PAdV-5, it was also described as PAdV-5 (Kadoi et al. (1995)). 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.

Porcine adenoviruses (PAdV) 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. (1995)) and the sequence analyses of the early regions E3, the site found most suitable for foreign gene insertion, (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)).

Further, recently PAdV-3 was developed into helper-dependent (Reddy et al. (1999a)) and -independent expression vectors (Reddy et al. (1999b); Hammond et al. (2000)). The use of helper independent 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)). Both recombinant viruses expressed the foreign gene to the some extent, proving that PAdVs could be used as such vectors. Despite these results, the widespread occurrence of serotype 3 in the swine populations and the pre-existing PAdV-3 specific virus neutralizing antibodies may restrict the use of this serotype as a vector vaccine. Consequently, what is needed is an improved vector for immunization of swine. SUMMARY OF THE INVENTION

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)).

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. In a preferred embodiment, the nucleic acid sequence of the PAdV-5 comprises:

(a) a nucleic acid sequence as shown in Figure 7 (SEQ.ID.NO.:l), wherein T can also be U; (b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a);

(c) a 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

(e) a 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.

Accordingly, in another aspect, 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. Preferably 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. Examples of modified forms include an isolated PAdV-5 wherein the E3 region has been deleted. According to one embodiment, the recombinant porcine adenovirus serotype 5 (PAdV-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.

In one embodiment, the recombinant PAdV-5 of the invention comprises a heterologous nucleic acid sequence that encodes an antigenic determinant from an infectious agent. In another embodiment, 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 (γlFN), porcine granulocyte macrophage colony stimulating factor (GM-CSF), or porcine granulocyte colony stimulating factor (G-CSF).

In another aspect, 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. Preferably the heterologous sequence encodes an antigenic polypeptide and /or an immuno-potentiating molecule, preferably 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 (γlFN), porcine granulocyte macrophage colony stimulating factor (GM-CSF), or porcine granulocyte colony stimulating factor (G-CSF).

In yet another aspect, 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. Preferably the at least one heterologous nucleotide encodes an antigenic polypeptide, more preferably antigenic determinants of infectious agents. Accordingly, 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.

In a further aspect 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. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which: 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 pViπ 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-ΔHH (B) genomic DNA in ethidium bromide stained 0.8% agarose gel.

Figure 7 (SEQ.ID.NO.:l) 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.

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 ΔRPAdV-2.2Sc. Figure 12 is a Western blot analysis the RPAdV-2.2S and ΔRPAdV-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

As stated above, 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.

The term "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.

The term "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, 5-trifTuoromethyl uracil and 5-trifluoro cytosine.

In a preferred embodiment, 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;

(b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a);

(c) a 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

(e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions. The term "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.:!). The term "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. For example, 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. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C.

The term "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 uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

Another example of 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. For example, 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. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, 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. Examples of 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. Accordingly, the present invention provides a modified PAdV-5 wherein the E3 region, or a portion thereof, has been deleted. In one embodiment, the E3 region that is deleted is as shown in Figure 13 (SEQ.ID.NO.:8) or Figure 14 (SEQ.ID.NO.:9). II. Recombinant PAdV-5

. 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. In particular, 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. Accordingly, in another aspect, 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. Preferably 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. When integrated 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. Preferably, 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).

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).

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.

The heterologous nucleic acid sequences incorporated may encode immuno-potentiator molecules such as cytokines or growth promoters, for example porcine interleukin 4 (IL4), gamma interferon (γlFN), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), FLT-3 ligand and interleukin 3 (IL-3).

It is to be understood that the 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. Preferably 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).

Instead of the porcine adenoviral major late promoter, any other suitable eukaryotic promoter may be used. For example, 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.

One skilled in the art will appreciate that other components or sequences may be included in the recombinant adenovirus and can be determined by one of skill in the art. Examples of methods for constructing an adenovirus vector encoding heterologous nucleic acid molecules are described in U.S.

Patent No. 4,920,209 and 6,086,890. III. Uses

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. Preferably 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. In order to direct the vaccine against a specific infectious organism, 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. If the vaccine is to be used to optimize protection against disease, suitable heterologous nucleotide sequences may also be those of immuno-potentiators such as cytokines or growth promoters.

In a further aspect of the invention, there is provided 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. Preferably 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 nucleotide sequence is conveniently foreign to the host vector Accordingly, 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.

The term "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 regima 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 10⁴ TCID₅₀ 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. Further, as will be readily understood by those skilled in the art, 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. Preferably, the vaccines 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. The expression "biologically compatible form suitable for administration in vivo" as used herein 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.

Accordingly, 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 administering a prophylactic or therapeutic amount of a recombinant porcine adenovirus serotype 5 comprising at least one heterologous nucleotide sequence, said heterologous nucleotide sequence preferably encoding and /or expressing an antigenic determinant of an infectious agent and /or an immuno-potentiating molecule. The vaccine of the invention may of course be combined with vaccines against other viruses or organisms such as parvovirus or Aujeszky's disease at the time of its administration.

In vaccines of the present invention 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. 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. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol( (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol( 974P, 934P and 971P). Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA( (Monsanto) which are copolymers of maleic anhydride and ethylene, linear or cross-linked, for example cross-linked with divinyl ether, are preferred. Reference may be made to J. Fields et al, Nature, 186: 778-780, 4 June 1960, incorporated herein by reference. 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). For example, 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. Preferably, 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.

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. IL-12 is usually released by APC upon interaction with infectious pathogens and is a crucial Thl differentiation and activation factor. Thus, 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)).

Recent studies have indicated that a 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. Alternatively, repeated vaccination with the same recombinant adenovirus could be used as the boost.

The vaccines or 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

Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the 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. In this regard, reference is made to U.S. Patent No. 5,843,456, incorporated herein by reference, and directed to rabies compositions and combination compositions and uses thereof.

The utility of the 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. For example, piglets may be vaccinated at 1 day old, breeders may be vaccinated regularly up to point of giving birth and thereafter.

Preferably 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.

In a preferred aspect of this embodiment of the invention, administration is by oral delivery or intra-nasally.

The following non-limiting examples are illustrative of the present invention: EXAMPLES

MATERIALS AND METHODS FOR EXAMPLES 1-4 Viruses and viral DNA The origin of the PAdV-5 prototype strains HNF-61 and HNF-70 has been described (Tuboly et al., 1995). Both strains were propagated in a continuous swine testicle (ST) cell line (McClurkin et al., 1966) and the intracellular viral DNA was extracted at the peak of infection by the method of Hirt (1967).

DNA cloning and sequence analysis

The cloning of the Mlul and Hindlll generated genomic DNA fragments of HNF-61 and HNF-70 into the ρGEM-7Zf(+) plasmid (Promega) has been described (Tuboly et al, 1995). Nested set deletions of 300-400 basepairs (bp) of the H dHI F and D fragments were generated in both orientations by using the ExoHI/Sl Deletion Kit (MBI, Fermentas, Lithuania) providing 200- 300 bp overlaps. 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.).

Homology search of the 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

-. ST cells were grown to confluence in 3 cm Petri dishes and infected with 10 m.o.i. (multiplicity of infection) of the HNF-61 or HNF-70 strain of PAdV-5 and incubated for 2-24 hours at 37°C. Samples were collected every 2 hours. For DNA dot blot analysis the supernatant was removed and 0.5 ml distilled water was added to the cells and frozen to -70°C. Total cellular RNA was extracted every 2 hours post infection with an RNA extraction kit (RNeasy, QIAGEN). RNA and DNA from mock-infected ST cells were similarly collected. RNA and DNA samples were also collected from cells treated with 50 μg/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 μl 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.

The time course of 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 [α32P]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 BamΗI-HinάTΩ. digested pWE15 cosmid vector (Stratagene, modified by Ojkic et al, unpublished). The resulting clone was used in co- transformation of competent E. coli BJ 5183 cells (Hanahan, 1983) together with HNF-70 genomic DNA as described (Degryse, 1996). The clones carrying the entire PAdV-5 genome were selected and transformed into DH5α cells for large scale DNA preparation with the Concert Nucleic Acid Purification System (Life Technologies) according to the manufacturers' instructions. In order to introduce deletions into the E3 region the cloned Mlul B fragment of HNF-70 (Tuboly et al, 1995) was used. Two deletion clones were generated, namely Mlul B-Δ(992-2497) and MZttl B-Δ(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-ΔPH and pR-ΔHH). 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).

EXAMPLE 1 Sequence analysis

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

) these had a capacity of more than 40 aa and none of them exceeded 90 aa (not shown). Four complete ORFs of the r strand had a coding capacity of more than 40 aa (Figure IC). The Blast analysis of these ORFs revealed sequence homologies of different percentages with adenovirus sequences present in

Genbank. ORFs 1-4 of the right strand had theoretical coding capacities of

25.3, 14.3, 68.4 and 5.5 kDa, respectively. The nucleotide sequence at the end 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.

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. There was only a 2 aa difference between the predicted ORF2 sequences of the 2 PAdV-5 strains at aa positions 31 and 35. The alignment of the nucleotide sequences of the HNF-61 and HNF-70 strains showed 7 mismatches in this region. A comparison of the predicted amino acid sequences of PAdV-5 HNF-70 ORF2 and the homologous ORFs of the most closely related animal adenoviruses is shown in Figure 2.

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. The last 40 aa of the C-terminal exhibited again a high sequence identity (91.9%). 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.

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. A Genbank search identified no related adenovirus gene. According to the sequence comparisons, the ORF starting at 81.7 m.u. (ORF5, from nt 2709 of HNF-61 and nt 2690 of HNF-70) was the beginning of the fiber protein coding region.

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.

One TATA box was identified (TATAAAA, SEQ.ID.NO.:ll) at nt 351 of both strains. No typical CCAAT was found upstream of the TATA box but a CAGTT sequence was located at nt 322. CAAT sequences were identified further upstream of the TATA box at nt 153 on both strains and also at nt 116 on HNF-61. A GC box was located starting at nt 322 (TTGGCGGGC, SEQ.ID.NO.:12). Other 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. Canine adenoviruses showed 58.9% (CAdV-1) and 61.2% (CAdV-2) similarity. PAdV 1-3 serotypes clustered closely to PAdV-5, with approximately 64% similarity, but PAdV-4 showed only 47.1% similarity. EXAMPLE 3

DNA replication and transcription To demonstrate that the putative E3 region, determined by sequence analysis, between ORFl (pVTfl) and ORF5 (fiber) was indeed expressed at early times of infection, DNA and RNA blotting assays were carried out. Results for the HNF-70 strain are presented in Figure 5, the analysis of HNF- 61 gave similar results. The dot blot assay showed that DNA replication started between 12-14 hours post infection (Figure 5A). No DNA replication could be detected in AraC treated infected cells. The Northern blot analysis indicated that RNA transcription within EcoRI G started between 4 and 6 hours post infection and the peak was reached at 12 hours from which point it gradually declined (Figure 5B). At least five mRNA species could be detected. 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

Two genomic clones with deletions in the E3 region (pR-ΔPH and pR- ΔHH) were generated by homologous recombination using the Mlul B-Δ(992- 2497) and Mlul B-Δ(1260-2497) clones, respectively (Fig. ID) and the DNA was transfected into ST cells. The E3 portion deleted from pR-ΔPH started at nt 992 downstream from the ORFl ATG and ended at nt 2497. This part of the E3 region contained the last 57 bp of ORF2, the entire ORF4 and most of ORF3. The deletion in pR-ΔHH 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. After transfection into ST cells only the pR-ΔHH clone resulted in infectious virus, pR-ΔPH did not generate CPE in the cell culture even after repeated attempts of transfections and several blind passages of the transfected cells. The pR-ΔHH generated virus (R-ΔHH) 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-ΔHH DNA. The original Hpal site was fused to the HincT site in R-ΔHH (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-ΔHH. 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-ΔHH as expected. Similarly, the size of the 4.2 kb H diπ D fragment was decreased to 3.0 kb by the 1.2 kb fragment deleted from R-ΔHH. DISCUSSION OF EXAMPLES 1-4

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).

The sequenced region presented here comprised the entire pVTH gene and the 5' portion of the fiber gene. By analogy with well characterized adenoviruses it is suggested that the region of PAdV-5 between the pVHI and fiber sequences is the E3 region. There are differences in size of the E3 region of different mammalian adenoviruses. HAdVs in general have a longer E3 region than animal adenoviruses. 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. The pVHI gene was followed by an ORF (ORF2) that was homologous to the 12.5 kDa HAdV E3 protein and its counterparts in PAdVs and several other mammalian adenoviruses.

The long 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. A large variation exists in HAdV E3a sequences among the different subgenera and also among members of the same subgenus, but the E3b region is usually more conserved (Bailey and Mautner, 1994). Porcine adenoviruses however, 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). Despite the differences detected in the ORF3 sequences of the PAdV-5 prototype strains, both viruses proved to be genetically stable when subjected to several tissue culture passages (results not shown). The viruses were isolated at the same time and from the same herd and it was speculated earlier that the differences in the RE pattern were due to rapid genetic changes of the virus rather than a concurrent infection with two different viruses of the same serotype (Tuboly et al, 1995). ORF4 appeared to be unique to the PAdV-5 genome, but the putative protein product has an unknown function.

The phylogenetic analysis of the established pVHI protein sequences showed that PAdV-5 is more closely related to BAdV-1 than to any of the known PAdVs. The 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. These data suggested that 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.

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. In order to increase the vector capacity two genomic clones (pR-ΔPH and pR-ΔHH) with different sizes of E3 deletions were generated and the DNA transfected into ST cells. The transfection with the clone where a 1505 bp part was deleted (pR-ΔPH) did not result in infectious virus particles after several repeated attempts. The deletion removed part of the 12.5 kDa homologue (ORF2) of the E3 region. This ORF is not essential for replication in cell culture in HAdVs (Hawkins and Wold, 1992) and BAdV-1 (Evans et al, 1998). The deletion of part of the 13.3 kDa protein coding gene and its fusion with the downstream ORF3 sequences in the attenuated CLL strain of CAdV-1 (Dragulev et al., 1991) does not seem to affect the virus replication in vitro. On the other hand the insertional inactivation of the PAdV-3 ORFl indicated that this gene may be essential for replication of porcine adenoviruses (Reddy et al., 1999). These ORFs were identical in the otherwise variable E3 regions of both prototype strains indicating that at least in vivo ORF2 might have an important role during the infection cycle. The smaller E3 deletion of 1237 bp did not influence the virus replication. The viruses generated by the pR-ΔHH transfection replicated to as high titers as the wild type virus. The size of the deletion was double that described for PAdV-3 (Reddy et al, 1999) and the deleted 1.2 kb fragment increased the theoretical vector capacity (105%) of PAdV-5 to 2.9 kb. EXAMPLE 5: THE COMPLETE NUCLEOTIDE SEQUENCE OF PAdV-5

The source and propagation of the PAdV-5 HNF-70 strain, extraction and cloning of the viral DNA fragments have been described (Tuboly et al, 1995). Nested set deletions were generated by exonuclease III and SI nuclease digestions (Henikoff, 1984), and the clones were sequenced in both directions with the SP6 and T7 promoter specific primers and primers based on the obtained sequence data by the dideoxy nucleotide chain termination technique. The contiguous sequence was assembled from overlapping sequences using the Lasergene software package.

Homology search of the GenBank database for the deduced amino acid sequence of each open reading frame (ORF) was done with the aid of the BLAST program (Altschul et al., 1990). The promoters and splice sites were predicted at the Berkeley Drosophila Genome Project website (Ohler & Reese, 1998; Reese et al., 1997). Polyadenylation (poly(A)) site prediction was done with the POL YAH program (Salamov & Solovyev, 1997). Protein sequence alignments were generated with CLUSTAL W (Thompson et al, 1994). The distance matrix analysis was carried out with the PHYLIP (v.3.5) program package (Felsenstein, 1989).

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

(19.88 kDa) and E1A 67R (7.76 kDa) encoded by two overlapping ORFs were similar in length to the canine adenovirus serotype 1 (CAdV-1) E1A proteins, although in CAdV-1 these ORFs are not overlapping (Morrison et al, 1997). The retinoblastoma susceptibility protein (pRb) binding motif LXCXE (Defeo- Jones et al, 1991) with a slight difference (⁹⁷LDYPE), and the zinc finger motif (¹⁰⁷CX₂CX₁₃CX₂C; 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 (DBP) 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 ( ⁰PKPKK (SEQ.ID.NO.:17) and ⁴⁷RRRK (SEQ.ID.NO.:18)) which were similar to those predicted for BAdV-3 (²⁹PRKK (SEQ.ID.NO.:19) and ³⁵RKRR (SEQ.ID.NO.:20)) and PAdV-3 (⁴⁷RRKR (SEQ.ID.NO.:21), ⁷⁷RRK (SEQ.ID.NO.:22)) (Reddy et al, 1998a, b). Two conserved zinc binding motifs were present: one at ¹⁹⁹CXHX₅₂CX₁₅C and the other at ³¹¹CXCX₅₁CX₁₅C exactly as in the corresponding proteins of HAdV-2 (Tucker et al, 1994) and PAdV-3 (Reddy et al, 1998a). According to amino acid sequence alignments with known terminal protein precursors (pTP), ORF₉₄₄₀.₇₅₄₂ encoded the main body of the pTP in the E2B region with a predicted splice acceptor site at nt 9348. All known pTPs have the sequence motif YSRLRYT (SEQ.ID.NO.:23) involved in protein primed DNA replication initiation (Hsieh et al, 1990). In PAdV-5 the ⁸⁶YSRLKYT (SEQ.ID.NO.:24) motif was identified at the same location. The nuclear localization signal (NLS) RLPI(R)₄PRI of the pTP of PAdV-5 was similar to that of PAdV-3 (RLPL(R)₄PRP) (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 ⁵¹²NSGD in the PAdV-5 pTP.

ORF₇₇₁₀.₄₃₀₉ 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)₇HTC(X)₁₀HH and CETRCDKC(X)₂₃CSVC of PAdV-5 pol were also present in PAdV-3 (Reddy et al, 1998a).

Based on the available sequence data, 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).

Intermediate regions. In 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). 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. 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 α_vβ₃ and _vβ₅ (Wickham et al, 1993) was missing from the predicted penton protein of PAdV-5. However, the entire LDV motif, which interacts with integrin α₄β_x (Komoriya et al, 1991), was present at ²⁶⁴LDV. The fibre-interacting domain is highly conserved in the penton base proteins of adenoviruses (Caillet-Boudin, 1989). In PAdV-5 the ²³⁰SRLNNLLGIRKR (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 ²⁰MYGGA (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 ³⁸MSGGF (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 (³⁰MNGGAFNW (SEQ.ID.NO.:29) and ²¹⁹IVGLGVRS (SEQ.ID.NO..-30)) which corresponded to the consensus protease cleavage site sequences (Russell & Kemp, 1995). In HAdV-2 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). In pVI of PAdV-5 this peptide sequence was well conserved at ²²²LGVRSVKRRRCF (SEQ.ID.NO.:32).

The predicted L5 region was characterized by a ρoly(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). After alignment to the corresponding BAdV-3 protein (Reddy et al, 1998b), two putative protease cleavage sites were found in the pVIII protein at ¹⁰⁸LAGGGRTT (SEQ.ID.NO.:33) and at ¹⁴⁸LAGGSRSS (SEQ.ID.NO.:34). Figure 9 shows the phylogenetic analysis of the putative pVHI protein compared to representative adenoviruses. PAdV-1-3 had an inferred common ancestor, whereas PAdV-4 and PAdV-5 were in two additional separate lineages. Similar relationships were noted for the hexon proteins. It seemed that 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 (Benkδ 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 (²KRAKR (SEQ.ID.NO.:35)) motif (Hong & Engler, 1991) and a penton base interacting "FDPVYPYG (SEQ.ID.NO.:36) sequence (Caillet-Boudin, 1989). Nineteen so-called pseudorepeats (Green et al, 1983) were observed in the shaft region of the PAdV-5 fibre protein. The sequence of the last complete motif before the head (KLGXGLXFD/N) (Chroboczek et al, 1995) was well conserved at ³¹⁵KLGAGLIFD (SEQ.ID.NO.:37). Interestingly, the TLWT motif, which in most cases indicates the N-terminus of the fibre head (Chroboczek et al, 1995) was not found in the PAdV-5 fibre.

Virus-associated RNA (VA RNA). Some adenoviruses encode low molecular weight RNAs (VA RNAs) transcribed by RNA polymerase-πi, required for the efficient translation of viral mRNAs late after infection (Larsson et al, 1986). In the mammalian adenoviruses studied by Ma and Mathews (1996), there are either one or two genes for VA RNA, known as VA RNA_X and VA RNA_π, located between the pTP and the 52 kDA ORFs. Based on sequence analysis and RNA secondary structure prediction only, the presence of VA RNAs in this region could not be predicted for PAdV-5 as was also observed for non-primate mastadeno viruses.

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). The TGEV S gene cDNA synthesis and cloning have been described elsewhere (Tuboly et al., 1995).

Transfer vector construction

Full length genomic PAdV-5 clones of the HNF-70 strain were constructed by homologous recombination in E. coli BJ 5183 (Hanahan, 1983) cells as described (Degryse, 1996). The strategy for the construction of the recombinant transfer vectors is summarized in Figure 10. Plasmid Rpac+ was generated by replacing the 1.9 kb Sall-Hpal fragment (spanning part of the pVEI protein and the majority of the E3 coding region) with a unique Pad restriction enzyme (RE) site. The Mlul B fragment of PAdV-5 (Tuboly et al., 1995) was used for the insertion of the S gene. Five different S gene- containing Mlul B fragments were generated: one construct contained the entire E3 region, and in four constructs a 1.2 kb piece was deleted in the E3 region between the H cE and the Hpal sites, i) MZwE3-2.2S: the 2.2 kb 5'-end of the S gene was inserted into the Hpal site of the E3 region in left to right (1- r) orientation; ii) Δ uIB-2.2Sc: the 1.2 kb HincT-Hpaϊ fragment of the E3 region was replaced by the 2.2 kb 5' S fragment, also in 1-r orientation; iii) ΔM/ttIB-2.2Sr: the same deletion in the E3 as in ii) construct but the 2.2 S gene was inserted in the reverse, r-1 orientation; iv) ΔMZwE3-2x2.2S: the 2.2 kb S gene was inserted in 1-r and subsequently in r-1 orientation; v) ΔMZwIB-4.4S: the entire 4.4 kbp S gene was inserted into the E3 region in 1-r orientation.

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.

The cell culture supernatant from each well was collected at the peak of CPE, about 6-7 days after inoculation and stored at -70°C until the next round of plaque purification. Cells were harvested for DNA RE analysis and for Western blotting. Only those viruses -one from each lineage- were included in further rounds of plaque purification that contained the entire expected S gene insert. Viruses, selected after three rounds of such plaque purification, were designated as RPAdV-2.2S, ΔRPAdV-2.2Sc, RPAdV-2.2Sr, ΔRPAdV- 2x2.2Sc and ΔRPAdV-4.4S, and were used for large scale virus propagation. Western blot analysis of recombinant S proteins

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. S gene mRNA time course ST cells grown in 6 well dishes were infected at a multiplicity of infection of 10 (M.O.I.) with the wild type and the recombinant viruses. RNA was extracted with the total RNA extraction kit (RNeasy, QIAGEN) every 4 hours between 2-24 hours post infection (pi.) and frozen to -70°C until use. RNA from mock-infected ST cells were also collected. Equal amounts of the total RNA extracted from each time point were separated on 1.1% formaldehyde- agarose gels, transferred to Nytran membranes (Sambrook et al., 1989) and immobilized by UV crosslinking (UV Crosslinker, Fisher Scientific). Prehybridization, hybridization in the presence of 50% formamide and washing of the blots were carried out as described by Sambrook et al. (1989). The cloned 2.2 kb TGEV S gene was released from the plasmid, labelled with [³²P]dCTP (ICN Pharmaceuticals) by the random primer method (Random Primer Labeling Kit, Life Technologies) and used as a probe. Animal experiments

Fifteen Yorkshire piglets from a TGEV- and PAdV-5-seronegative herd were weaned 21 days after birth and divided into 5 groups and housed separately. One group received uninfected ST cell supernatant, one group was immunized with wild type PAdV-5 and 3 groups were immunized with the selected recombinant viruses (RPAdV-2.2S, ΔRPAdV-2.2Sc and ΔRPAdV- 2.2Sr). Each pig received a single oral dose of 1 ml, with a virus titer of 5xl0⁶ p.f.u./ml (repeated vaccination with the same recombinant adenovirus could be used as the boost). 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).

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

Five full-length genomic PAdV-5 clones were generated by recombination in E. coli strain BJ5183 cells, each one carrying either the full or a partial S gene. The structure of these vectors, derived from the MZwIB and ΔMZwIB clones is shown in Figure 10A. The detailed RE analysis of the transfer vectors indicated that the orientation, location and size of the inserts were as expected.

Construction of recombinant 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)

70% of the plaques carried the 2.2 kb S gene in the correct orientation and expressed the S gene. Stable virus clones could be selected after the first plaque purification, with neither the insert nor parts of the E3 region being lost. Those recombinant viruses that carried a single copy of the same 2.2 kb insert in either orientation (ΔRPAdV-2.2Sc and ΔRPAdV-2.2Sr) produced plaques immediately after the transfection and were all positive in the RE digestion. Western blots showed that the S protein was expressed only in those viruses in which the S gene was inserted in left to right orientation (ΔRPAdV-2.2Sc). These viruses remained stable during further plaque purification.

Transfection with the vector containing two duplicate 2.2 kb S genes inserted in opposite orientations (ΔRPAdV-2x2.2S), carrying altogether a 4.4 kb foreign DNA insert, yielded eight positive plaques in the first round of purification. All of which expressed the S protein and the insert and S gene expression remained stable during subsequent purification.

Only 20% of the viruses in which the complete S gene was inserted into the PAdV E3 region (ΔRPAdV-4.4S) retained the S gene after the transfection, and the ratio remained low throughout further plaque purifications. In contrast with the rest of the recombinant viruses, the RE digests of the DNA always indicated that only part of the virus population from a single plaque carried the entire S gene, smaller DNA bands also appeared even after the third plaque purification (not shown). Similarly, many bands were observed in the Western blots of cells infected by these virus clones (not shown). EXAMPLE 7

Expression of the S gene and S protein

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 ΔRPAdV-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 ΔRPAdV-2.2Sc infected cells, at 14 hours p.i. (Figure 11).

No S gene specific mRNA detected in ΔRPAdV-2.2Sr infected cells (data not shown). The temporal pattern of transcription in ΔRPAdV-2x2.2S infected cells was similar to that for ΔRPAdV-2.2Sc. However Northern blot analysis of replicates of ΔRPAdV-4.4S infected ST cell cultures indicated different sizes of transcripts and the ratios of the transcripts were not consistent (data not shown).

For Western blot analysis, cells infected with the different recombinant viruses were collected at 24 hours p.i. RPAdV-2.2S and ΔRPAdV-2.2Sc expressed the S protein of the expected size, 110 kDa (Fig.3; lanes 2 & 3) and a similar result was obtained with the ΔRPAdV-2x2.2S recombinant virus (Figure 12; lane 4). No S protein was detected in cells infected with ΔRPAdV- 2.2Sr virus. S protein specific bands with a wide range of sizes (30 - 220 kDa) were seen on the blots of samples collected from ΔRPAdV-4.4S infected cells (Figure 12, lane 5). EXAMPLE 8

Animal experiments

Pigs orally inoculated with the recombinant viruses, namely RPAdV-2.2S, ΔRPAdV-2.2Sc and ΔRPAdV-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 detected from day 1 to 7 but was not detected in any of the samples by day 8 p.i. 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.

All three types of recombinant viruses isolated at day 5 pi. were tested by RE analysis of the extracted DNA with several of the characteristic REs. The DNA fragment patterns of viruses recovered from inoculated pigs were indistinguishable from those observed for the inocula before the animal "passages" (data not shown) indicating that the recombinant viruses were stable. ELISA and VN assays were conducted to detect TGEV- and PAdV-5- specific antibodies in the infected pigs. The results are shown in Table 3.

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 ΔRPAdV-2.2Sc. No TGEV specific VN antibodies were detected in the samples from pigs injected with ΔRPAdV-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 ΔRPAdV-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. DISCUSSION OF EXAMPLES 6 TO 8

Recombinant 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. However the wide prevalence of PAdV-3 may be a limiting factor in their use as recombinant vaccines because of widespread preexisting PAdV-3 neutralizing antibodies.

In contrast, 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. Two of these viruses carrying the foreign gene in left to right orientation expressed the protein in vitro and induced humoral immune response in pigs, not, only systemic but also of a local nature. The same approach was used for the construction of all the recombinant adenoviruses. One of the purposes was to test whether it is necessary to include foreign promoter sequences upstream of the insert or if the PAdV promoters are sufficient to express the gene. In one construct (RPAdV-2.2S) no E3 sequences were removed and the 2.2 kb S gene fragment was inserted in a left to right orientation near the 3' end of the E3 region, more than 1.8 kb downstream of the putative E3 promoter (see Example 1). S gene specific transcripts in Northern blots were detected from 18 hours pi., reaching the peak between 18 and 24 hours p.i. The S protein in Western blots was detected indicating that the native adenovirus promoters were sufficient for foreign gene expression (Torres et al, 1996). Although, together with the 2.2 kb foreign gene, the genome size of RPAdV-2.2S was 106.6% of the original wild type genome, neither the insert nor parts of the E3 region were lost during the plaque purifications or the several replication cycles in the pig intestine. The stable insertion of such a large foreign DNA is in accordance with the findings of Hammond et al. (2000) who increased the genome size of PAdV-3 to 106.8% of the original, despite earlier findings of a maximum of 105% for human adenoviruses (Bett et al., 1993). As a result of the 1.2 kb deletion of the E3 region, the other recombinant viruses carried the S gene closer to the E3 promoter than in RPAdV-4.4S (only 594 bp downstream of the putative E3 promoter. Those recombinant viruses that had the insert in left to right orientation (ΔRPAdV-2.2Sc, ΔRPAdV-2x2.2S, ΔRPAdV-4.4S) started to express the gene at the end of early times, between 14 and 18 hours pi., as detected by Northern blot analysis, whereas the virus with the S gene in reverse orientation (ΔRPAdV-2.2Sr) showed no signs of S gene expression in vitro (Northern and Western blot analyses) or in vivo as judged by the lack of TGEV specific antibodies in the immunized pigs.

Those ΔRPAdVs that carried a single copy of the 2.2 kb S gene appeared to be stable right after the transfection and all of the plaques tested had the inserted gene of the expected size at the expected position. ΔRPAdV-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 ΔRPAdV-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 ΔRPAdV-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, ΔRPAdV-2.2Sc and ΔRPAdV-2x2.2S was 110 kD. The ΔRPAdV-4.4S virus preparation also expressed the expected 200 kD protein but smaller S protein fragments were also detected. Direct measurement of the amount of recombinant proteins was not carried out but from comparisons to known amounts of baculovirus and transgenic plant expressed S proteins (Tuboly et al., 1994; Tuboly et al., 2000) it was estimated that approximately 5-10 μg protein/ 10⁶ cells can be obtained at 24 hours p.i. This figure is in accordance with that of Torres et al. (1995) for the expression of TGEV S gene and gene fragments in a human adenovirus vector without the use of additional external promoters.

•^•ι Three recombinant viruses were tested for their ability to induce TGEV specific immune responses in pigs. Those that carried the 2.2S gene in a left to right orientation induced a TGEV-specific response. It was concluded that a single oral dose of the recombinant virus was sufficient to induce both a systemic and a local humoral immune response. The efficiency of live vaccines is dependant on their ability to induce the required immune response in one dose, as the second injection may be less effective due to the immune response induced by the first injection. The antibodies induced by the recombinant viruses were capable of neutralizing both the PAdV-5 and the TGEV. The presence of TGEV specific IgA antibodies in the small intestine indicated that a local immune response was also induced. This is very important for TGEV and other viruses with replication localized to surfaces. A local immune response is particularly important against TGEV where the survival of the piglets depends on the slgA and slgA secreting plasma cells in the colostrum of the sow. Although challenge experiments were not done, it was concluded that recombinant PAdV-5 carrying the 2.2 kb S gene fragment could be a useful tool in the protection of swine herds against TGEV.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Table 1. Analysis of the recombinant viruses.

^aDNA profile based on Restriction endonuclease digestion,

"Expression of the S protein analyzed by Western blot, ^cNo. of S gene (protein) positive plaques/No. of plaques tested.

Table 2. Shedding of wild type and recombinant PAdV-5 viruses.

Virus Pig ;# Virus titers

Rectal swabs (days p.i ..) Int. Lung

1 2 3 4 5 6 7 8 9 1 3 weeks

0 p.i.

1 1 3 2 1 1 1 0 0 0 0 0 0

RPAdV-2.2S 2 1 3 2 2 2 1 0 0 0 0 0 0

3 2 3 3 2 1 0 0 0 0 0 0 0

4 1 3 2 1 0 0 0 0 0 0 0 0

ΔRPAdV-

2.2.Sc 5 2 2 1 1 1 1 0 0 0 0 0 0

6 2 2 1 1 1 1 0 0 0 0 0 0

7 1 3 2 1 1 1 0 0 0 0 0 0

ΔRPAdV-

2.2Sr 8 2 2 1 1 1 1 1 0 0 0 0 0

9 1 3 2 1 1 1 0 0 0 0 0 0

10 2 2 3 2 2 1 0 0 0 0 0 0

PAdV-5 11 3 4 3 2 * 2 2 1 0 0 0 0 0

12 2 1 2 2 1 1 0 0 0 0 0 0

Titers as loglO dilutions in 0.1 ml pi.= post infection

Table 3. TGEV and PAdV-5 specific antibody titers of pigs immimized with recombinant PadV and measured by virus neutralization and ELISA.

Virus Pig# VN TGEV specific ELISA

TGEV PAdV-5 Serum IgG Intestinal IgA Lung

IgA 5 6 7 2 1 RPAdV-2.2S 6 7 7 2 0 5 6 8 3 2

4 4 7 7 2 1

ΔRPAdV-2.2Sc 5 5 8 6 2 1 6 5 6 7 2 1

0 6 0 0 0

ΔRPAdV-2.2Sr 0 4 0 0 0

0 6 0 0 0

10 0 8 0 0 0 PAdV-5 11 0 7 0 0 0 12 0 7 0 0 0

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.

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DETAILED LEGENDS FOR VARIOUS FIGURES

Figure 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-ΔPH and pR-ΔHH genomic clones.

Figure 2. 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.

Figure 3. 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-ΔHH (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.

Claims

We Claim:

1. An isolated porcine adenovirus serotype 5 (PAdV-5) comprising a nucleic acid sequence comprising: (a) a nucleic acid sequence as shown in Figure 7 (SEQ.ED.NO.:l), wherein T can also be U;

2. An isolated PAdV-5 according to claim 1 comprising a nucleic acid sequence shown in Figure 7 (SEQ.ID.NO.:!) or a homolog or analog thereof.

3. A modified porcine adenovirus serotype 5 (PAdV-5) wherein a non- essential region has been deleted.

4. A modified PAdV-5 according to claim 3 wherein the E3 region has been deleted.

5. A modified PAdV-5 according to claim 4 wherein the deleted E3 region is as shown in Figure 13 (SEQJD.NO.:8) or Figure 14 (SEQ.ED.NO.:9).

6. A recombinant porcine adenovirus serotype 5 (PAdV-5) comprising a heterologous nucleic acid sequence that is stably integrated into the recombinant porcine adenovirus genome.

7. A recombinant PAdV-5 according to claim 6 comprising (a) a PAdV-5 according to any one of claims 3 to 5 and (b) a heterologous nucleic acid sequence integrated into a non-essential region of the PAdV-5.

8. A recombinant PAdV-5 as claimed in claims 6 or 7 wherein said recombinant porcine adenovirus comprises a live porcine adenovirus having virion structural proteins unchanged from those in a native porcine adenovirus from which said recombinant porcine adenovirus is derived.

9. A recombinant PAdV-5 as claimed in any one of claims 6 or 8 wherein said heterologous nucleic acid sequence encodes an antigenic polypeptide.

10. A recombinant PAdV-5 as claimed in claim 9 wherein said heterologous nucleic acid sequence encodes antigenic determinants of infectious agents causing intestinal or respiratory diseases in pigs.

11. A recombinant PAdV-5 as claimed in any one of claims 6 or 8 wherein said heterologous nucleic acid sequence encodes an immuno-potentiating molecule.

12. A recombinant PAdV-5 as claimed in claim 9 wherein said heterologous nucleic acid sequence encodes one or more antigenic determinants selected from the antigenic determinants of porcine parvovirus, mycoplasma hyopneumonia, porcine influenza virus, transmissible gastroenteritis virus (TGEV, porcine coronavirus), porcine rotavirus, hog cholera virus (classical swine fever), swine dysentery, African swine fever virus, pseudorabies virus (Aujeszky's disease virus), porcine respiratory and reproductive syndrome virus (PRRSV) and porcine circovirus (postweaning multisystemic wasting syndrome).

13. A recombinant PAdV-5 as claimed in claim 9 wherein said heterologous nucleic acid sequence encodes an antigenic determinant of transmissible gastroenteritis virus (porcine coronavirus).

14. A recombinant PAdV-5 as claimed in claim 11 wherein said heterologous nucleotide sequence encodes interleukin 3 (IL-3), interleukin 4 (E.4), gamma interferon (γlFN), porcine granulocyte macrophage colony stimulating factor (GM-CSF), and porcine granulocyte colony stimulating factor (G-CSF).

15. A recombinant PAdV-5 as claimed in any one of claims 6 to 14 wherein said heterologous nucleic acid sequence encodes an antigenic polypeptide and an immuno-potentiating molecule.

16. A recombinant PAdV-5 as claimed in any one of claims 6 to 15 wherein the heterologous nucleic acid sequence is stably integrated into non-essential regions of the porcine adenovirus genome.

17. A recombinant PAdV-5 as claimed in claim 16 wherein the nucleic acid of interest is stably integrated into the right hand end of the genome.

18. A recombinant PAdV-5 as claimed in claim 16 wherein the nucleic acid of interest is stably integrated into the E3 region of the genome.

19. A recombinant PAdV-5 as claimed in claim 18 wherein the nucleic acid of interest is stably integrated into the E3 region of the genome between map units at about 75 and about 82.

20. A method of producing a recombinant porcine adenovirus serotype 5 (PAdV-5) vector for use as a vaccine comprising inserting into a non-essential region of a porcine adenovirus genome, at least one heterologous nucleic acid sequence.

21. A method as claimed in claim 20, wherein the at least one heterologous nucleotide sequence is in association with an effective promoter sequence.

22. A method as claimed in claim 20, wherein prior to insertion of said heterologous nucleotide sequence, a restriction enzyme site is inserted into said non-essential region of said porcine adenovirus genome.

23. A use of a recombinant PAdV-5 according to any one of claims 6 to 15 as a vaccine.

24. A use according to claim 23 to prevent or treat an infectious organism in pigs.

25. A use according to claim 24 wherein said infectious organism is selected from the group consisting of porcine parvovirus, mycoplasma hyopneumonia, porcine influenza virus, transmissible gastroenteritis virus (TGEV, porcine coronavirus), porcine rotavirus, hog cholera virus (classical swine fever), swine dysentery, African swine fever virus, pseudorabies virus (Aujeszky's disease virus), porcine respiratory and reproductive syndrome virus (PRRSV) and porcine circovirus (Postweaning multisystemic wasting syndrome).

26. A use according to claim 24 wherein said infectious agent is TGEV (porcine coronavirus).